REPRODUCTION - Facultad de Ciencias Veterinarias

REPRODUCTION 

10

Abstract 

822 


Oxidative stress is implicated in loss of embryos through apoptosis. The results of study demonstrates that proportion of 

embryos exhibiting apoptotic morphology is more at advanced stages of development with relative abundance of Bax is 

higher than Bcl XL and cysteamine supplementation did not have significant effect on the apoptosis and relative 

abundance of Bax and Bcl XL. 

INTRODUCTION 

Apoptosis and effect of cysteamine 

supplementation in IVM and IVC media 

during in vitro development of buffalo 

(Bubalus bubalis) embryos 

The process of in vitro embryo production faced many changes since the birth of first in vitro produced (IVP) buffalo 

calf; however the final yield of blastocysts is low. Embryos in in vitro development are vulnerable to oxidative stress 

thereby the in vitro embryos suffer supra physiological oxygen tension and toxicity elicited by ROS. Supplementation of 

thiols such as cysteamine and Beta mercaptoethanol in embryo culture medium protected embryos from oxidative stress 

and improved the production efficiency 1,2,3 . The aim of the present study was to investigate the process of apoptosis in 

buffalo in vitro produced embryos and the effect of cysteamine on the development of in vitro buffalo embryos with 

respect to apoptosis. 

MATERIALS AND METHODS 

Anand V 1 ; Singh KP 1 ; Palta P 1 ; Manik RS 1 ; Singla S K 1 ; Chauhan MS 1,2 

1 Animal Biotechnology Centre, National Dairy Research Institute, 

karnal-132001, INDIA 

E- mail: chauhan_abtc@gmail.com 

Production of in vitro fertilized buffalo embryos 

Keywords: apoptosis - cysteamine - In Vitro - buffalo 

In vitro production of buffalo embryos was done as described in 1 .For experimental purpose in vitro maturation and in 

vitro cultre media was supplemented with cysteamine at 50ìM and 100 ìM respectively. 

Assessment of apoptosis by Acridine orange and Ethidium bromide (AO/EB staining) 

Assessment of apoptosis in different stages of embryos wad done as described in 4 . Embryos treated with 100 ìM H2O2 was 

used as control. Embryos are examined under fluorescence microscope (Excitation wavelength 450-490 nm; barrier filter 

515nm) after washing them in culture medium. 

Proceedings 9 th World Buffalo Congress

Semi quantitative RT PCR 


Total RNA isolation and cDNA preparation from embryos at different stages of development was done using “cell-to-cDNA 

kitII” (Ambion, Austin, TX) In all experiments 18S rRNA was used as an internal control. The mRNAs of Bax, BclXL and 18S 

rRNA were detected by PCR with specific primers for Bax Bcl XL and 18SrRNA with amplicon size 427,505 and 337 bp 

respectively using Taq DNA polymerase (Promega, Madison WI) The intensity of each band after 2% gel electrophoresis 

was assessed by densitometry using AlphaDigiDoc AD-1201 image analysis program. 

RESULTS 

The temporal course of apoptosis during in vitro embryo development is shown in Table1&Figure 1 Relative 

abundance of Bax transcripts was significantly higher (p

824 

Fig. 1 

Fig. 3 


Fig. 4 

Fig. 2 

Fluorescent microscopic images of buffalo 

embryos at various stages of development 

stained with AO/EB. Images obtained by 

conventional epifluorescent microscopy. 

(A)

DISCUSSION 


The results of the present study were consistent with other mammalian species that cell proliferation in IVP buffalo 

embryos is associated with apoptotic cell death. Apoptosis was first observed at 8- to 16-cell stage in embryos which 

otherwise had normal morphology The Bcl-2/Bax ratio appears to determine the fate of a cell 5 .RT-PCR analysis of 2- to 12cell 

human embryos revealed transcripts for both Bax and Bcl-2 throughout these early cleavage stages 6 . The detection of 

apoptosis related genes in buffalo pre implantation embryos indicates that buffalo embryos are capable of undergoing 

programmed cell death and constitutively express genes that are required to execute the death program. High expression 

of cell death gene (Bax) may confirm that early embryos were inherently biased towards PCD 7 .Though Bax was highly 

expressed in preimplantation buffalo embryos, the fate (survival or death) of embryos is finally determined along with 

other factors such as external stimuli, internal defects and activation of other apoptotic genes .ROS derived oxidative 

stress during in vitro culture of embryos induce mediate mitochondria dependent apoptotic response 8 . Addition of 

antioxidants to the culture media can decrease apoptosis in embryos during culture by preventing oxidant-mediated 

damage 9 . The results of the present study show that supplementation of cysteamine did not have significant effect on 

the incidence of apoptosis detected by AO/EB staining. Enhanced development of buffalo IVM-IVF embryos following 

the addition of cysteamine to a maturation and culture medium containing serum has been reported 1,3 but the mechanism 

through which cysteamine brings about this development is not clear. We hypothesized that cysteamine would have 

promoted development of embryos through anti- apoptosis but there was no significant difference between the level of 

expression of Bax and Bcl XL between control and cysteamine treated groups. Fetal bovine serum supplementation would 

have masked the effect of cysteamine. Study with serum free medium may give the clear picture of mechanism of 

cysteamine. 

In conclusion, the results of the present study suggest that the incidence of apoptotic morphology was significantly 

higher in produced buffalo embryos at advanced stages of embryos, the relative abundance of pro-apoptotic Bax 

transcripts was significantly higher than the anti-apopotic Bcl XL transcripts in all the developmental stages and supplementation 

of cysteamine in IVM and IVC media did not have any significant effect on the apoptosis and the relative 

abundance of Bax and Bcl XL transcripts. 

Acknowledgement. This work was supported by Niche project on buffalo production and reproduction genomics 

REFERENCES 

1. Anand T., D.Kumar, M.S. Chauhan, R.S. Manik and P.Palta. 2008. Cysteamine supplementation of in vitro maturation medium, in vitro culture 

medium or both media promotes in vitro development of buffalo (Bubalus bubalis) embryo. Rep Fert Dev. 20:253-257 

2. de Matos, D.G., B. Gasparrini, S.R. Pasqualini and J.G.Thompson. 2002. Effect of glutathione synthesis stimulation during in vitro maturation 

of ovine oocytes on embryo development and intracellular peroxide content. Theriogenology.57: 1443-1451 

3. Gasparrini, B., H.Sayoud, G. Neglia, D.G. de Matos, I. Donnay and L. Zicarelli. 2003. Glutathione synthesis during in vitro maturation of buffalo 

(Bubalus bubalis) oocytes: effects of cysteamine on embryo development. Theriogenology. 60:943-952. 

4. Velez-Pardo, C., Morales, A.T., Del Rio, M.J. and Olivera-Angel, M. 2007. Endogenously generated hydrogen peroxide induces apoptosis via 

mitochondrial damage independent of NF- B and p53 activation in bovine embryos. Theriogenology, 67: 1285-1296 

K 

5. Wyllie, AH. Kerr, J.F., Currie, A.R. 1980. Cell death: the significance of apoptosis. Int Rev Cytol, 68:251-306. 

6. Warner, C. M., Cao, W., Exley, G. E., McElhinny, A. S.,Alikani, M., Cohen, J., Scott, R. T. & Brenner, C. A. (1998).Genetic regulation of egg and 

embryo survival. Hum. Reprod. 13 (Suppl. 3), 178–196. 

7. Oltvai ZN,Milliman CL, Korsmeyer SJ. 1993. Bcl-2heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell 

death. Cell 74:609–619. 

8. Herrera, B., A.M. Alvarez, A. Sanchez, M. Fernandez, C. Roncero, M. Benito and I.Fabregat. 2001. Reactive oxygen species (ROS) mediates the 

mitochondrial-dependent apoptosis induced by transforming growth factor (beta) in fetal hepatocytes. FASEB J. 15:741–751. 

9. Uhm, S.J., M.K. Gupta, J.H. Yang, S.H. Lee and H.T. Lee. 2007. Selenium improves the developmental ability and reduces the apoptosis 

inporcine parthenotes. Mol Reprod Dev. 74:1386–1394. 

Buenos Aires, Abril 2010 825

ABSTRACT 

826 


Assessment of Buffalo Semen 

with MTT Reduction Assay 

M. Iqbal † , M Aleem † , A. Ijaz ‡ *, H. Rehman ‡ and M. S. Yousaf ‡ 

Department of Theriogenology † , Department of Physiology and Biochemistry ‡ , 

University of Veterinary and Animal Sciences, Lahore-54000, Pakistan 

MTT [3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay is commonly used to validate the viability 

of metabolically active cells. This study was conducted to evaluate and validate the MTT test to assess the spermatozoa 

viability of Nili-Ravi buffalo bulls and compare the efficiency of the test with the supra-vital staining technique 

(eosino-nigrosine) and hypo-osmotic swelling test (HOST). Fresh semen samples from breeding Nili-Ravi buffalo bulls (n 

= 20) were collected using an artificial vagina. After assessing the quality of the semen for routine parameters, the MTT 

assay was carried out in phosphate buffer saline. Results revealed a high significant correlation (r = 0.995) between the 

viability of spermatozoa and the rate of reduction of MTT. The other proportions of same semen samples showed a poor 

relationship between the eosine-nigrosine method (r = -0.32), the hypo-osmotic swelling test (r = -0.12) and spermatozoal 

motility (r = -0.08). However, MTT was found to be superior over other tests as it was able to determine those 

spermatozoa which were more than 90% viable. In conclusion, it can be said that the MTT is a simple, robust test that 

can be used to select the Nili-Ravi buffalo bulls on the basis of spermatozoal quality. 

INTRODUCTION 

Keywords: MTT, Nili-Ravi, Buffalo, Spermatozoa, Viability 

It is very important to have high quality semen as quality of spermatozoa is positively correlated with the fertility in 

bovines (Garner et al., 1997). Most of the methods that are currently used to evaluate the quality of semen such as supravital 

staining and HOST are subjective in nature and can easily be influenced by the experience of the analyst (McNiven 

et al., 1992). On the other hand, computer-assisted analysis of spermatozoa requires special instruments and softwares 

that provide rapid and objective evaluation of the semen quality. However, these techniques are expansive and sometimes, 

not readily available in an average breeding farms. Therefore, it becomes imperative to look for some other 

techniques that may be cheap, objective and can easily be performed without the assistance of any sophisticated 

expansive instrument. 

In this regard, various analytical techniques have been developed to evaluate the quality of spermatozoa in bovines. 

Among others, assessment of the metabolic status of spermatozoa is one of the techniques that can provide valuable 

information regarding the spermatozoa characteristics. Reduction activity of spermatozoa depends upon the ability of 

metabolically active spermatozoa to reduce the specific stains. The ability of spermatozoa to reduce the resazurin redox 

dye (Foote, 1999; Zrimsek et al., 2004) and methylene blue dye (Chandler et al., 2000) had been employed to evaluate 

the semen quality in boar and bulls respectively. MTT [3-(4, 5-dimethylthiazol-2-y1)-2, 5-diphenyltetrazolium bromide], 

a yellow water-soluble tetrazolium salt dye, is converted to water-insoluble purple formazan by the succinate dehydrogenase 

system of an active mitochondria by the reductive cleavage of its tetrazolium ring (Slater et al., 1963). The amount 

of formazan formed can, thus, be determined spectrophotometrically and it serves as an estimate of the number of active 

mitochondria and hence the viable cells in a sample (Denizot and Lang, 1986; Song et al., 2007). 



Although MTT assay has successfully been evaluated in different animal species (Aziz et al., 2005; Aziz, 2006; Byun et al., 

2008) but literature pertaining to the use of this technique for buffalo semen is still lacking. Therefore, we evaluated the 

viability of buffalo spermatozoa using the MTT reduction assay and compared it with eosin-nigrosin staining (E-N), HOST 

and spermatozoal motility. 


Nili-Ravi buffalo bulls, maintained at the Semen Production Unit, Qadirabad, Punjab-Pakistan were used in the study. 

The bulls were routinely used for collection of semen, which was supplied to various Artificial Insemination Centers 

throughout the Punjab, Pakistan. The breeding bulls were divided into 2 age groups. The age of group A bulls (n = 13) 

was about five years, while the bulls in group B (n = 7) were ranged between 6-8 years. The circumference of both testes 

of the breeding buffalo bulls was recorded. The experimental bulls were maintained under naturally prevailing climatic 

conditions, with free access to drinking water. Each bull was fed seasonal green fodder (Berseem) and wheat straw. 

Semen Collection and Evaluation 

Semen from the experimental bulls was collected once a week with two ejaculates per collection using an artificial vagina. 

Each bull was given sufficient time for sexual arousment before semen collection, with one to two false mounts being 

allowed for sexual stimulation. Each collection that was comprised of two ejaculates was pooled and divided into various 

aliquots. Following the determination of the ejaculate volume and the concentration of spermatozoa in each semen 

sample, spermatozoal motility, plasma integrity of spermatozoa in terms of HOST, percentage of live and dead spermatozoa 

and MTT reduction assay of each ejaculate was determined. 

Hypo-osmotic Swelling Assay (HOST) 

Plasma membrane integrity of fresh spermatozoa was assessed using a HOST assay as described earlier (Ijaz et al., 2009; 

Adil et al., 2009). Briefly, the hypo-osmotic solution was prepared by dissolving 0.735 g of sodium citrate and 1.351 g 

fructose in 100 ml distilled water (osmotic pressure = 190 mOsm/kg). For the assay, 50 ìl of semen was mixed with 500 ìl 

of the pre-warmed (37 °C) HOST solution and incubated at 37 °C for 45 minutes. Two slides of each sample were prepared 

and examined under a phase contrast microscope (X 40). Two hundred spermatozoa were microscopically counted per 

sample and the number of spermatozoa showing characteristic swelling of tail, an indicative of intact plasma membrane, 

was recorded. 

Percentage of Live/Dead Spermatozoa 

To determine the viability of spermatozoa, a drop of semen sample was mixed with a larger drop of the eosin (1%; Merck, 

Germany) and nigrosin (5%; Merck, Germany) stains on a pre-warmed slide using applicator stick and a thin smear was 

made using another slide (Khan and Ijaz, 2008). After air-drying, the smear was observed under a phase contrast 

microscope (X 400) for unstained heads of the spermatozoa (live) and stained/partial stained heads of the spermatozoa 

(dead). A total of two hundred spermatozoa were counted to determine the percentage of live and dead spermatozoa. 

MTT Reduction Assay 

The MTT assay was performed according to the method of Mosmann (1983). The semen samples were diluted using a 

phosphate buffer saline (PBS) solution to obtain a concentration of 30 × 10 6 spermatozoa/ml. Six wells of the 96-well 

microplate were used. A 100 ìl of semen sample plus 10 ìl of MTT stock solution (5 m MTT/ml of PBS) was placed in each 

well. Six replicates of each collection were placed in six different wells of the same plate. The rates of MTT reduction were 

recorded immediately and after incubation at 37 o C for one hour (Aziz, 2006; Naser-Esfahani et al., 2002) using a 


828 


spectrophotometer (MS2 Reader) at a wavelength of 550 nm. A small drop of the semen sample after the measurement of 

second optical density (OD) was observed under the microscope (X 1000) for the viable cells that developed colored 

formazan following reduction by the MTT assay. MTT reduction rate (optical density) for each sample was calculated by 

concurring the difference between the first and second reading of the spectrophotometer. 

Statistic Analysis 

Statistical program SPSS for window (version 10.0.1, SPSS Inc., Chicago, Illinois, USA) was used for data analysis. The 

Kolmogorov Smirnov test was used to test the normal distribution of the data. Results are expressed as Means ± S.E. 

Pearson correlation coefficients and Regression analysis was used to evaluate the efficacy of the MTT assay for the 

assessment of sperm viability of the buffalo semen. An independent Student’s t test was used to compare the means 

between two groups for each parameter. A probability value at P < 0.05 was considered to be significant. 

RESULTS 

The mean volume of semen collected was more (P < 0.05) in group A (7.12 ± 0.5) compared to bulls of group B (5.29 ± 

0.6). However, the concentration of spermatozoa (562.5 ± 31.6 vs. 642.2 ± 66.4 millions/ml) and testes circumference 

of the bulls (34.7 ± 0.14 vs. 34.47 ± 0.2 cm) were similar (P > 0.05) in both groups. There was no significant difference 

between two groups for the remaining spermatozoal variables recorded. Data were, therefore, pooled for both groups (n 

= 20) for subsequent analyses. The means of percentages of live spermatozoa for MTT, E-N, HOST and motility were 74.7 

± 1.9, 68.8 ± 0.7, 59.5 ± 1.0 and 72.3 ± 0.5 respectively. After incubation of the semen samples for an hour, a regression 

equation of the relationship between the MTT reduction rate and the percentage of viable spermatozoa was calculated (y 

= 90.403x -0.109). The corresponding curve is presented in Figure 1 and this was later used to determine the viability 

of the spermatozoa based on the other diagnostic tests. The OD was significantly (P < 0.001) correlated (r = 0.995) with 

the percentage of viable spermatozoa. Results also showed a very low negative correlations (P > 0.05) between the results 

of the MTT reduction rate and the percentage of viable spermatozoa as determined by either eosin or nigrosin staining 

(r = -0.32, Figure 2), HOST (r = -0.12; Figure 3) or percentage spermatozoal motility (r = -0.08; Figure 4). The distribution 

of the experimental breeding bulls based on the various percentages of viable spermatozoa as determined by 

different diagnostic tests is set out in Table 1. Most of the animals exhibited the presence of viable spermatozoa were 

between a range of 60-80% in all the conducted tests. However, MTT was able to determine the >90% viable spermatozoa 

in two bulls (Table 1). 

Figure 1 Relationship between MTT reduction rate and 

percentage of viable spermatozoa. The regression curve 

shown is y = -0.109+90.402x; r =0.995; n = 20; OD = 

optical density 


percentage of viable spermatozoa (eosin and nigrosin 

staining). The regression curve shown is y = 77.15- 

10.15x; r =- 0.32; n = 20; OD = optical density 


DISCUSSION 


The data generated in this trial provide an evidence of a relationship between MTT reduction and semen quality. Different 

tests or methods have been developed for the differentiation and selection of viable spermatozoa (WHO, 1992). Some 

of the tests have only a diagnostic value like dye exclusion tests while others have a more clinical application such as 

HOST. The dye exclusion tests like eosin–nigrosin are based on the cell permeability and, therefore, the viable spermatozoa 

remain colorless. The non-viable spermatozoa, on the other hand, either stain red. However, in HOST, the spermatozoa 

are exposed to a hypo-osmotic condition, thus an influx of water results in the swelling of the cytoplasmic spaces 

causing curling of viable sperm tail fibers (Hossain et al., 1998). Therefore, we investigated the diagnostic value of the 

MTT to select the viable spermatozoa in Nili-Ravi buffalo bulls and compared it with E-N, HOST and motility of spermatozoa. 

Mosmann (1983) used MTT tetrazolium salt for the assessment of cellular viability, proliferation and cytotoxicity 

assay of lymphocytes. Additionally, MTT assay has also been used in many studies to evaluate the viability of different 

cells (Carmichael et al., 1987; Campling et al., 1988; Freimoser et al., 1999). 

This study hopes to present a new diagnostic test using the MTT for sperm viability test in Nili-Ravi buffalo bulls. 

Formation of MTT formazan granules or spikes around the sperm mid-piece showed that sperm mitochondria to contain a 

succinate dehydrogenase system that convert MTT to formazan. The presence of formazan granules in this mid-piece 

region identifies the viability of spermatozoa. Results indicate a high correlation (r = 0.995) between the MTT reduction 

rate and spermaotozoal viability (Figure 1). A high correlation between MTT and spermatozoal viability has also been 

found in bovine (Aziz, 2006), stallion (Aziz et al., 2005), boar (Byun et al., 2008), fowls (Hazary et al., 2001) and human 

(Naser-Esfahani et al., 2002). The MTT reduction rate in this trial was determined after an hour of incubation that was 

shorter than the method of Mosmann (1983). This seems logical as spermatozoa are highly active cells and contain more 

mitochondria. Therefore, less time may be needed for the reduction of the dye. A similar observation was also noted in 

bovines (Aziz, 2006) and equines (Aziz et al., 2005) where the optimal time for formazan reading was one hour. Naser- 

Esfahani et al. (2002) found that the optimal time for formazan reading was between 1.5 and 2.5 h after the addition of 

spermatozoa to MTT. 

As E-N and HOST tests are based on the functional aspect/integrity of spermatozoa, while MTT is based on the mitochondrial 

activity of the spermatozoa, a high significant correlation coefficient was expected between these tests and 

motility. However, very low relationships were noted between the MTT and the other tests (Figures 2, 3 and 4). There 

exists a dearth of reports regarding the relationship between the MTT and the other tests used in the present study. A 


percentage of viable spermatozoa (HOST). The regression 

curve shown is y = 64.74-6.36x; r =-0.12; n = 20; OD = 

optical density 


percentage of viable spermatozoa (motility). The regression 

curve shown is y = 73.56-1.592xl; r =-0.08; n = 20; 

OD = optical density 


830 


high correlation (P < 0.001) between MTT test and E&N was noted in boar semen extended with Beltsville thawing 

solution. On the other hand, a weak correlation (r = 0.4) was noted between these two variables in the semen samples of 

human in HAM’S F10 solution instead of PBS that was used in the current study. It seems that the nature of the medium 

in which the MTT is carried out also affects the outcomes of the test. Similarly, lack of correlation between MTT and HOST 

might also be due to the nature of the HOST test. It has been suggested that during HOST procedure, hypo-osmotic 

shock on its own induces membrane damage and, therefore, increases the percentage of false positive spermatozoa, 

which means that the percentage of dead spermatozoa is higher than the expected value (Ramirez et al., 1992). 

The results of the present study also suggest that the MTT assay can be used to evaluate the spermatozoal quality in Nili- 

Ravi buffalo bulls. An additional advantage of this test is that it can determine the spermatozoa having more than 90% 

viability that could not be observed with other traditional tests used for the evaluation of the buffalo semen (Table 1). 

This characteristic may be exploited to increase the doses of the semen to be used for insemination. 

Conclusion 

Table 1 Distribution of breeding bulls based upon the percentage of viable spermatozoa 

as determined by different diagnostic tests 

MTT test was found to be as a reliable method for the evaluation of semen in buffalo bulls and can used successfully in 

routine analysis, where practical aspects like time, costs and practicability are important. 



REFERENCES 

Adeel, M., A. Ijaz, M. Aleem, H. Rehman, M. S. Yousaf, and M. A. Jabbar. 2009. Improvement of liquid and frozen-thawed semen quality of Nili-Ravi buffalo 

bulls (Bubalus bubalis) through supplementation of fat. Theriogenology, 71:1220-1225. 

Aziz, D. M. 2006. Assessment of bovine sperm viability by MTT reduction assay. Anim.Reprod. Sci. 92:1-8. 

Byun, J. W., S. H. Choo, H. H. Kim, Y. J. Kim, Y. J. Hwang, and D.Y. Kim. 2008. Evaluation of boar sperm viability by MTT reduction assay in Beltsville 

thawing solution extender. Asian-australas. J. Anim. Sci. 2:494-498. 

Campling, B.G., J. Pym, P. R. Galbraith, and S. P. Cole. 1988. Use of the MTT assay for rapid determination of chemo sensitivity of human leukemic 

blast cells. Leuk. Res. 12:823-831. 

Carmichael, J., W. G. DeGraff, A. F. Gazdar, J. D. Minna, and J. B. Mitchell. 1987. Evaluation of a tetrazolium-based semi automated colorimetric assay: 

assessment of radio sensitivity. Cancer Res. 47: 943-946. 

Chandler, J. E., C. M. Harrison, and A. M. Canal. 2000. Spermatozoal methylene blue reduction: an indicator of mitochondrial function and its 

correlation with motility. Theriogenology. 54:261–271. 

Denizot, F., and R. Lang. 1986. Rapid colorimetric assay for cell growth and survival. J. Immunol. Methods. 89: 271-277. 

Foote, R. H. 1999. Resazurin reduction and other tests of semen quality and fertility of bulls. Asian J. Androl. 1:109-114. 

Freimoser, F. M., C. A. Jakob, M. Aebi, and U. Tuor. 1999. The MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide] assay is a fast 

and reliable method for colorimetric determination of fungal cell densities. Appl. Environ. Microbiol. 65:3727-3729. 

Garner, D. L., C. A. Thomas, H. W. Joerg, J. M. DeJarnette, and C. E. Marshall. 1997. Fluorometric assessments of mitochondrial function and viability 

in cryopreserved bovine spermatozoa. Biol. Reprod. 57:1401-1406. 

Hossain, A. M., B. Rizk, S. Barik, C. Huff, and I. H. Thorneycroft. 1998. Time course of hypo-osmotic swellings of human spermatozoa: Evidence of 

ordered transition between swelling subtypes. Hum. Reprod. 13:1578-1583. 

Ijaz, A., A. Hussain, M. Aleem, M. S. Yousaf, and H. Rehman. 2009. Butylated hydroxytoluene inclusion in semen extender improves the post-thawed 

semen quality of Nili-Ravi buffalo (Bubalus bubalis). Theriogenology, 71:1326-1329. 

Khan, M. I. R., and A. Ijaz. 2008. Effects of osmotic pressure on motility, plasma membrane integrity and viability in fresh and frozen-thawed buffalo 

spermatozoa. Animal. 2:548-553. 

McNiven, M. A., R. K. Gallant, and G. F. Richardson. 1992. In vitro methods of assessing the viability of trout spermatozoa. Theriogenology. 38:679- 

686. 

Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Meth. 

65:55-63. 

Naser-Esfahani, M. H., R. Aboutorabi, E. Esfandiari, and M. Mardani. 2002. Sperm MTT viability assay: a new method for evaluation of human sperm 

viability. J. Assoc. Reprod. Genet. 19:477-482. 

Ramirez, J. P., A. Carreras, and C. Mendoza. 1992. Sperm plasma membrane integrity in fertile and infertile men. Andrologia. 24:141-144. 

Slater, T. F., B. Swyer, and U. Straeuli. 1963. Studies on succinate-tetrazolium reductase systems. III. Points of coupling of four different tetrazolium 

salts. Biochim. Biophys. Acta. 77:383-393. 

WHO. 1992. Laboratory Manual for the Examination of Human Semen and Sperm Cervical Mucus Interaction. 3rd ed. Cambridge University Press, UK. 

Zrimsek, P., J. Kunc, M. Kosec, and J. Mrkun. 2004. Spectrophotometric application of resazurin reduction assay to evaluate boar semen quality. Int. 

J. Androl. 27:57-62. 


832 


Calving interval in water buffaloes 

Murrah in Zona da Mata 

de Pernambuco 

Sérgio Augusto de Albuquerque Fernandes 1 , 

Glesser Porto Barreto 2 , Severino Benone Paes Barbosa 2 , Kleber Regis Santoro 2 

1 Universidade Estadual do Sudoeste da Bahia, Brazil - 2 Universidade Federal Rural de Pernambuco, Brazil 

fernandes_pe@hotmail.com 

he aim of the study was analyze calving interval (CI) of buffalo’s Murrah herd. Data from a herd of 75 Murrah dams 

breeds, collected from 1982 to 1996. Animal were raised in the extensive system in native pasture, with supplementation 

in the dry period and mineralization in the Zona da Mata de Pernambuco. It was determined the 

calving interval (CI) of Murrah buffaloes. The statistical analyses procedure was carried out by GLM model (SAS), fitting 

mixed linear model. Was not observed sex influence in the CI. The average adjusted for the CI was 424.50 days (male) and 

for was 422.63 (females), with no significant statistical difference. IP average observed was 413.3 days. Was observed 

calving concentration in autumn/winter (69.9%). The season of calving did not influence the CI (autumn /winter - 

411.57 days, spring / summer - 435.57 days). The buffaloes have seasonal reproductive behavior. 

Keywords: breeding, seasonality, seasonality 



Comparative Efficacy of GnRH, lens esculents 

moench and randia dumetorum for the Treatment 

of Anoestrus in Nili-Ravi Buffalo 

Abstract 

M. S. Akhtar, A. A. Farooq 1 , S. A. Muhammad 2 , L. A. Lodhi 3 , C. S. Hayat 1 and M. Aziz 4 

The Islamia University of Bahawalpur, Pakistan. 1 Bahaud Din Zikriya University, Multan, Pakistan. 

2 College of Veterinary Sciences, Jhang, Pakistan. 3 University of Agriculture, Faisalabad, Pakistan. 

4 Buffalo Research Institute, Pattoki, District Kasur, Pakistan 

The present study was accomplished on with the objective to determine the effectiveness of various ethno-veterinary 

practices for the treatments of anoestrus in Nili-Ravi buffalo and to compare them with hormonal treatment. A total of 60 

Nili-Ravi buffalo with the history of anoestrus maintained at Buffalo Research Institute, Pattoki, District Kasur were 

divided into four groups (A, B, C, D). Group A (n=15) was given orally 800 grams lens esculents moench daily for three 

days where as group B (n=15) was given orally about 15 grams of randia dumetorum daily for four days. Group C (n=15) 

was given a single intra-muscular injection of GnRH at the dose rate of 100 µg where as group D (n=15) was given no 

treatment and served as control. The blood samples from each buffalo were collected before the start of treatments and 

after the treatments, samples were taken after every three days interval from all experimental buffaloes for progesterone 

(P 4 ) estimation. In group A, the percentage of buffaloes showing estrus was 46.66% whereas in animals of group B, the 

respective value was 66.66%. The percentage of buffaloes exhibiting estrus in group C was 73.33% and in the control 

group (group D) was zero %. The estrus showing animals percentages were higher with GnRH treatment than lens esculents 

moench and randia dumetorum treated buffaloes. In all four groups, serum progesterone concentration was below 0.25 

ng/ml before the start of treatments. At estrus, the progesterone concentration was 0.33, 0.34 and 0.38 in animals of 

group A, B and C respectively. It was concluded that the use of GnRH treatment is more effective as compared to lens 

esculents moench and randia dumetorum for the treatment of anoestrus buffaloes. 

INTRODUCTION 

Key words: buffalo, anoestrus, lens esculents moench, randia dumetorum, GnRH. 

Buffalo is of high economic importance for farmers in Pakistan. The reproductive performance is poor due to various 

diseases of reproductive system and low reproductive efficiency remains a major economic problem. The failures of 

ovarian follicle to reach mature size and ovulate (anoestrus) or abnormal over development of follicles on the ovary 

(cystic follicles; Kesler and Garverick, 1982) represent significant sources of reproductive inefficiency in cattle experiencing 

negative energy balances (Bauman and Currie, 1980). The reproductive efficiency of buffalo is affected by 

inherent late maturity and prolonged intercalving interval (Singh et al., 2000). The longer calving interval is due to the 

long service period, which is affected by various factors, including postpartum anoestrus. The incidence of anoestrus in 

buffalo kept under rural managemental conditions in Pakistan was 35% (Anwar et al., 2003). In buffaloes, under good 

management and adequate nutrition, the ovarian activity commences within 60 days after calving (Usmani et al., 1985). 


834 


In the last many years, the farmers experimented several natural remedies in animal reproduction and developed an 

intrinsic and invaluable knowledge from generation to generation. These invaluable practices have been passed from one 

generation to another by word to mouth. Various ethno-veterinary practices have also been used in the field for the 

treatment of anoestrus in Nili-Ravi buffalo but the effectiveness of these treatments have never been verified scientifically. 

There is an urgent need to evaluate the efficacy of these ethno-veterinary treatments. The main objective of this 

investigation was, therefore, to determine the effectiveness of various ethno-veterinary practices for the treatments of 

anoestrus in Nili-Ravi buffalo and to compare them with hormonal treatment. 


The present study was accomplished on 60 Nili-Ravi buffalo with the history of true anoestrus and maintained at Buffalo 

Research Institute, Pattoki, District Kasur. To confirm no corpus leutum on ovaries, these buffaloes were examined two 

times through rectal palpation at 11 days interval. These animals were divided into four groups (A, B, C, D) comprising 

of 15 animals in each group. Group A was given orally 800 grams lens esculents moench daily for three days where as group 

B was given orally about 15 grams of randia dumetorum daily for four days. Group C was given a single intra-muscular 

injection of GnRH at the dose rate of 100 µg where as group D was given no treatment and served as control. All buffaloes 

were kept under similar managemental conditions. Detection of estrus was started after the treatments in each group. All 

buffalo were observed for estrus twice daily (0700 and 1800) for approximately 1 h during the experiment. A buffalo was 

recorded to have shown estrus if she stood to be mounted by a herd mate. 

The blood samples from each buffalo were collected before the start of treatments and after the treatments, samples were 

taken after every three days interval from all experimental buffalo for progesterone (P 4 ) estimation. 

About 20 ml of blood from each animal was collected in a clean sterilized glass test tube through venipuncture of jugular 

vein using a sterile 16 gauge needle. The test tubes containing blood were placed in slanting position for one hour to 

let the serum ooze out and was aspirated carefully placed in glass vials, labeled and stored at -20 o C till analysed. 

Progesterone was analyzed through ELISA. The data thus collected was analyzed statistically. 

Table 1: Mean (±SD) values of progesterone (ng/ml) in anoestrus 

Nili-Ravi buffaloes before and after the different treatments. 

Values sharing different superscripts in a row differed significantly (P

RESULTS 

DISCUSSION 


In group A, the percentage of buffaloes showing estrus was 46.66% whereas in animals of group B, the respective value 

was 66.66%. There was high number of buffaloes showing estrus in group A and B as compared to control. The percentage 

of buffaloes exhibiting estrus in group C was 73.33% and in the control group (group D) was zero %. Group C animals had 

higher estrus as compared to group A and B. The mean time interval from treatment to estrus was around 12 to 15 days 

in group A, B and C buffaloes. The estrus showing animals percentages were higher with GnRH treatment than lens 

esculents moench and randia dumetorum treated buffaloes. 

The mean serum progesterone concentration before and after treatments is shown in table 1. In all four groups, 

serum progesterone concentration was below 0.25 ng/ml before the start of treatments. After treatments the progesterone 

concentration was increasing gradually. At estrus, the progesterone concentration was 0.33, 0.34 and 0.38 in 

animals of group A, B and C respectively. 

In the present study two ethno-veterinary treatments (lens esculents moench and randia dumetorum) for anoestrus 

condition in buffaloes have been compared with hormonal treatment (GnRH). The main aim of these treatments was to 

induce ovulation and estrus by stimulating maturation of ovarian follicles and then to compare the efficacy of ethnoveterinary 

treatments with hormonal treatment. 

Since no work has been previously on lens esculents moench and randia dumetorum to bring animals into estrus, so no 

comparable data is there. There were high percentage of buffaloes showing estrus in group C compared with group A and 

B. This shows that GnRH effectively stimulated the maturation of ovarian follicles. The animals of group B showed higher 

estrus percentage than A. The estrus percentage together with progesterone concentrations at estrus in group B and C 

buffaloes is comparable. It seems that randia dumetorum may have potential to induce follicular maturation in true 

anoestrus buffaloes. The main factors delaying the increase in LH pulse frequency are suckling, lower energy intake, 

inadequate body reserves, increased portioning of energy to milk production and peri-partum diseases (Rhodes et al., 

2003). 


836 


Based on the information obtained from this study, it was concluded that the use of GnRH treatment is more effective as 

compared to lens esculents moench and randia dumetorum for the treatment of anoestrus buffaloes. It is suggested that 

randia dumetorum maybe given with different doses to optimize its use in anoestrus buffaloes. 

Acknowledgements. 

The authors wish to thank Mr. Abdul Latif and Mr. Muhammad Rashid for assistance in giving treatments to animals and 

the collection, analysis of serum samples. 

REFERENCES 

1. Anwar, M., N. Ullah, A. Mehmood and S. M. H. Andrabi, 2003. Postpartum anoestrus of Nili-Ravi buffaloes maintained under rural and peri-urban 

management around Islamabad, Pakistan Vet. J., 23:114-117. 

2. Bauman, D. E. and W. B. Currie, 1980. Partitioning of nutrients during pregnancy and lactation: A review of mechanisms involving homeostasis 

and homeorhesis. J. Dairy Sci., 63: 1514. 

3. Kesler, D. J. and H. A. Garverick, 1982. Ovarian cyst in dairy cattle. A review. J. Anim. Sci., 55: 1147. 

4. Rhodes, F. M., S. McDougall, C. R. Burke, G. A. Verkerk and K. L. Macmillan, 2003. Invited Review: Treatment of cows with an extended 

postpartum anoestrus interval. J. Dairy Sci., 86: 1876-1894. 

5. Singh, J., A. S. Nanda and G. P. Adams, 2000. The reproductive pattern and efficiency of female buffaloes. Anim. Reprod. Sci., 60: 593-604. 

6. Usmani, R. H., M. Ahmad, E. K. Inskeep, R. A. Dailey, P. E. Lewis and G. S. Lewis, 1985. Uterine involution and postpartum ovarian activity 

in Nili-Ravi buffaloes. Theriogenology, 24: 435-44 


Abstract 


Correlations between uterine involution, 

first estrus post calving and milk production 

on Murrah buffaloes 

Snel-Oliveira, M.V. 1 ; Fidelis, A.A. 2 ; Borges, P.H.R. 2 ; Doroteu, E.M. 1 ; Valadares, 

M.L. 1 ; Wetzel-Gastal, D. 3 ; Sartori, R. 4 ; Neves, J.P. 5 

1 UPIS-FAVET, Brasília, DF; 2 Private Veterinarian; 3 UTL-FMV, Lisboa; 

4 ESALQ-USP, Piracicaba, SP; 5 UnB - FAV, Brasilia, DF. 

This work aimed to correlate the uterine involution period (UIP) with the interval calving/ first estrus (ICFE), the UIP 

with the milk production during the first sixty lactation days (MP60d) and ICFE with MP60d. Data from 5 primiparous 

murrah buffaloes and 9 multiparous with normal calving during autumn in central-west Brazil, body condition score of 

3.5 to 4.0, raised in pasture and milked without calf. The Pearson correlation was used to assess the correlations between 

MP60d, UIP and ICFE. Significant differences were observed for MP60d (p0.05) between the multiparous and primiparous 

for IUP (25.78±6.14; 25.40±3.85 44 days, respectively), neither for ICFE (42.78±7.98, 37.40±9.91 days, respectively). 

There were no significant correlations between UIP and MP60d (r=0.23, p>0.05), nor between IPPE and MP60d 

(r=0.28, p>0.05). There was a significant correlation between UIP and ICFE (r=0.60, p

838 


with the females throughout the experiment. The animals were milked once a day with milking machine, without calf. The 

milk was automatically measured (De Laval milking Marc 5), weekly, from the first week after calving until the end of 

lactation. For this study only the production of the first 60 days of lactation was considered. To assess the correlations 

between MP60d, UIP and ICFE analysis of the Pearson correlation was performed. 

RESULTS AND DISCUSSION 

As there were no significant differences between primiparous and multiparous cows in mean IUP and mean ICFE (p = 

0.05) (Table 1), all animals were considered independently of the group for analysis of correlation. The IUP and ICFE were 

correlated (r=0.61, P < 0.05), indicating that the animals with a higher IUP had a greater ICFE. Similar correlation (r = 

0.53, P 0.05). These results are consistent with those reported for Murrah buffaloes with low milk 

production 6 . However, in a review 7 , studies were reported without and with correlation between these parameters where 

animals with higher milk production showed a longer UIP than those with lower production. There were no significant 

correlations between MP60d and ICFE. (r = 0.10, p >0.05), it agrees with those reported for Murrah buffaloes with low 

milk production 4 . In review 8 there was reported a correlation between these two parameters only when the milk yield was 

up to 8kg/day. 

In conclusion, there was a significant correlation between UIP and ICFE. This correlation is moderate, due the 

great variability in the range of animals presenting the first estrus, indicating that there are other factors involving this 

parameter. There was no correlation between MP60d and the UIP, as well as no correlation between the MP60d and the 

ICFE under the conditions of this work. 




For financial support to Brazilian Agricultural Research Corporation (Embrapa), Animal Biotec Network and UPIS-Faculdades 

Integradas. 

REFERENCES 

1. Abdalla EB. 2003. Improving the reproductive performance of Egyptian buffalo cows by changing the management system. Anim Reprod Sci 

75(1-2):1-8. 

2. Bahga CS, Gangwar PC. 1988. Seasonal variations in plasma hormones and reproductive efficiency in early postpartum buffalo. Theriogenology 

30(6):1209-1223. 

3. Barkawi AH, Khattab RM, El-Wardani MA. 1998. Reproductive efficiency of Egyptian buffaloes in relation to oestrous detection systems. Anim 

Reprod Sci 51(3):225-231. 

4. Baruselli PS. 1992. Atividade ovariana e comportamento reprodutivo no período pós-parto em búfalos (Bubalus bubalis). MSc Thesis, São Paulo 

University, São Paulo, Brasil, p.71. 

5. Baruselli PS. 1994. Sexual behaviour in buffaloes. Proceedings of the IV World Buffalo Congress, São Paulo, Brasil, vol. I, p.158-173. 

6. Baruselli PS, Mucciolo RG, Viana WG, Castro Junior FG, Reichert RH, Alvarez RH. 1996. Involução uterina no período pós-parto em fêmeas 

bubalinas (Bubalus bubalis). B Indústr Anim 53:51-55. 

7. El-Wishy AB. 2007. The postpartum buffalo: I. Endocrinological changes and uterine involution. Anim Reprod Sci 97(3-4): 201–215. 

8. El-Wishy AB. 2007. The postpartum buffalo II. Acyclicity and anestrus. Anim Reprod Sci 97(3-4):216–236. 

9. Perera BMAO, de Silva LNA, Kuruwita VY, Karunaratne AM. 1987. Postpartum ovarian activity, uterine involution and fertility in indigenous 

buffaloes at a selected village location in Sri Lanka. Anim Reprod Sci 14(2):115-127. 

10. Piapon EC, Alonso JC, Hincapie JJ, Garcia L, Faure O, Fernandez, O. 2002. Seazonal influence on uterine involution and postpartum ovarian 

activity in river buffaloes. Proceedings of the Ith Buffalo Symposium of Americas, Belém, Brasil, p.456-459. 

11. Ribeiro HFL, Andrade VJ, Marques Jr AP, Vale, WG. 1997. Effect of body score condition at calving interval to first oestrus of buffalo cows. R Bras 

Med Vet 19(5):213-218. 

12. Usmani RH, Dailey R A, Inskeep EK. 1990. Effects of limited suckling and varying prepartum nutrition on postpartum reproductive traits of 

milked buffaloes. J Dairy Sci 73(6):1564-1570. 

13. Usmani, RH, Ahmad N, Shafiq P, Mirza MA. 2001. Effect of subclinical uterine infection on cervical and uterine involution, estrous activity and 

fertility in postpartum buffaloes. Theriogenology 55:563-571. 

14. Vale WG, Ribeiro HFL, Sousa JS, Ohashi OM. 2002. Involucion uterina y actividad ovarica post-parto en búfalas, Bubalus bubalis lin. Memorias 

del Curso Internacional de Reproducción Bufalina. Impresos Caribe, Medellín, Colombia, p. 59-63. 

Keywords - reproduction; buffaloes; uterine involution; first postpartum estrus; milk production; Bubalus bubalis 


840 


Cryopreservation of Buffalo (Bubalus Bubalis) 

Spermatogonial Stem Cells 

Abstract 

Jayanti Tokas 1 and Gautam Kaul 2 

1 Seth Jai Parkash Mukund Lal Institute of Engineering and Technology (JMIT), Radaur-135133 (Yamunanagar). 

2 National Dairy Research Institute (NDRI), Karnal-132001 - Email – jiyandri@gmail.com 

A protocol was developed for cryopreservation of buffalo (Bubalus bubalis) spermatogonial stem cells (SSCs). Spermatogonia 

isolated by serial enzymatic digestion and then enriched by differential plating and percoll gradient were suspended 

in freezing media (DMEM/F12 + BSA) to examine the effect of cryoprotectants under two freezing regimes (slow and fast 

freezing). DMSO was found to be a better cryoprotectant than glycerol. A controlled slow freezing regime resulted in a 

significantly higher (p

MATERIAL AND METHODS 


Spermatogonia were isolated from the testes of 5-9 months old buffalo (Bubalis bubalis) calves (Idgah slaughterhouse, 

New Delhi) by enzymic digestion procedure [collagenase (2mg/ml), Trypsin (1mg/ml) and Dnase I] 8 . After further removal 

of somatic cells, the isolated spermatogiona were purified by discontinuous percoll density gradient purification with 

suitable modifications 2 . 

The enriched population of spermatogonia cell suspension in DMEM/F12+BSA supplemented with sodium bicarbonate, 

penicillin, streptomycin were cryopreserved in eight different combination of cryoprotectants : A) 10% FCS B) 20% FCS 

C) DMSO +10% FCS D) DMSO + 20% FCS E) DMSO + 10%FCS + 0.07 M sucrose F) DMSO + 10%FCS + 0.14 M sucrose G) DMSO 

+ 10% FCS + 0.21 M sucrose H) Glycerol + 10% FCS. In the two freezing media, DMSO+10%FCS & Glycerol+10%FCS, effect 

of two freezing regimes was evaluated on viability of spermatoginia after cryopreservation viz., i) fast freezing (direct 

immersion of the cryovials into the liquid nitrogen (-196 o C) for storage), (ii) Slow freezing (placing cryovials into an 

ambient temperature freezing chamber containing isopropyl alcohol and then into a -70 o C freezer for 18 h before plunging 

into liquid nitrogen. 

Frozen spermatogonia were recovered through two thawing methods (a) rubbing of frozen vial between palms into a back 

and forth motion until the cell suspension just thawed and placing the vials into crushed ice before further processing, 

(b) immersing a vial of frozen cell suspension in a 37 o C water bath until the cell suspension just thawed. Cell viability of 

recovered spermatogonia was assessed with trypan blue staining. 

The donor cell suspension was washed twice in serum-free medium, resuspended in loading buffer (Sigma) and stained 

with lipophilic dye, PKH-26 (0.5µl PKH-26 (1mM in ethanol) in 200µl of loading buffer) and the reaction was stopped 

with 500µl FCS to bind most of the serum proteins to the dye. Cell pellet was obtained by centrifugation and washed 

twice with 40ml of DMEM/F12 medium containing 10% FCS to remove all of the PKH-26. The cells were then resuspended 

@1 x 10 6 cells/ml and cultured for a month with four subculture passages in sterile 96 well gelatin coated culture plates. 

Cell proliferation was assessed by CD9 labelling 8 . 

Buffalo spermatogonial stem cells were transplanted into seminiferous tubules obtained from of mice treated with Busulfan 

@36 mg/kg body weight for four weeks as per the standard procedures 2 . Successful transplantation of the donor cells 

into the mouse seminiferous tubules was monitored by PKH-26 and then CD9 staining respectively. 

The data was subjected to analysis of variance using SYSTAT 7.0 statistical software. Significance was considered at P< 

0.05 or mentioned otherwise. 


We examined eight different freezing media to define the best freezing media for the cryopreservation of buffalo spermatogonial 

stem cell. The viabilities of spermatogonia in different freezing media are presented in Table 1. Viability of 

freshly isolated buffalo spermatogonia was 80%. Freezing media composition influenced the viability of cells after 

freezing and thawing. Increasing FCS to 20% did not improve cell survival. Only 30% of the cells frozen in DMEM/F-12 

containing 10% FCS survived after cryopreservation. However, a significantly higher percentage of living cells was 

observed when cells were frozen in medium containing 10% FCS and 1.4 M glycerol or DMSO as compared to medium with 

only FCS. DMSO provided significantly better cell survival (P < 0.001) than glycerol and the viability of cells was significantly 

high in slow freezing than fast freezing (Table 2). Addition of sucrose (0.07M, 0.14M, or 0.21 M) in freezing 

media significantly improved the viability of cells (Table 1). Amongst the three concentrations of sucrose studied, 0.07M 

was found to be the most effective. Survival and proliferation in culture, was significantly higher in spermatogonia that 

had been frozen in the media containing sucrose than those without sucrose. This was similar to that of freshly cultured 

cells, indicating that the proliferative activity of spermatogonia in culture was not influenced by the freeze/thaw 


procedure. 

842 


A comparable survival rate was reported for the cryopreservation of hematopoietic stem cells using DMSO as cryoprotectant 

9-10 and bovine spermatogonial stem cells 11 . Almost 60% of the original cell population survived cryopreservation 

procedure in the presence of sucrose. Sugars being non-penetrating cryoprotectants, have been used successfully in 

cryopreservation protocols of many cell types, including embryos 12-13 and gametes 14-17 . 

An important parameter for cell survival is the cooling rate. Though slow cooling and faster cooling procedures have their 

own advantages and limitations, slow cooling procedure provided better viabilities in our experiments than the faster 

cooling rates. The concentration of liquid water progressively decreases as much of the extracellular water is transformed 

into ice leading to an extensive dehydration of the cells 18 . A too high cooling rate as in snap freezing causes excessive 

lethal intracellular ice formation leading to cell damage 18-20 . Two reports describe the use of uncontrolled-rate freezing 

method for non-purified spermatogonial populations, which were assumed to contain a small percentage of spermatogonial 

stem cells 21-22 . However, there is scanty information on the optimal cooling rate for spermatogenic stem cells. In our 

study, enriched populations of buffalo spermatogonia containing more spermatogonial stem cells were frozen successfully 

with the controlled slow cooling rate. Reduced cell recovery following the freeze/thaw procedure was also reported by 

other investigators studying cryopreservation of non-pure spermatogonia from other species, including rodents 21-22 and 

domestic animals 23 . Enriched spermatogonia consisted of a mixture of a few spermatogonial stem cells and spermatogonia 

A that were already destined to develop into spermatozoa. 

To test the functionality of spermatogonial stem cells among these cells after cryopreservation, cells were injected into 

mouse testes. They were able to produce a mixture of spermatogonial cells and proliferate in mouse testes. Both freshly 

isolated as well as cryopreserved bovine spermatogonial stem cells did not produce more advanced germ cells in the 

recipient mouse testes. This is probably due to the phylogenetic distance between the donor and the recipient species, 

as has been reported by other investigators working on xenogeneic spermatogonial transplantation 23-24 Hence, spermatogonial 

stem cells were found to maintain their functionality after cryopreservation. Similar results have been obtained 

earlier in bovine 1 . 

In conclusion, to the best of our knowledge this study demonstrates, for the first time, a protocol for successful 

cryopreservation of buffalo spermatogonia as well as spermatogonial cells without any detrimental effect on their viability 

and function. The technique would have has immense potential for application in preserving the germ line of proven 

sires which inturn can be used for producing progenies of required traits through breeding and assisted reproductive 

technologies. 

Table 1. Effect of different freezing media on percent viability of spermatogonia during cryopreservation and in culture 

post cryopreservation 



Table 2. Effect of slow and fast freezing of spermatogonia on their percent viability post cryopreservation 

REFERENCES: 

1. Izadyar, F., J. Matthihs-Rijsenbilt, K. Ouden, L. Creemers, H. Woelders, and Rooij, D.G. 2002b. Development of a cryopreservation protocol 

for type A spermatogonia. J. Androl., 23: 537-545 

2. Oatley, J.M., Reeves, J.J. and Mclean, D.J. 2004. Biological activity of cryopreserved bovine spermatogonial stem cells during in vitro culture. 

Biol. Reprod., 71: 942-947. 

3. Brinster, R. and Zimmermann, J.W. 1994. Spermatogenesis following male germ-cell transplantation. Proc. Natl. Acad. Sci. USA, 91: 11289–11302. 

4. Nagano, M. and Brinster, R.L. 1998. Spermatogonial transplantation and reconstitution of donor cell spermatogenesis in recipient mice. Acta 

Pathol. Microbiol. Immunolog. Scand., 106: 47-57 

5. Kanatsu-Shinohara M, Tomomi Muneto4, Jiyoung Lee, Manami Takenaka, Shinichiro Chuma, Norio Nakatsuji, Toshitaka Horiuchi and 

Takashi Shinohara. 2008. Long-Term Culture of Male Germline Stem Cells From Hamster Testes. Biology of Reproduction April 1, vol. 78 no. 4 611-617 

6. Lee DR, Kaproth MT, Parks JE. 2001. In vitro production of haploid germ cells from fresh or frozen-thawed testicular cells of neonatal bulls. Biol 

Reprod 65:873-878 

7. Izadyar, F., Den Ouden, K., Creemers, L.B., Posthuma, G., Parvinen, M. and Rooij, D,G. 2003. Proliferation and differentiation of bovine type 

A spermatogonia during long term culture. Biol. Reprod., 68: 272-281. 

8. Jayanti Tokas, Gautam Kaul. 2009. Detection of CD9 antigen on buffalo spermatogonial stem cells. Ind. Vet. J., Vol.86: 1135-1137 

9. Grilli, G., Porcellini, A. and Lucarelli, G. 1980. Role of serum on cryopreservartion and subsequent viability of mouse bone marrow hemopoietic 

stem cells. Cryobiol., 17:516–520 

10. Donaldson, C., Armitage, W.J., Denning-Kendall, P.A., Nicol, A.J., Bradley, B.A., Hows, J.M. 1996. Optimal cryopreservation of human 

umbilical cord blood. Bone Marrow Transplant., 18:725–731 

11. Izadyar, F., Spierenberg, G., Creemers, L., Ouden, K. and De Rooij, D. 2002a. Isolation and purification of type A spermatogonia from the 

bovine testis. Reprod., 124: 85-94 

12. Lazar, L., Spak, J. and David, V. 2000. The vitrification of in vitro fertilized cow blastocysts by the open pulled straw method. Theriogenol., 

54:571–578. 

13. Oberstein, N., O’Donovan, M.K., Bruemmer, J.E., Seidel, G.E., Carnevale, E.M., Squires, E.L. 2001. Cryopreservation of equine embryos by 

open pulled straws, cryoloop, or conventional slow cooling methods. Theriogenol., 55:607–613 

14. Yildiz, C., Kaya, A., Aksoy, M., Tekeli, T. 2000. Influence of sugar supplementation of extender on motility, viability and acrosomal integrity 

of dog spermatozoa during freezing. Theriogenol., 54:579–585. 

15. Fabbri, R., Porcu, E., Marsella, T., Rocchetta, G., Venturoli, S., Flamigni, C. 2001. Human oocyte cryopreservation: new perspectives regarding 

oocytes survival. Hum. Reprod., 16:411–416. 

16. Park, S.E., Chung, H.M., Cha, K.Y., Hwang, W.S., Lee, E.S., Lim, J.M. 2001. Cryopreservation of ICR mouse oocytes: improved post-thaw 

preimplantation development after vitrification using Taxol, a cytoskeleton stabilizer. Fertil. and Steril., 75:1177–1184 

17. Sztein, J.M., Nobel, K., Farley, J.S., Mobraaten, L.E. 2001. Comparison of permeating and non permeating cryoprotectants for mouse sperm 

cryopreservation. Cryobiol., 42:28–39. 

18. Mazur, P., Leibo, S.P. and Chu, E.H.Y. 1972. A two-factor hypothesis of freezing injury. Exp. Cell Res.,71:345–355. 

19. Mazur, P. 1963. Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J. Gen. Physiol., 47:347–369. 

20. Mazur, P. 1977. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiol., 14:251–272. 

21. Avarbock, M., Brinster, C., and Brinster, R.L. 1996. Reconstitution of spermatogenesis from frozen spermatogonial stem cells. Nature Med., 

2(6): 693-696. 

22. Brinster, R.L. 1998. Spermatogonial stem cell transplantation, cryopreservation and culture. Semin. Cell Dev. Biol., 9:401–409. 

23. Dobrinski, I., Avarbock, M.R. and Brinster, R.L. 2000. Germ cell transplantation from large domestic animal into mouse testes. Mol. Reprod. 

Dev., 57:270-279 

24. Dobrinski, I., Ogawa, T., Avarbock, M.R. and Brinster, R.L. 1999. Computer assisted image analysis to assess colonization of recipient 

seminiferous tubules by spermatogonial stem cells from transgenic donor mice. Mol. Reprod. Dev., 53: 142-148. 


Abstract 


Deep freezing buffalo semen - 

state of art 

Prof. Dr. William G. Vale 

Universidade Federal do Oeste do Pará - UFOPA 

Santarém-Pará, BRAZIL 

Email: wmvale@hotmail.com wmvale@ufpa.br Cell: +55 91 8114-9806 

The domestic buffalo is an important species in many parts of the world. It is most numerous in India, Pakistan, China 

and South East of Asia however it has spread its numbers in other countries of the world due its superiority production 

of milk, meat and other by-products like power, leather and dung. Major focus for increasing animal production must be 

on enhancing productivity per animal and per unit of land. Selective breeding is highly effective and sustainable 

approach for increasing animal productivity in the long-term. Reproductive technologies such as artificial insemination 

(AI) allow each animal to have multiple progeny, hence reducing the number of parent animals required. This significantly 

increases the intensity of selection as well as pace of genetic improvement. However in some countries of the world, 

limited selection of buffaloes has been applied at institutional herds and it seems that there is no official selective 

breeding policy for different breeds. For majority of the breeders’ associations, criteria for selective breeding are not 

focused which could introduce superior genes for milk and meat production traits. Although AI has been used in 

buffaloes in some countries like India and Pakistan in other countries it is of very limited use as a tool for genetic 

improvement of buffalo herds. This technology is quite feasible in Latin America condition and the fertility rates have 

achieved more than 80 per cent of conception rates, when used throughout correct technical criteria. Hence urgent need 

is felt to undertake necessary steps to set up programs of deep freezing semen of this species as the main tool for breed 

improvement mainly for milk and meat production. 

INTRODUCTION 

Key words: Artificial insemination, biotechnology, buffalo, deep freezing semen, reproduction. 

The world’s buffalo population is estimated at 177.247 million of heads FAO STAT (2008). Since long buffaloes have 

become an integral part of rural agriculture in India sub-continent and South-East Asia and spread to many other 

countries outside Asia, towards Middle East, Europe, Latin America, North America and Africa. Buffalo is reared for milk, 

meat and draught power, while it also provides manure, fuel and hides. In many rural farming systems, it is also a source 

of economic security and social status. 

Proceedings 9th In spite of its unquestionable contribution to animal industry, only recently this species has started receiving its well 

deserved recognition by farmers, producers, consumers and research workers. Publication of specialized chapters in the 

textbooks ‘The Semen of Animals and Artificial Insemination’, by JP Maule, 1962; ‘The Artificial Insemination of Farm 

Animals’, by EJ Perry 1968; ‘The Husbandry and Health of the Domestic Buffalo’, FAO, edited by W Ross Cockrill, 1974 and 

‘Buffalo Production’ - World Animal Science Series, 1992 by NM Tulloh and JHG Holmes, on many aspects of buffalo 

biology, physiology, nutrition, management, husbandry, production, reproduction and biotechnology provided substantial 

information about buffalo. Proceedings of the World Buffalo Congresses and the specialized issues of Buffalo 

844 

World Buffalo Congress


Journal as well as other scientific and technical journals around the world have contributed a rich literature on buffalo. 

Thus, intention of the present paper is to highlight the recent advances that influenced the Artificial Insemination in 

this species in Latin America. 

A short history on the deep freezing buffalo semen 

The use of frozen semen for artificial insemination (AI) in buffaloes was used for the first time by Bhattacharya & 

Srivastava (1955) in India. Later, several reports e.g. Roy et al. (1956), Basirov (1964), Sahani & Roy (1972), however, 

did not report encouraging success rates in the absence of appropriate technology concerning diluters, percentage of 

glycerol, equilibrium time, freezing methods and the AI at field level. Maybe the main reason for unsuccessful results was 

the fact that the whole technology was based on the same methodology used for bovine (Abdou et al., 1978). Some 

authors suggested the low resistance of buffalo sperm cells as an intrinsic factor (Ibrahim et al., 1985; Bhoreskar 1993). 

Singh et al. (1970) reported that lower concentrations of citric acid, Na+, K+ and Mg++, higher amounts of Ca++ and 

bicarbonate, and similar levels of fructose, inorganic phosphorus (Pi) and total antioxidant substances in buffalo semen 

as compared to the bovine. 

An important step to consolidate the feasibility of buffalo semen diluters was through the program “Deep freezing 

preservation of water buffalo semen” developed at the ‘Semen Production Unit’ in Qadirabad, Pakistan, with the support 

of the GTZ (German Agency for Technical Cooperation) and the Clinic for Andrology and Artificial Insemination, Hannover 

College of Veterinary Medicine, Germany and the University of Minnesota, USA through the presence of the Prof. Dr. Bo 

Crabo as a FAO-Consultant. This resulted in adoption of different diluters for use in deep freezing buffalo semen all over 

the world (Heuer, 1980). 

In some parts of the world, buffaloes are considered seasonal but the male seems to be less susceptible to environmental 

variations; though thermal stress affects the quality of the semen, apart from inappropriate semen handling and poor 

nutrition (Misra & Sengupta, 1965; Vale et al., 1984; Vale, 1994; 1997). After the Seminar on Buffalo Reproduction and 

Artificial Insemination, promoted by FAO and the Swedish and the Indian Governments, in Karnal (India), 1979, progress 

was achieved at different AI laboratories of the world, culminating with claims of superior fertility indexes upto 65 per 

cent calving crop (Sengupta & Sukhija 1988). Several diluters were examined, mainly TRIS, TES, Skim milk, LAICIPHOS 

478, milk-citrate-lactose, lactose, citrate, citric acid-serum (Roy & Ansari 1973; Anand, 1979; Guenzel et al. 1979; 

Heuer, 1980; Avenell 1982; Vale et al., 1984, 1991; Tayel et al. 1988; Stoyanova 1991; Vale, 1994). 

In Latin America, Brazil took the lead in the development and practice of AI in buffaloes as the first attempt to use AI in 

buffaloes was made in early 1980s by Vale and collaborators at Universidade Federal do Pará, Belém, Pará state, Brazil, 

with the guidance of Prof. Dr. Hans Merkt and Prof. Dr. Anne-Rose Guenzel from the Clinic for Andrology and Artificial 

Insemination, Hannover Veterinary College, Germany, through initiating a “pilot project” on deep freezing of buffalo 

semen. Vale et al. (1984) used TRIS and TES for successful deep freezing of buffalo semen and made the first inseminations 

with frozen semen, obtaining fertility indexes upon 50% based on non return rates at 60 days post insemination. 

Later, superior indexes to 70 per cent of calving were obtained by the same authors. The practice spread out to other 

countries and today AI is considered as an important tool for genetic improvement of buffalo in the continent. Yet, in 

Latin America the practice of AI is not so widely adopted for buffaloes as for other farm animals, especially bovine. The 

reasons include lack of serious support by the local governments, difficulty in assessing the existing AI programs by 

small-holders and medium farmers and identifying constraints and formulating and assisting in the implementation of 

remedial measures. 

In Latin America is a continent where buffaloes are raised in all countries. Although there are no official buffalo population 

records for this continent, more than 5 millions buffalo heads of six different breeds are estimated viz. Murrah, 

Mediterranean, Jafarabadi, Nili-Ravi, Carabao (swamp type) and Bufalipso – the last one concentrated in Trinidad- 

Tobago. The first four breeds are traditionally regarded as milk animals, though with pronounced meat characteristic. The 

Swamp buffalo is mainly a work animal but it can make the transition to meat producer as well. Nowadays there is great 

enthusiasm among breeders and livestock associations for buffalo, which is justifiably regarded as the animal of the 



future. Many traditional bovine farmers are changing to buffalo considering that buffalo is more profitable. For deep 

freezing semen and the use of AI, there are Centers and Laboratories in almost all countries with positive and negative 

experiences. One of the serious constraints in some areas is the supply and cost of liquid nitrogen. Such problem has 

limited the use of AI in some areas (Vale, 1994; Baruselli & Vale, 2008). 

Selection and training of males for semen collection 

The first step to decide if a buffalo male will be a semen donor is to know his pedigree concerning milk or meat 

production since there are very few progeny tested animals in Americas and as well as in other regions of the world. Main 

objective is to improve production per unit of land or animal, using the available resources in a sustainable manner. 

Ideally animal recording schemes (milk recording for dairy animals and performance recording for beef animals) should be 

in place. This will allow for the selection of sires used in artificial insemination (AI) based on Estimated Breeding Values 

(EBVs) of the sire’s parents or, in the case of beef breeds, his own EBVs. 

Although the sexual behavior at artificial service is weak and the thrust not as strong and evident as in European cattle, 

buffalo can be conditioned to jump on a male or a female, although the reaction time is long when compared to bovine 

(Vale et al., 1994). Nevertheless, male buffalo is perhaps the easiest domestic specie to be trained to serve the artificial 

vagina and generally it will ejaculate into the artificial vagina at the first attempt. The artificial vagina is generally 

accepted as the preferred method of semen collection in order to ensure optimal semen quantity, quality, hygiene and 

thermal protection. Young males at puberty start to mount other males and Flehmen’s response is observed when the bull 

sniffs the other herd mates, male genitalia or urine (Vale et al., 1984; Vale,1991;1994;1997). 

Deep freezing semen according to the Brazilian federal legislation 

Quarentine 

Once selected as semen donor, the male is subjected to a general clinical examination and a Breeding Soundness Examination 

(BSE) before shifting to AI centre. In Latin America, special attention needs to be paid to hereditary diseases 

which can cause any morpho-functional disturbance related to the genital system such as gonodal hypoplasia, epididymal 

disfunction, arrested development of mesonephric ducts, absence or weak libido, due to inbreeding (Vale et al., 

2008). 

Then the buffalo male is transferred the animal will be put in a quarantine system which consist of an isolation in an 

special barn when blood samples and clinical procedures will be done to check his health status. 

The following steps are performed: 

Blood serum: Brucellosis and bovine viral diarrhea (BVD). 

Preputial smegma in swab: (three exams with an interval of one week) Bovine Genital Campylobacteriosis 

Preputial washing specimen: (three exams with an interval of one week) Trichomonosis. 

Intradermal tuberculin inoculation double compared test: bovine and avium strains. 

Treatment is carried out against ectoparasites in case of infestation after faeces exam. The material for clinical surveys are 

sent on the same day of collection to the Biological Institute of São Paulo state. 

Once, after the arrival of non-reactive results of every tests required the animal expend at least 28 days to be able to 

integrate the block stalls of the animals in the semen collection system. 

Nutrition and health management of buffalo bulls submitted to deep freezing semen 

Proceedings 9th Every animal while housed in specialized AI center, remain in individual stalls with exercise area of adequate size with 

shade and free access to mineral and water source, and supply of Napier grass (Pennisetum purpureum) ad libitum 

shopped into the trough twice a day (morning and afternoon). A balanced ration should be fed, either home grown or 

purchased or both. Care should be taken not to over-feed bulls as fat deposition in the inguinal canal negatively affects 

846 



fertility. Condition score is an important guide to nutritional requirements. Working buffaloes should have a score of 3 

to 3.5 on a scale of 1-5, which also provides nutritional assessment. Allow ample green grass or good silage, mineral 

mixture and water source ad libitum and controlled ingestion of protein supplement rations. 

Also before arriving in the AI center, individual buffalo vaccinated animals should be integrated into the routine of the 

same health program, including routine prophylactic immunization against the following diseases: 

Leptospirosis: yearly vaccinations four times (February, May, August and November) 

Foot and Mouth: yearly vaccinations two times (May and November) 

IBR/IPV and BVD: same as for Leptospirosis 

Treatment against ectoparasites in the period of greatest infestation. 

As the majority of Latin American countries are located in the tropical or semi-tropical areas with warm and humid 

temperatures housing may be open, semi-open and rarely closed. Moreover, buffalo bulls raised in tropical and subtropical 

conditions require protection from heat and adequate ventilation. Shady trees/thatch roof are effective. Fine water 

sprays with fans or air conditioning stall can be used to cool buffalo bulls under extremely hot conditions. Buffaloes 

should be housed securely so there is no chance of escape and interact with other males, staff and the general public. 

Handling 

The establishment of a firm relationship between the personnel involved in buffalo management, mainly the handler and 

the bull need not be overemphasized. The buffaloes should be at ease when handled and the handler should not feel 

threatened. The application of a buffalo nose ring is required sometimes. However, our experience has demonstrated the 

use of nose ring sometimes is a disaster and no semen can be collected from some old buffalo bulls. The use of small sugar 

tablets, banana and comfort, cause a positive impact in the buffalo male behavior. Buffaloes should always be handled in 

such a manner that semen production is optimized. This includes taking note of all aspects of the physiology of male 

sexual behaviour. Negative stimuli such as noise, low hygienic conditions and stress of any origin, should be avoided in 

the collection area (Vale et al., 1984; Vale, 1991; 1994; 1997). 

Semen collection and technology 

At Central for Animal Reproductive Biotechnology-CEBRAN, Castanhal county, Pará State, Brazil, as well as in other places 

that I have worked on deep freezing semen technology, the following procedures has been recommended. 

Semen collection area should be as close as possible to the semen laboratory. The collection area should be sheltered and 

must have adequate ventilation and light. For teaser buffalo restraint a stanchion made from strong metal bars or smooth 

treated wooden poles and timber is recommended. The floor of the collection site should not be slippery. It can be made 

of rough concrete or a dug-out pit filled with sand and sprinkled with water to avoid dust. Rubber mats can also be used. 

As teaser, a buffalo female in estrus or a male can be used. For older buffalo males, it is necessary to the use of a female 

buffaloes in estrus and usually it is necessary to have a small herd with females for estrus induction. Well trained buffalo 

males can serve in the other young male as a dummy. 

Preparation of buffaloes 

Semen donor bulls are housed under clean dry conditions, washed and cleaned before they arrive at the collection area. 

The washing area should not be more than 20 m from the serving area and should be made of rough concrete with a 

slanting floor to facilitate drainage of water, dung and urine. Adequate clean water with reasonable pressure should be 

provided through a hose pipe. Prior to cleaning, the preputial hair should be cut short, leaving a tuft of 2 cm length. 

Ordinary washing soap and a soft brush should be used to clean the bulls. During cleaning, emphasis should be put on the 

lower abdomen and the preputial area. If necessary, washing of the preputial sheath with normal saline solution can be 

done once every week or fortnight to reduce bacterial contamination of semen. Disinfectants should not be used. Clean, 

dry paper towels should be used after washing to remove excess water. If the teaser buffalo or dummy is dirty, its back 

should be cleaned with water and soap and dried thoroughly. An apron may be used if necessary. There is little risk of 


848 


contamination of the penis or the semen if the teaser is clean and collection technique is good, allowing no or little 

contact of the penis with the teaser. 

Artificial vaginas 

Ensure that all equipment used for semen collection, evaluation and processing are clean and sterilized. An outer rubber 

barrel (usually 35-40 cm long) with rough inner rubber liner that is non-spermiotoxic is recommended. The inner liner 

should periodically be checked for possible leakage. The rubber cones should also be non-spermiotoxic and a correctly 

labeled collection tube should be attached. A jacket should be provided for the cone to prevent breakage and avoid 

direct exposure to sunlight. Rubber bands for holding on the cones and the two ends of the reflected inner lining onto 

the outer barrel should be strong. Water for the outer jacket filling should be warmed to 60oC. Enough of this should be 

poured into the inner chamber to provide the required pressure. This quantity may range from 400-600 ml. For buffaloes, 

the inner temperature after lubrication should range between 44-46oC. Assembled AVs should be kept in incubators at 

55-60oC. If there is a delay between preparation of the AV and collection, the temperature should be checked. Just 

before collection, excess water must be taken out from the AV and enough air blown in to provide adequate internal 

pressure. 

The collector 

A collector should be selected on the basis of his/her ability, enthusiasm and experience to work with livestock. Protective 

gear should include gum boots with steel or wooden-toed caps, apron, head cap and thin half length plastic hand 

gloves. 

Collection procedure 

In tropical areas, semen collection must be performed early in the morning or at night. Buffaloes should be led, preferably 

using a halter, to the teaser in a gentle friendly manner by the handler, who should pay attention to the temperament of 

the particular male. The buffalo bull should be allowed to watch other buffaloes mounting before collection and led 

around behind the teaser and may be allowed to mount other young buffaloes in case of using male as dummy. Two false 

mounts are usually given. These measures improve the sexual excitement, which enhance the quality of semen by cleansing 

the urethral passage and increasing the amount of semen collected. Then, the bull is allowed to mount for the first 

collection. The penis should not be touched. The handler may rest his shoulder against the bull’s flank and move with the 

movement of the buffalo as he thrusts. The AV should be held so that the bull withdraws as it dismounts, and should not 

be pulled away from the penis. After this, the collection tube is separated from the rubber cone, grasped by the hand and 

transferred for the personnel inside the laboratory to evaluation. 

Semen evaluation 

Semen evaluation should be performed in a laboratory with a environmental temperature around 20o C. 

Macroscopic examination: just after the collection, the ejaculate should be transferred to a water bath maintained at 

35oC. An initial visual evaluation for volume, color, consistency/density, odor and observation for presence of foreign 

material (urine, blood, pus cells, dung, hair etc.) shall be made and recorded. If dung or hair is found in the ejaculate, 

it must be immediately discarded. 

Microscopic examination: microscopic evaluation is done using a simple or phase contrast microscope for mass activity 

(wave motion) and individual motility. Determination of concentration is done with a hemocytometer or a calibrated 

photometer. At this point, if required, smears can be made for morphological studies. Buffered Eosin-Nigrosin solution or 

Eosin alcohol five per cent solution, must be mixed with a drop of semen and smeared on a glass slide for live / dead 

count as well as for morphological examination. It should be dried and examined under oil immersion. Automated 

computerized machines for recording motility and concentration and calculating the required extension rate are now 

frequently used in AI laboratories that can afford. Semen used for artificial insemination should be of high quality. At 

CEBRAN and other laboratories which we have supervised, the following are guides to the values of semen characteristics 

in the bull that indicate good reproductive function: 



Motility (moving actively forward): >60%; Concentration: ?600 million /ml; Live sperm: >70%; Abnormal sperm:

* Thermo Resistance Test 

850 


surface; in the absence of freezing machines this step can be done in a large semen storage tank or a big polystyrene 

container, containing liquid nitrogen); Place racks in liquid nitrogen at -196oC; Collect straws with a gloved hand and 

store in goblets in liquid nitrogen; Wash and sterilize glassware for the next day. The common types of packaging used for 

processed semen are: French Cassou straws packaged and sealed in straws, mini (0.25 ml) or medium (0.5 ml); German 

minitub-packaged and sealed with small glass or steel spheres (0.25 ml). In both systems, each straw must contain a 

minimum of 20–30 million spermatozoa per dose.In the Figure 1, it can be follow the different steps from the ejaculation 

throughout the thawing process. 

Diluters used for deep freezing semen 

There are many diluters available for deep freezing buffalo semen. Some drawn from international literature; others 

developed at regional level. Basically egg yolk and / or milk are the basic ingredients while glycerol is the main cryoprotective 

agent used @ 7 per cent. Some vegetables derivate compounds, like coconut water and soybean milk have also 

been used on experimental basis. The main diluters used in our work in Brazil and in other countries of Latin America have 

its composition as follow: 

Composition of TES (hydroxy-methyl-amino-ethan) diluter 

Solution A - 500 mL (distillate or deionised water) 

TES (hydroxy-methyl-amino-ethan)…………………………………………….24.5 g 

TRIS (hydroxy-methyl-amino-methan).………………………………….. ...........5.3 g 

D (-) Fructose …………………………………………………………………….1.08 g 

Streptomycin …………………………………………………………………….700mg 

Penicillin…………………………………………………………………………..70mg 

Solution B - 100 mL (distillate or deionised water) 

Skim milk…………………………………………………………………………..11g 

Note: maximum heated to 80 ° C to not denature the protein contend 

Preparation of the final diluter – 100 ml: 

Solution A ……………………………………………………………………..36.5 mL 

Solution B………………………………………………………………………36.5 mL 

Glycerol………………………………………………………………………… 7.0 mL 

Egg yolk……………………………………………………………………… 20.0 mL 

Osmolarity to 290 ± 5mOsm 

pH= 6.8 – 7.0 



Composition of TRIS (hydroxy-methyl-amino-methan) diluter 

Solution A - (500mL) 

TRIS (hydroxy-methyl-amino-methan)………………………………………..19.05 g 

Citric acid………………………………………………………………………. 9.85 g 

D (-) Fructose ………………………………………………………………….. 7.75 g 

Streptomycin sulphate………………………………………………………… 700 mg 

Penicillin-G-potassium………………………………………………………… 70 mg 

Final Solution 

Solution A………………………………………………………………………73.0 mL 

Glycerol………………………………………………………………………….7.0 mL 

Egg yolk………………………………………………………………………. 20.0 mL 


pH 6.8 to 7.0 

Composition Lactose/TRIS diluter 

Solution A - (500mL) 

Lactose………………………………………………………………………...55.0 g 

TRIS (hydroxy-methyl-amino-methan)………………………………………19.05 g 

Streptomycin sulphate……………………………………………………….. 700 mg 

Penicillin-G-potassium…………………………………………………………70 mg 


Solution A……………………………………………………………………73.0 mL 

Glycerin ………………………………………………………………………7.0 mL 

Egg yolk……………………………………………………………………. 20.0 mL 


pH 6.8 to 7.0 


Composition Skim Milk diluter 

Solution A (500 mL) 

852 


Skim milk………………………………………………………………………..55g 



Skim milk…………………………………………………………………....73.0 mL 

Egg yolk……………………………………………………………………..20.0 mL 

Glycerol……………………………………………………………………….7.0 mL 

Streptomycin sulphate………………………………………………………..700 mg 

Penicillin-G-potassium………………………………………………………...70 mg 


pH 6.8 to 7.0 

Composition of CEBRAN-1 diluter (Coconut water)* 

Solution A - 125 mL (distillate or deionised water) 

Coconut water……………………………………………………………50 mL 

Sodium citrate 5%..................................................................................... 25 mL 

*Coconut fruit must be green, not ripe 

Solution B – 125 mL (distillate or deionised water) 

Solution A……………………………………………………………….. 90.0 mL 

Egg yolk………………………………………………………………… 10.0 mL 

Streptomycin sulphate……………………………………………………..700mg 

Penicillin-G-potassium…………………………………………………….70 mg 

Final solution 

Solution B…………………………………………………………………93.0 mL 

Glycerol…………………………………………………………………….7.0 mL 


pH 6.8 to 7.0 


Composition of CEBRAN II diluter (Ringer-Lactate) 

Solution A 


D-Fructose ……………………………………………………………………1.08 g 

Penicillin G potassium…………………………………………………………70mg 

Streptomycin sulphate………………………………………………………..700mg 

Ringer Lactate…………………………………………………………………500mL 

Solution B (100mL) 

Skim milk………………………………………………………………………..11g 



Solution A………………………………………………………………….36.5 mL 

Solution B………………………………………………………………….36.5 mL 

Glycerol……………………………………………………………………..7.0 mL 

Egg yolk……………………………………………………………………20.0 mL 


pH 6.8 to 7.0 

Freezing process 

Cryopreservation of semen is established following a gradual process of lowering the temperature where the semen already 

diluted and added to the equilibrium curve and the freezing of the diluted and loaded semen is subjected to the 

following procedures: 

Semen remains at 5 ° C for a period of 3-4 hours, for the equilibrium time, when occur the entry by diffusion of 

cryoprotectants contained in the diluent into the sperm cell. 

Then, the loaded semen is put on a 5 cm distance of liquid nitrogen vapour for 20 minutes, when it will reach a 

temperature of -36 º C to then dump them in a polystyrene box containing liquid nitrogen, whose temperature is at -196 

degrees C. 

Preservation and storage 

Frozen semen is preserved in liquid nitrogen at –196oC. Transferring of semen must be done quickly. Canisters containing 

packages when raised from the container should remain in the neck of the container for less than 10 seconds. Liquid 

nitrogen is dangerous and must be handled carefully. 

Post packaging quality control 

The motility of samples from processed batches of semen should be checked before dispatch. The post thaw motility 

should be 40% or more. All semen storage containers should be regularly checked for liquid nitrogen level and replenished 

as required. 



It also accomplished the thermal resistance test (quick TTR), where the sample is placed in a water bath at 46 º C for 30 

minutes to then be examined. 

Field Practices 

AI in buffaloes is an effectible reproductive biotechnology; however it requires the effective and continuous supervision 

of a Veterinarian with specific training in buffalo reproduction. Veterinary services must be offered as a task force. It is 

important to figure out that one of main constrain of the use of AI in buffaloes is the absence of Veterinary services and 

continuous supervision in the program. Thus, the main impediments to the use of AI in buffaloes in the Brazil as 

elsewhere in Latin America, at the present time are inadequate nutrition, poor management and handling, a lack of 

rational breeding policies, and diseases (not necessarily all occurring simultaneous) and difficulty in liquid nitrogen 

supply. The AI in buffaloes as technology is quite feasible in Latin America condition and the fertility rates have achieved 

more than 80 per cent of conception rates, when used throughout correct technical criteria. 

CONCLUSION AND RECOMENDATION 

The AI in buffalo is a concern for each regional country and reflects the growing interest in the species as an important 

constituent of local agriculture and substantial contributor to country’s economy. The AI programme goes side by side 

with improvement of the management, nutrition, diseases control with an overall impact on reproduction and milk / 

meat production. In some areas, the supply and cost of liquid nitrogen can limit the use of this technique. Moreover, it 

is the only alternative to avoid inbreeding which is affecting some buffalo breeds in this continent. The technology has 

unmistakable advantages for animal husbandry but unfortunately may be disadvantageous if used uncontrolled leading 

to inbreeding and lethal malformations. Thus, it is necessary to establish efforts to discuss such problems at international 

level in order to avoid the dissemination of many of these inheritance defects. This biotechnology is quite feasible in 

buffaloes and the fertility rates have achieved more than 80 per cent of conception rates, when used throughout correct 

technical criteria. On this concern it seems that a wide discussion among buffalo breeders and technicians should be 

established to find out a way to introduce “new blood” in the form of semen, from selected animals from India and 

Pakistan. 

Acknowledgements 

I would like to express my sincere gratitude to: To Prof. Dr. José Seixas Lourenço, Dear of the Universidade Federal do 

Oeste Paraense – UFOPA and FAPESPA – Fundação de Amparao e Desenvolvimento a Pesquisa do Estado do Pará, for the 

support provided. To Prof. Dr. Gustavo Crudelli and Dr. Marco Zava for the kind invitation to participate of this meeting. 

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Vale, W.G.; Nahúm, B. de S.; Silva, A.O.A.; Sousa, J.S.; Souza, H.E.M.; Ohashi, O.M.; Ribeiro, H.F.L. 1999. Inseminação artificial em bubalinos com 

sêmen congelado em diluente a base de água de côco (Cocus nucifera). Rev. Bras. Reprod. Animal. 23(3):354-356. 

Vale, W.G. & Ribeiro, H.F.L. 2005. Características reprodutivas dos bubalinos: puberdade, ciclo estral, involução uterina e atividade ovariana no pósparto. 

Rev Bras Reprod Anim, Belo Horizonte, v.29, n.2, p.63-73, abril/jun. Disponível em www.cbra.org.br 

Vale, W.G., Ribeiro, HFL, Sousa, J.S., Silva, A.O.A., Barbosa, E.M., Rolim Filho, S.T. 2008. Selection and breeding soundness evaluation in the male 

buffalo. Rev. Bras. Reprod. Anim. 32(2):141-155, 2008, www.cbra.org.br 


856 


Developmental Rates of Vitrified Buffalo Oocytes 

Following Parthenogenetic Activation and 

Intracytoplasmic Sperm Injection 

Yuanyuan LIANG 1 , Tatsanee PHERMTHAI 2 , Takashi NAGAI 3 , Tamas SOMFAI 3 , Rangsun PARNPAI 1 

1 Embryo Technology and Stem Cell Research Center and School of Biotechnology, Suranaree University of Technology, Nakhon 

Ratchasima, 30000, Thailand - 2 Department of Obstetrics and Gynecology, Faculty of Medicine Siriraj Hospital, 

Mahidol University, Bangkok 10700, Thailand. 3 National Institute of Livestock and Grassland Science, Ikenodai 2, 

Tsukuba, Ibaraki 305-0901, Japan - E-mail: rangsun@g.sut.ac.th 

Abstract 

The objective of this study was to investigate the potential of swamp buffalo oocytes vitrified-warmed at the MII stage 

to develop to the blastocyst stage after parthenogenetic activation (PA) and intracytoplasmic sperm injection (ICSI). In 

the first experiment, we examined the toxic effects of cryoprotectants on in vitro development. In vitro matured oocytes 

were placed in 10% dimethylsulfoxide (DMSO) + 10% ethylene glycol (EG) for 1 min and then exposed to 20% DMSO + 

20% EG + 0.5 M sucrose for 30 sec (1+30), 45 sec (1+45) or 60 sec (1+60). The oocytes were exposed to warming 

solution (0.5M sucrose) for 5 min and then washed in TCM199 HEPES + 20%FBS for 5 min. Oocyte viability was assessed 

by fluorescein diacetate (FDA) staining. Surviving oocytes were parthenogenetically activated and cultured for 7 days. 

The viability in all groups of CPA treatment and the control were 100%. The development rates to the blastocyst stage 

among CPA exposed 1+30 (17%), control (23%) and fresh control (control without FDA assay) (27%) did not differ 

significantly, but they were significantly higher than those in CPA exposed 1+45 (9%) and 1+60 (1%) groups. In the 

second experiment, we examined the effect of two CPA exposure times, 1+30 and 1+45 on the in vitro development for 7 

days after PA of oocytes vitrified by the microdrop method. The viability in vitrified 1+30, 1+45 and the control groups 

was not different (97%, 95% and 100%, respectively). The development of surviving oocytes to blastocyst stage in the 

vitrified 1+30 group (8%) was significantly higher than that in the vitrified 1+45 group (4%) and significantly lower 

than those in control and fresh control groups (24% and 26%, respectively). In the third experiment, we examined the 

effect of two CPA exposure times, 1+30 and 1+45 on in vitro development after ICSI of vitrified oocytes. The FDA viability 

in vitrified 1+30, 1+45 and control groups (100%) was not different (96%, 91% and 100%, respectively). After ICSI 

vitrified-warmed oocytes were activated and oocytes with the 2 nd polar body were cultured for 7 days. The development 

of ICSI oocytes to the blastocyst stage in the vitrified 1+30 group (11%) was significantly higher than that in vitrified 

1+45 (7%) and significantly lower than those in the control and fresh control (21% and 23%, respectively). In conclusion, 

our study demonstrated that the 1+30 CPA treatment regimen could yield the highest blastocyst rates for oocytes 

vitrified by the microdrop method and that the FDA viability test had no effect on the embryo development. 

INTRODUCTION 

Nowadays, buffalo is the major milk and meat producing farm animal in many developing countries. Buffalo oocytes 

obtained from slaughterhouse ovaries and matured in vitro are useful sources for reproductive procedures such as in vitro 

fertilization (IVF) and ICSI, in which cryopreserved spermatozoa are used. Cryopreservation of oocytes also has great 

importance to preserve female gamete for future use. Efficient oocyte cryopreservation protocols will widen and improve 

the strategic implementation of reproductive technologies in the buffalo species. 

Keywords: buffalo oocytes; microdrop; vitrification; ICSI. 



Cryopreservation of mammalian oocytes has become more successful using vitrification as an alternative to cryopreservation 

compared with slow cooling methods in recent years (Chian et al., 2004; Vajta and Nagy, 2006). Vitrification is the 

process that induces a glass-like solidification of living cells at low temperatures. The unique advantage of the vitrification 

process is the elimination of ice crystal formation, the most dangerous cause of cryoinjury. Insufficient cooling rate 

of oocytes was considered as one of the obstacles in vitrification technology (Vajta, 1997). In order to overcome this 

problem, several methods have been proposed that use very small amounts of solution. There are some improved vitrification 

methods which has been successfully used for oocyte cryopreservation such as cryotop (Kuwayama and Kato, 

2000), cryoloop (Lane et al., 1999), open pulled straw (OPS; Vajta et al., 1998), glass micropipette (GMP; Hochi et al., 

1994), microdrop (Papis et al., 2000), electron microscope grids (EMG; Martino et al., 1996) and solid surface vitrification 

(SSV, Dinnyés et al., 2000). 

Since the first report on buffalo oocytes vitrification by using French Straw (Dhali et al., 1999), there are some reports 

regarding the cryopreservation of buffalo oocytes (Wani et al., 2004; Gasparrini et al., 2007; Muenthaisong et al., 2007; 

Boonkusol et al., 2007; Sharma and Loganathasamy, 2007; Gautam et al., 2008; Mahmoud et al., 2008). The first 

successful production of buffalo blastocyst derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of 

vitrified-warmed oocytes (Wani et al., 2004). 

Among the various methods of vitrification, the microdrop method is considered easy and cheap, as it excludes the use 

of any specialized device to introduce oocytes into liquid nitrogen. The microdrop method is a simplest way of vitrification 

by dropping oocyte containing solutions directly into liquid nitrogen. This method was first proposed for mouse 

embryos (Landa and Tepla, 1990), and then successfully applied for in bovine embryos, zygotes and oocytes (Riha et 

al.,1991; Yang and Leibo,1999; Papis et al., 2000). During cryopreservation or treatment with cryoprotectants structural 

changes in the zona pellucida (ZP) has been shown to reduce fertilization rates (Carroll et al., 1990; Vincent et al., 

1990). Although the mechanism of ZP hardening of the cryopreserved oocytes is unclear, it seems to be caused by the 

premature release of cortical granules (Vincent et al., 1990) resulting in lower incidences of sperm entry into oocytes. 

This consequence of zona hardening could be overcome by micromanipulation techniques such as ICSI (Carroll et al., 

1990; Kazem et al., 1995; Karlsson et al., 1996; Porcu et al., 1997). 

One essential factor in cryosurvival is permeation of a centain amount of cryoptotectant into the oocytes, which increase 

with the duration of exposure; however, the toxicity of the cryoprotectant must be avoided, with an optimum exposure 

time being favorable for this purpose. The aim of this study was to investigate the exposure time in vitrification solution 

on the post-thaw viability and the developmental competence of vitrified-warmed swamp buffalo oocytes after parthenogenetic 

activation (PA) or ICSI. Differential cell staining was applied to assess the qualitative aspects of blastocysts that 

were derived from fresh or vitrified-warmed oocytes. 


Chemicals and media 

All reagents were purchased from Sigma Chemical Company (St. Louis, MO, USA) unless otherwise stated. The medium used 

for IVM was TCM199 supplemented with 25 mM HEPES, 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA), 

0.02 AU/mL FSH (Antrin, Denka Pharmaceutical, Tokyo, Japan), 50 iu/mL hCG (Chorulon, Intervet, Boxmeer, Netherlands) 

and 1 µg/mL estradiol-17ß. The Emcare holding medium (ICP Bio, Auckland, New Zealand) was used as the basal medium 

throughout the process of ICSI and parthenogenetic activation. The medium for embryo culture (IVC medium) was 

modified synthetic oviduct fluid supplemented with amino acids (mSOFaa) (Gardner et al., 1994) and 0.3% fatty acidfree 

BSA. 


Oocyte collection and in vitro maturation 

858 


Buffalo ovaries were obtained from a slaughterhouse and transported to the laboratory within 4 h at room temperature. 

The ovaries were placed in 0.9% NaCL during transport to laboratory. Cumulus-oocyte complexes (COCs) were collected 

from the 2–8 mm follicles using a 21-gauge needle and were in vitro cultured in IVM medium for 22 h. After maturation, 

cumulus cells were removed by gentle pipetting with a fine glass pipette, and the oocytes were subsequently washed 3 

times in Emcare medium. Oocytes with a visible first polar body were selected for the following experiments. 

Vitrification and warming 

The M-II oocytes were vitrified-warmed by the Microdrop method originally reported by Papis et al. (2000). Groups of 5 

oocytes were washed in TCM199-Hepes + 20% FBS before placed in TCM199-Hepes + 20% FBS + 10% DMSO + 10% 

ethylene glycol (EG) for 1 min, and then exposed in TCM199-Hepes + 20% FBS + 20% DMSO +20% EG + 0.5 M sucrose for 

30 (1+30), 45 (1+45) or 60 sec (1+60) at 22-24 °C. The oocytes were then directly dropped with about 2 µL vitrification 

solution into liquid nitrogen. For storage, vitrified microdrops were placed in a pre-cooled cryovial filled with liquid 

nitrogen using a pre-cooled forceps and kept for 1-2 weeks. The vitrified microdrops were warmed by directly immersing 

into 3 mL of 0.5 M sucrose in TCM199-Hepes + 20% FBS at 38.5 °C for 5 min, and then transferred to the TCM199-Hepes+ 

20% FBS for 5 min. Then oocytes were kept in the TCM199-Hepes + 20% FBS under a humidified atmosphere of 5% CO 2 in 

air at 38.5 °C for 1 h. Some oocytes were treated with cryoprotectants and warming solution without the cooling and 

warming process (“CPA-exposed” group). 

Evaluation of oocyte viability 

Oocyte viability was evaluated by FDA staining according to the method previously described by Mohr and Trounson 

(1980). Briefly, oocytes were treated with 2.5 µg/mL FDA in PBS supplemented with 5 mg/mL BSA at 38.5 ºC for 2 min in 

a dark room and then washed three times in PBS supplemented with 5 mg/mL BSA and evaluated under an epifluorescence 

microscope with UV irradiation using a U-MWIB3 filter with excitation wavelength of 460–495 nm and emission at 510 

nm. Oocytes expressing a bright green fluorescence were regarded as living ones and were used subsequently. 

Parthenogenetic activation (PA) 

The MII oocytes were subjected to the PA treatment as described previously (Laowtammathron et al., 2005). Briefly, the 

oocytes were first treated with 7% ethanol in the Emcare holding medium for 5 min, and then incubated with 10 µg/mL 

cycloheximide (CHX) and 1.25 µg/mL cytochalasin D (CD) in mSOF medium + 10 % FBS for 5 h at 38.5 °C under humidified 

atmosphere of 5% CO 2 in air. 

ICSI 

Straws of frozen spermatozoa were thawed in a 37°C water bath for 30 sec. Thawed spermatozoa were gently placed to the 

bottom of the 1mL BO medium supplemented with 1 mM caffeine for sperm swim up for 30 min and then the supernatant 

was collected and centrifuged at 500×g for 5 min. The sperm pellet was washed twice with 1mL of BO-medium by 

centrifugation at 500×g for 5 min. The sperm pellet was resuspended in the BO-medium and diluted approximately 1:5 

with polyvinyl pyrrolidone (PVP). Sperm injection was performed with inverted microscope (IX71, Olympus). The inner 

diameter of the sperm injection needle was 8-10 µm, and the inner diameter of the holding pipette was 20 µm. Three 

droplets of media covered with mineral oil were prepared on 60 mm petri dish cover for the ICSI procedure; the first 

droplet was 10% PVP medium for washing pipette, the second droplet was the sperm suspension diluted with 10% PVP 

medium (1:5), and the third droplet was Emcare holding medium for the ICSI procedure. A single, motile buffalo sperma- 



tozoon was immobilized against the bottom of the PVP droplet, loaded tail first with a minimum volume of medium into 

the injection pipette and then injected into the cytoplasm of a buffalo oocyte. Within 1 h of the injection, the injected 

oocytes were activated by exposure to 7% EtOH in Emcare medium for 5 min, and then subsequently cultured in TCM199 

+ 10% FCS for 3 h to allow extrusion of the second polar body. With the purpose of producing haploid activated oocytes 

for ICSI, the injected and activated oocytes which extruded the second polar body were selected and transferred to mSOF 

medium supplemented with 10 µg/mL CHX and cultured for 5 h at 38.5 °C under humidified atmosphere of 5% CO 2 in air. 

In vitro culture and differential cell staining 

The PA and ICSI oocytes were cultured in 100 µL microdrops (20 oocytes per microdrop) of the IVC medium covered by 

paraffin oil under a humidified atmosphere of 5% CO 2 , 5% O 2 , 90% N 2 at 38.5 °C for 2 days. Thereafter, embryos at the 8cell 

stage were selected and co-cultured with bovine oviductal epithelium cells (BOEC) under a humidified atmosphere of 

5% CO 2 in air at 38.5 °C for 5 days, as reported previously (Parnpai et al., 1999). Half of the medium was removed every 

day from each drop and replaced with fresh medium. The cleavage rates were recorded on Day 2, the development of 

embryos to the blastocyst stage was recorded on Day 7 (the day of PA or ICSI was considered Day 0). 

The blastocysts harvested on Day 7 were stained to distinguish cells of the inner cell mass (ICM) and trophectoderm (TE), 

as reported previously (Suteevun et al., 2006). Briefly, zona pellucidae of blastocysts on Day 7 were removed by 0.5% 

protease. After washing with mSOFaa medium, the zona-free blastocysts were incubated in 100 µL of 10% rabbit antibuffalo 

spleenocyte antibodies for 45 min, and then transferred into a 100 µL mixture of 10% guinea pig complement, 

75 mg/mL propidium iodide (PI) and 100 µg/mL Hoechst 33258 for 45 min. The ICM (blue) and trophectoderm cells 

(red) were counted under a fluorescent microscope at 330–380 nm, allowing determination of the total number of cells 

for blastocysts and the percentage of ICM cells based on the total number of blastocyst cells. 

Experimental design 

Experiment 1 was performed to test the toxicity of Vitrification solution. After treatment with the equilibration solution 

for 1 min oocytes were exposed to vitrification solution for 30 (1+30), 45 (1+45) or 60 sec (1+60) and then to warming 

solution. Surviving oocytes selected by the FDA test were then subjected to PA and then in vitro cultured. The development 

of oocytes exposed to the vitrification solution for were compared with that of untreated oocytes (Control). To test 

the possible effects of FDA staining some oocytes without CPA and FDA treatment were also activated and cultured (Fresh 

control). 

Experiment 2 was performed to assess the developmental ability of vitrified-thawed oocytes induced by PA. On the basis 

of the results from Experiment 1, only the 1+30 and 1+45 CPA treatment regimens were used to vitrify M-II oocytes. 

Vitrified oocytes were stored in liquid nitrogen containers for several days or weeks. After warming all surviving (FDA 

positive) oocytes were subjected to PA and their in vitro development was compared to those of activated Control and 

Fresh control oocytes. 

Experiment 3 was performed to assess the development of ICSI embryos generated from vitrified-thawed oocytes. Oocytes 

vitrified by the 1+30 and 1+45 CPA treatment regimens were warmed and all of the surviving (FDA positive) oocytes were 

subjected to ICSI. Their in vitro development was compared to those of Control and Fresh control oocytes following ICSI. 

Statistical analysis 

The experiments were replicated at least three times in each treatment group. Data were analyzed by one-way ANOVA using 

the statistical analysis systems (SAS). The differences between groups were considered to be statistically significant 

when P < 0.05. 


RESULTS 

860 


Experiment 1. Parthenogenetic development of CPA-treated oocytes. 

Survival and in vitro development of buffalo MII oocytes following CPA-exposed and PA treatment is summarized in Table 

1. The viability of oocytes determined by the FDA test in all groups of CPA treatment and the Control were 100%. The 

cleavage rate and embryo development in the 1+30 and 1+45 groups were significantly higher than in the 1+60 group, 

but lower than in the Control and Fresh control groups. The development of oocytes after PA to the blastocyst stage did 

not differ significantly among the 1+30 (17%), Control (23%) and Fresh control groups (27%) but were significantly 

higher than those in 1+45 (9%) and 1+60 (1%) groups. The number of total cells and ICM cells were similar among the 

1+30, 1+45, Control and Fresh control groups and were higher than those in 1+60 group (Table 1). 

Experiment 2. Parthenogenetic development of vitrified oocytes. 

In vitro development of oocytes following vitrification and PA treatment is summarized in Table 2. The rates of live 

oocytes among the vitrified 1+30 (97%), 1+45 (95%) and the Control groups (100%) were not different. The cleavage 

and blastocyst rates were influenced when the oocytes were subjected to vitrification. The development of PA oocytes to 

the blastocyst stage in the vitrified 1+30 group (8%) was significantly higher than that in the vitrified 1+45 group (4%) 

and significantly lower than those in the Control (24%) and Fresh control (26%). The total cell numbers of blastocysts in 

the 1+30 (71.6±18.3) and 1+45 (69.9±19.6) groups were lower than that in the Control (78.5±24.6) and Fresh control 

(80.0±23.1) groups. There was no difference in the ICM cell numbers among the four groups. 

Experiment 3. In vitro development following vitrification and ICSI. 

In vitro development of buffalo MII oocytes following vitrification and ICSI treatment is summarized in Table 3. The rates 

of live oocytes among the vitrified 1+30 (96%), 1+45 (91%) and Control (100%) groups were not different. After 

activation of ICSI oocytes by ethanol the 2 nd polar body extrusion rate in the 1+30 (43%) group was significantly higher 

than that in the 1+45 group (35%) and significantly lower than those in the Control (56%) and Fresh control groups 

(59%).The cleavage and blastocyst rates in the 1+30 group (67%, 11%) were also significantly higher than that in the 

vitrified 1+45 group (50%, 7%) and significantly lower than those in the Control and the Fresh control groups (86%, 

21% and 86%, 23%, respectively). There was no significant difference in total cell numbers among the four groups. 

However, the ICM number of Fresh control embryos was higher than those of the other three groups (Table 3). 

DISCUSSION 

The major finding of this study is that buffalo MII oocytes could be cryopreserved by vitrification with microdrop 

method and the oocytes were suitable for ICSI produce. Vitrification is a simple, rapid and cost-effective method of 

cryopreservation mammalian cells. Using this method cryopreserved cells are less likely to experience solution effects and 

intracellular ice formation (Fahy et al., 1984). However, a negative consequence of this strategy is the increased probability 

of nearly all forms of injury except for those caused by ice crystal formation. To achieve vitrification of solutions, 

a radical increase of both the cooling rates and the concentration of cryoprotectants are required. 

Microdrop method involves dropping an oocyte-containing solution directly into liquid nitrogen, the success of this 

method is due to elimination of the insulation effect of the container wall. Warming of the oocytes is equally rapid when 

vitrified samples are directly dropped into a warm solution (Papis et al., 2000). One essential factor in cryosurvival is the 

permeation of a certain amount of cryoprotectant into the oocyte, which increases with the duration of exposure. 

However, the toxicity of the cryoprotectant must be avoided, with an optimum exposure time being favorable for this 

purpose. 

In the present study, we investigated the effect of different vitrification solution exposure time. The 1+30 group showed 

higher survival rate and embryos developmental rate than 1+45 or 1+60. A toxicity test of the different vitrification 

solution exposure time showed that the embryo development rate decreased with the exposure time extended. The very 



short exposure to a high concentration of EG before cooling and after thawing reduced its toxic effects; moreover, the 

toxic effects during thawing were minimized by the direct dilution method. We attempted to handle oocytes in solutions 

at or close to body temperature of buffalo, followed by quick cooling. 

This study has demonstrated that ICSI into vitrified-warmed oocytes decreased the rates of 2 nd polar body formation, 

cleavage and blastocyst development when compared with the fresh oocytes. The 2 nd polar body formation was judged as 

the ability of oocytes accomplishing meiosis. Our study revealed that the vitrification and warming procedure could 

reduce the oocytes ability to accomplish the second meiosis. A possible reason may be the damage of meiotic spindle 

which has been frequently observed in cryopreserved oocytes (Chen et al., 2003). 

Developmental rate into blastocyst in the 1+30 group was significantly higher than that of the 1+45 group and significantly 

lower than control groups, but did not differ between the Control and Fresh control groups. Similar observation 

were made in total cell number of day 7 blastocysts. This indicated that the vitrification procedure resulted in retarded 

embryonic development and that 30 sec exposure to the vitrification solution was the most effective regimen for buffalo 

MII oocyte vitrification. 

In our study, oocyte viability was determined by FDA staining which in stained cells indicates normal esterase enzyme 

activity and oolemma integrity. We compared the Fresh control and Control oocytes treated with FDA to determine the 

toxicity of FDA staining procedure. Our results showed no difference in the the 2 nd polar body extrusion rate, the embryo 

development and the blastocyst cell number between Fresh control and Control oocytes. This indicates that FDA staining 

does not exert any negative effect upon embryo development and thus it can be safely used for viability testing of 

oocytes. 

In conclusion, our study demonstrated that buffalo oocytes vitrified by the microdrop method with the 1+30 CPA 

regimen could yield high blastocyst rates after ICSI and that the FDA viability checking had no effect on embryo 

development. 

Table 1. In vitro development of CPA-exposed buffalo MII oocytes and quality of blastocysts 

following parthenogenetic activation. 

a,b,c Means within columns with different superscripts differ (P

862 


Table 2. In vitro development of buffalo MII oocytes and quality of blastocysts following vitrification 

and parthenogenetic activation. 

a,b,c Means within columns with different superscripts differ (P


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Abstract 

864 


Effect of different egg yolk levels 

on post thaw quality of Kundhi 

buffalo bull semen 

Fatih, A; M.U. Samo 1 ; I.B. Marghazani * ; Nasrullah; M.A. Kakar; A.Nawaz; 

Livestock and Dairy Development Department, Balochistan, Quetta, Pakistan. 

1 Department of Animal Reproduction, Sind Agriculture University, Tandojam, Pakistan. 

E-mail: marghazani76@hotmail.com 

The study was conducted to evaluate the effect of different levels of egg yolk (20, 15 and 10%) in diluents on post-thaw 

quality of Kundhi buffalo bull semen. It was observed that all the ejaculates were creamy white in color. The mean volume, 

pH, mass activity, sperm concentration and motility % was 2.95+0.07 ml, 6.63+7.30, 3.64+.05, 1283.7+12.12 x10 6 / 

ml and 80.05+3.79%, respectively for fresh semen. The effect of different levels of egg yolk in diluents indicated that 

15% egg yolk resulted significantly highest (P< 0.05) mean post-thaw motility %, livability index and membrane integrity 

of the cells. However, a significant (P

Table-1: Composition of dilution protocol 


Sperm concentration was measured using Hemocytometer. 3 Data thus obtained were analyzed statistically for all the 

parameters. Analytical Computer Software Version 8.1 (for Windows) was applied to develop analysis of variance to 

workout differences between the treatment groups. 


Volume of the fresh semen (2.95±0.07) was recorded in Kundhi buffalo bulls. However, ejaculate volume (4.92±0.23ml) 

of Nili-Ravi 4 and Indian (5.67±0.07ml) Surti buffalo bull 2 were higher than ejaculate volume recorded during current 

study. The discrepancy might be due to the breed variation or age of the bulls as the bulls of current study were at early 

puberty stage. The pH value (6.81±0.04) of fresh semen is in line with the study 5 in which 6.74 pH of bull semen was 

recorded. The mass activity and ejaculation concentration of the sperms in Kundhi buffalo bull were 3.64±0.05 and 

1283.7±12.219x10 6 respectively. Comparatively the mass activity (2.65±1.14) of Nili-Ravi 6 is lower than mass activity 

recorded during current study. 

The post thaw motility, livability index and sperm membrane integrity (%) are given in table-2. The sperm 

mottility (80.05±3.79) in the present study is similar to other reported work 7 which falls in the same range. Egg yolk level 

(15 %) yielded higher post-thaw motility % in buffalo bull semen. However, 18% egg yolk level has also been used for 

dilution in buffalo bull semen 8 . Similar to our study, other 9 found that moderate level of egg yolk was better for membrane 

integrity of sperm cell. 

Table 2: Mean (± S.E) post thaw motility, livability index and sperm membrane integrity (%) of Kundhi buffalo bull semen 

*Different superscripts within the column are significantly (P

REFERENCES 

866 


1. Watson PF. 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. 

Reprod Fertil Dev. 7: 871-891. 

2. Gokhale SB, Joshi PH, Mushtaque M, Mokashi SP. 2002. Studies on the effect of hydrogen ion concentration (pH) of extender on semen 

characters of Surti buffalo bulls. Buffalo Bulletin, 21 (2) : 2-3. 

3. Henery JB. 1991. Clinical Diagnosis & Lab. Management Methods (18th edi.) Co. Sudner, p.499. 

4. Andrabi SMH, Ansari MS, Ullah N, Anwar M, Mehmood A, Akhter S. 2008. Duck egg yolk in extender improves the freezability of buffalo bull 

spermatozoa. Anim Reprod Sci. 104 (2-4): 427-33. 

5. Bracken BN, Bouman LK, Martinez VR. 2003. Comparison between milk and CUE as extenders for bull semen. Connecticut Agr. Exp. Sta. Bull 676. 

6. Javed MT, Khan A, Kausar R. 2000. Effect of age and season on some semen parameters of Nili-Ravi buffalo Bubalus bubalis bulls. Vet. Arhiv, 70 

: 83-94. 

7. Benjamin BR. 1988. Assessment of reeducates activity and motility of Taurine and Bubaline spermatozoa in extenders containing different levels 

of egg yolk and without yolk. Proc. II World Buffalo Congress, ICAR, Vol.1:14-30. 

8. Maxwell WMC, Curnock RM, Longue DW, Reed HCB. 1980. Fertility of ewes following A.I. with semen frozen in pellets or straws a preliminary 

report. Theriogenology. 14(2):83-89. 

Sindhu KS, Guraya SS. 1985. Buffalo Bull semen Morphology, Biochemistry, Physiology and Methodology, USA publisher and distributor 

Ludhiana, India, p. 1 


Abstract 


Effect of different gauge needle 

on transvaginal oocyte 

retrieval in bovine 

Sahatpure, S.K.,C.H.Pawshe and A.S.Ninawe 

Department of Gynaecology 

Post Graduate Institute of Veterinary and Animal Sciences, Akola (M.S.) 

The disposable needles were found to have several advantages over the non disposable type. They are short and easy to 

manipulate, they are easy and less costly to replace, allowing a new needle to be used in each cow, they have a least dead 

volume, which makes them suitable for collecting follicular fluid from individual follicles and unlike the non disposable 

needle, the disposable one did not lose its sharpness after the few aspirations. In the present study, the oocytes were 

aspirated from live animals ovaries using three different needle gauge (18, 19 and 20) to study the effect of different 

needle gauge on the recovery rate and morphology of the cumulus oocytes complex of the oocytes. A total of 36 sessions 

were carried out, in which total 134 animals were aspirated by using different needle gauge. By using 18-gauge needle, 

total 29 animals were aspirated in 9 sessions, in which total 74 follicles (2.55 follicles/animal and 8.22 follicles/session) 

were aspirated. The mean number of large, medium and small follicles were 2.11, 4.11 and 2.00, respectively. A total of 50 

oocytes (1.72 oocytes/animal and 5.56 oocytes/session) were collected, in which the mean number of A, B, C and D 

quality oocytes were 2.44, 0.78, 1.11 and 1.22, respectively. The average oocytes recovery by using 18-gauge needle was 

67.57%. By using 19-gauge needle, total 84 animals were aspirated in 21 sessions, in which total 248 follicles (2.95 

follicles/animal and 11.81 follicles/session) were aspirated. The mean number of large, medium and small follicles were 

3.09, 4.95 and 3.76, respectively. The total 174 oocytes (2.07 oocytes/animal and 8.29 oocytes/session) were collected, 

in which the mean number of A, B, C and D quality oocytes were 2.62, 2.29, 1.71 and 1.67, respectively. The average 

oocytes recovery by using 19-gauge needle was 70.16%. 

By using 20-gauge needle, total 21 animals were aspirated in 6 sessions, in which total 57 follicles (2.71 

follicles/animal and 9.50 follicles/session) were aspirated. The mean number of large, medium and small follicles were 

2.00, 4.16 and 3.33, respectively. The total 27 oocytes (1.29 oocytes/animal and 4.50 oocytes/session) were collected, 

in which the mean number of A, B, C and D quality oocytes were 1.33, 0.83, 2.00 and 0.33, respectively. The average 

oocytes recovery by using 20-gauge needle was 47.37%. The results revealed that by using 19-gauge needle, 11.81 

follicles/session were aspirated. In which, average 8.29 oocytes/session were collected, so the total oocytes recovery 

rate was 70.16%, which was comparatively better than 18 and 20 gauge needle, which having 67.57 and 47.37% oocyte 

recovery, respectively. 


Abstract 

868 


Effect of different vacuum pressure 

(mmHg) on transvaginal oocyte retrieval 

in bovine 

Sahatpure, S.K.,C.H.Pawshe and A.S.Ninawe 

Department of Gynaecology 

Post Graduate Institute of Veterinary and Animal Sciences, Akola (M.S.) 

There is paucity of information concerning the aspiration vacuum which is inconsistent. To date, the aspiration 

vacuum has usually been expressed in millimeter of mercury and has varied between 40 to 400 mmHg. However, the exact 

aspiration vacuum at the top of the needle depends on the construction of the ovum pick up device, the length and the 

diameter of the tubing system and the diameter of the needle. To study the effect of vacuum pressure on recovery rate 

and quality of oocytes three different aspiration vacuum pressures (80, 90 and 100 mmHg) were applied. In the present 

study, the total 36 sessions were carried out in which total 134 animals were aspirated by using three different vacuum 

pressure (80, 90 and 100). By using 80-mmHg vacuum pressures, total 24 animals were aspirated in 7 sessions, in which 

total 59 follicles (2.46 follicles/animal and 8.43 follicles/session) were aspirated. The mean number of large, medium and 

small follicles were 2.29, 3.71 and 2.43, respectively. The total 32 oocytes (1.33 oocytes/animal and 4.57 oocytes/ 

session) were collected, in which the mean number of A, B, C and D quality oocytes were 1.85, 1.00, 1.57 and 0.14, 

respectively. The average oocytes recovery by using 80-mmHg vacuum pressures was 54.23%. By using 90-mmHg vacuum 

pressures, total 90 animals were aspirated in 23 sessions, in which total 262 follicles (2.91 follicles/animal and 11.39 

follicles/session) were aspirated. The mean number of large, medium and small follicles were 2.74, 5.09 and 3.57, respectively. 

The total 182 oocytes (2.02 oocytes/animal and 7.91 oocytes/session) were collected, in which the mean number 

of A, B, C and D quality oocytes were 2.61, 1.96, 1.65 and 1.67, respectively. The average oocytes recovery by using 

90mmHg was 69.46 %. By using 100-mmHg, total 20 animals were aspirated in 6 sessions, in which total 58 follicles 

(2.90 follicles/animal and 9.67 follicles/session) were aspirated. The mean number of large, medium and small follicles 

were 2.83, 3.83 and 3.00, respectively. The total 37 oocytes (1.85 oocytes/animal and 6.16 oocytes/session) were 

collected, in which the mean number of A, B, C and D quality oocytes were 2.00, 1.33, 1.50 and 1.33, respectively. The 

average oocytes recovery by using 100 mmHg was 63.79%. The results revealed that by using 90mmHg vacuum pressure, 

11.39 follicles/session were aspirated. In which, average 7.91 oocytes/session were collected, so the total oocyte 

recovery rate was 69.46%, was better than 80 and 90 mm Hg vacuum pressure, having 54.23% and 63.79% oocytes 

recovery, respectively. 



Effect of eCG Treatment 

On Anestrous Buffalo 

Carvalho, N.A.T. 1 , Gimenes, L.U. 2 ; Ayres, H. 2 ; Soares, J.G. 3 ; Souza, D.C. 2 ; Neves, K. 2 ; Baruselli, P.S. 2 

1 Unidade de Pesquisa e Desenvolvimento de Registro/Pólo Regional do Vale do Ribeira/APTA, Rod. Br116, Km435, Postal 

Code:122, PoBox:11.900-000, 2, Registro–SP, Brazil. 2 Departamento de Reprodução Animal, FMVZ-USP, São Paulo-SP, Brazil. 

3 Universidade Estadual do Maranhão, São Luís-MA, Brazil. - E-mail: nelcio@apta.sp.gov.br 

Abstract 

The aim of this study was to evaluate the effect of eCG treatment on follicular response, diameter of CL and P4 level on 

buffaloes synchronized with P4 device. Forty buffalo were assigned into two Groups (GnoeCG and GeCG) and received a P4 

device (DIB ® ) plus 2.0mg of estradiol benzoate (D0). On D9, the DIB ® was removed and a PGF 2 a was administered. On this 

day, buffalo in GeCG received 400IU of eCG. After two days (D11), each buffalo received 10µg of GnRH. Buffaloes were 

examined by ultrasonography on D -12, D0, D9, D11 to D14, D16, D20 and D24. All animals were bled in order to measure 

P4 level from D16 to D24. There were no significant differences between GnoeCG and GeCG on dominant follicle diameter, 

device removal/ovulation interval, ovulation rate, CL diameter and volume on D20 and D24. Notwithstanding, there were 

differences between GnoeCG and GeCG on CL diameter and volume on D16 and P4 level from D16 to D24. These results 

indicate that treatment with eCG can increase the CL diameter and volume and the P4 level on anestrous buffaloes 

synchronized with P4 device. The beneficial effect of eCG needs to be further investigated on pregnancy rate of anestrous 

buffaloes submitted to FTAI. 

INTRODUCTION 

Keywords: eCG, progesterone, anestrous, CL, ovulation synchronization, buffaloes. 

The pharmacologic base of the ovulation synchronization protocol which utilize the association of progesterone (P4) 

and estradiol (E2) is enough discussed at the literature for bovines, and the effects attributed to this hormones, for the 

simulation of the lutein phase, the follicular atresia, the emergency of the new growing follicular wave and the ovulation 

synchronization are reported by several researches 5, 6, 8, 10 . 

According to Cavalieri et al. 7 , the administration of equine Chorionic Gonadotropin (eCG) at the end of the treatment 

with P4 and E2 for the ovulation synchronization, increase the answer to the estrus of animals in anestrous. The eCG 

stimulate the development of the ovarian follicles 9 due its the only gonadotropin able to league to FSH or LH receivers 13 . 

At this manner, the aim of the present study was to evaluate the effect of the utilization of eCG on the follicular 

development, ovulation, CL development and P4 level of buffalo submitted to P4 and E2 treatment for the ovulation 

synchronization. 



870 


Forty milking buffalo (> 40 days after parturition) were assigned into two Groups (GnoeCG, n=20 and GeCG, n=20) 

according parity, days of parturition, body condition score, presence of dominant follicle and CL on Day -12 (D -12), D0 

and D9 (device removal). All buffaloes received an intravaginal progesterone device (1.0 g P4; DIB ® ) plus 2.0mg of 

estradiol benzoate intra-muscular (IM, RicBE ® ) at random stages of estrus cycle (D0). On D9, the DIB ® was removed and a 

luteolytic dose of PGF 2 a was administered (0.150 mg d-cloprostenol, Preloban ® ). On this day, buffalo in GeCG received 

400IU of eCG (Folligon ® ). After two days (D11), each buffalo received 10µg of GnRH (Conceptal ® ). Buffaloes were examined 

by ultrasonography (Mindray DP2200Vet, 7,5MHz) on D -12, D0 and D9 to verify the ovary status, from D11 to D14 

(12hs apart for 60 hours) to establish the moment of ovulation and on D16, D20 and D24 to measure the CL. All animals 

were bled in order to measure P4 level (RIA) on D16, D20 and D24. Data were analyzed by ANOVA. Results are presented as 

untransformed arithmetic means ± SEM. 


Results are shown in the table below. 

We evaluate the cyclic activities by the CL presence. It was verified by ultrasonographyc examination effected on D-12, 

D0 and D9 that approximately 50% of buffalo did’t has any CL. These females were considered in anestrous. According to 

Baruselli et al. 2 , the eCG effect is more pronounced as much as the incidence of anestrous, which was verified by the 

authors in Bos indicus. 

An the present study, no differences were found between groups regarding to the maximum diameter of dominate follicle, 

time and synchrony of ovulation, as that observed by Marques et al. 11 with cows, Baruselli et al. 3 with heifers and Porto 

Filho et al. 14 with buffalo. These results were different to the study of Cavalieri et al. 7 , that observed synchrony of 

ovulation in cows treated with P4+E2 and eCG. 

There were no differences between GnoeCG and GeCG on ovulation rate. However, the ovulation rate was numerically 

increased by eCG administration. In a previous investigation 3 , the employment of eCG increased the ovulation rate in 

heifers treated with P4 device. The low ovulation rate observed on GnoeCG is in agreement with the results obtained from 

buffalo in anestrous 1, 12, 16 . These authors didn’t use eCG associated to the ovulation synchronization protocol. Thus, 

although it constitutes in numeric increase, the utilization of eCG could promote better ovulation rates. The ovulation 

rates obtained by Singh et al. 15 with the utilization (58.3%) or not (33.3%) of eCG associated to P4 devices in buffalo 

corroborates this hypothesis. Moreover, Singh et al. 17, 18 reported ovulation rates of 65% and 100% in buffalo that 

received eCG. On these both experiments, the authors verified that, neither buffalo presented ovulation on the groups 

without eCG. 



Table. Ovary status and eCG effect on follicular development, ovulation, CL development and P4 level of buffalo submitted 

to ovulation synchronization. Registro – SP, 2009. 

The diameters and volumes of CL on D20 and D24 didn’t differ between the groups, such as verified by Porto Filho et al. 14 . 

Nevertheless, the diameters and volumes of CL on D16 and, the level of P4 from D16 to D24, were higher on GeCG than 

GnoeCG. Baruselli et al. 3 and Marques et al. 11 related increase on P4 level in heifers and cows treated with eCG associated 

to P4 device. Binelli et al. 4 attribute to eCG beneficial effects on the CL area and, consequently, on the P4 levels. 

These results indicate that treatment with eCG can improve the follicular response and increase the CL diameter 

and volume and the P4 level on anestrous buffaloes synchronized with intravaginal P4 device. The beneficial effect of the 

eCG administration needs to be further investigated on pregnancy rate of anestrous buffaloes submitted to FTAI. 


We would like to thanks the Intervet Shering-Plough Animal Health, for the supply of all the hormones utilized on the 

study and the Santa Helena farms. 


872 


REFERENCES 

1 - Bartolomeu CC. 2003. Estudo da dinâmica folicular durante o tratamento com CIDR-B e Crestar visando, a inseminação artificial em tempo fixo 

em fêmeas bubalinas (Bubalus bubalis). 149p. Tese (Doutorado em Medicina Veterinária) – FMVZ-USP. 

2 - Baruselli PS, Marques MO, Reis EL, Bó G.A. 2003. Tratamientos hormonales para mejorar la performance reproducitiva de vacas de cria en anestro 

em condiciones tropicales. Proc. 5o Simp. Intern. Reprod. Ani. Córdoba, Argentina. p.103-116. 

3 - Baruselli PS, Reis E.L, Carvalho NAT, Carvalho JBP. 2004. eCG increase ovulation rate and plasmatic progesterone concentration in nelore (Bos 

indicus) heifers treated with progesterone releasing device. Proc. 15th Intern. Cong. Ani. Reprod. Porto Seguro. 1: 117. 

4 - Binelli M. Thatcher WW, Mattos R, Baruselli P.S. 2001. Anti-luteolytic strategies to improve fertility in cattle. Theriogenology. 56: (9): 1451- 

1463. 

5 - Bó GA, Caccia M, Martinez M, Adams GP, Pierson RA, Mapeltoft RJ. 1994. The use of estradiol and progestogen treatment for the control of 

follicular wave dynamics in beef cattle. Theriogenology. 41(1): 165. 

6 - Bó GA, Adams GP, Caccia M, Martinez M, Pierson RA, Mapletoft RJ. 1995. Ovarian follicular wave emergence after treatment with progesterone and 

estradiol in cattle. Ani. Reprod. Sci. 39: 193-204. 

7 - Cavalieri J, Rubio I, Kinder JE. 1997. Synchronization of estrous and ovulation and associated endocrines changes in Bos taurus indicus cows. 

Theriogenology. 47: 801-814. 

8 - Driancourt MA. 2000. Regulation of ovarian follicular dynamics in farm animals. Implications for manipulation of reproduction. Theriogenology. 

54 (6): 1211-1239. 

9 - Hafez ESE (Ed.). 1993. Reproduction in farm animals. 6th ed. Philadelphia: Lea & Febiger. 573p. 

10- Lane EA, Austin EJ, Roche JF, Crowe MA. 2001. The effect of estradiol benzoate on synchrony of estrus and fertility in cattle after removal 

progesterone-releasing intravaginal device. Theriogenology. 55 (9): 1807-1818. 

11- Marques MO, Reis EL, Campos Filho EP, Baruselli P.S. 2003. Efeitos da administração de eCG e de benzoato de estradiol para sincronização da 

ovulação em vacas Bos taurus X Bos indicus no período pós-parto. Proc. 5o Simp. Intern. Reprod. Ani. Cordoba. p.392. 

12- Moura AJDR. 2003. Sincronização da ovulação com dispositivo intravaginal de progesterona (CIDR-Bâ) associado a estrógeno e prostaglandina 

F2a em búfalas (Bubalus bubalis) tratadas em estações reprodutivas distintas. São Paulo, 129p. Tese (Doutorado). FMVZ-USP. 

13- Murphy BD, Martinuk SD. 1991. Equine chorionic gonadotropin. Endocrine Reviews. 12: 27-44. 

14- Porto Filho RM, Carvalho NAT, Nichi M, Viel Júnior JO, Vannucci FS, Reichert RH, Baruselli PS. 2004. Follicular responses according eCG dosage 

in buffalo treated with progesterone intravaginal device during the off breeding season. Proc. 2nd Buffalo Symposium of Americas. Corrientes 

(CDroom). 

15- Singh G, Singh GB, Sharma RD, Nanda AS. 1983. Experimental treatment of summer anoestrus buffaloes with norgestomet and PRID. 

Theriogenology. 19 (3): 323-329. 

16- Singh G, Singh GB, Sharma RD, Nanda AS. 1984. Ovulation and fertility after PRID, PRID + GnRH and GnRH in anestrous buffaloes. Theriogenology. 

21(6): 859-867. 

17- Singh G, Dhaliwal GS, Sharma RD, Biswas RK. 1988. Treatment of summer anestrous buffaloes (Bubalus bubalis) with progesterone releasing 

intravaginal device plus pregnant mare serum gonadotropin. Theriogenology. 29 (5): 1201-1206. 

18- Singh G, Singh GB, Sharma RD, Nanda AS. 1988. Ovarian and uterine responses in relation to norgestomet - PMSG treatment in the true anoestrus 

buffalo. Ani. Reprod. Sci. 16 (2): 71-74. 


Summary 


Effect of post-thaw cotton incubation 

on semen quality of buffalo bulls 

Madhumeet Singh 

Department of Veterinary Gynaecology and Obstetrics 

College of Veterinary Science, Himachal Pradesh Agricultural University, 

Palampur (HP) INDIA 

Telephone: +919418091090, email: madhumeet2004@rediffmail.com 

In many geographical locations in India, due to difficult terrain and poor transport facilities, thawed semen straws are 

carried for insemination from artificial insemination (AI) stations to the farmers’ door, insulated in cotton. The aim of 

this study was to investigate the effect of this incubation method on post-thaw semen quality of buffalo bulls. Semen 

was collected from 4 Murrah buffalo bulls, stationed at Semen Processing Laboratory Palampur, India. Processing and 

freezing involved dilution in Tris extender and each 0.5 ml French straw contained 40 million spermatozoa. Ten different 

batches, comprising 5 straws per batch, were analysed. These straws were thawed at 40 o C for 14 seconds. Immediately 

after thawing, semen from one straw from each batch was examined for live sperm, progressive motility and acrosomal 

integrity. The remaining 4 straws were wrapped in cotton. Semen quality of each straw from each batch was again 

evaluated at hourly intervals. Mean post-thaw live sperm percentages were 61.7±1.7, 51.3±1.6, 37.8±1.7, 27.3±1.4 and 

24.5±1.3, progressive motility was 48.1±1.9, 29.4±1.2, 19.6±1.9, 13.5±1.8 and 6.5±1.7% and acrosomal abnormalities 

were 17.6±1.0, 21.9±1.2, 28.2±1.7, 35.8±1.7 and 37.7±1.5% immediately after thawing and following 1, 2, 3 and 4h 

incubation, respectively. This post-thaw deterioration in semen quality suggest that the practice of carrying frozen 

thawed straws insulated in cotton for insemination from AI stations to the farmers’ door is likely to adversely affect the 

fertility in buffaloes. 

INTRODUCTION 

Key words: Insemination, fertility, semen. 

Indian breeds of buffalo are one of the best in world and nearly half of the world population of buffaloes is in India. In 

order to achieve the maximum production from these animals, it is necessary that maximum number of females be served 

with the semen of the bulls of superior genetic constitution. Accordingly, buffalo bulls are maintained in many centres 

in the country for greater application of AI in this species. In many geographical locations in hilly state of Himachal 

Pradesh, India, due to difficult terrine and poor transport facilities, thawed semen straws are transported from the rural 

AI stations to the farmers’ door for insemination wrapped in cotton. 

The present study was aimed to investigate the effect of this incubation interval on semen quality of buffalo bulls 

maintained in geographical locations where the climate is more like temperate countries and its possible implication on 

conception in buffaloes. 



874 


Semen was collected From 4 Murrah buffalo bulls of known fertility. These bulls were stationed at Intensive Livestock 

Improvement Project (ILIP) Semen Processing Laboratory Palampur, Himachal Pradesh, India ((32.6 O N, 76.3 O E, altitude 

1290.8 m) and were being used routinely in the AI programme of the state government. Processing and freezing was done 

as per routine procedure. Semen was diluted in Tris extender with the final concentration of 80 million sperms per ml so 

that each 0.5 ml French straw contained 40 million spermatozoa. Ten different batches from each bull, comprising 5 

straws per batch, were analysed. These straws were thawed in a constant temperature water bath at 40 o C for 14 Seconds. 

Immediately after thawing (0 hr), semen from one straw from each batch was examined for progressive motility, live sperm 

percentage 2 and acrosomal integrity 7 . The remaining 4 straws from each batch were wrapped in cotton and stored at 

room temperature for 4 hr. Semen quality of each straw from each batch was again evaluated at hourly interval. 

RESULTS AND DISCUSSI0N 

Mean values for live sperm percentage, progressive motility and acrosomal abnormalities immediately post-thaw (0 hr) 

and at hourly interval following incubation in cotton in buffalo semen has been shown in Table 1. 

Table 1: Effect of post-thaw incubation in cotton on semen quality of buffalo bulls. 

In the present study, mean post-thaw live sperm percentage was 61.7±1.7 immediately after thawing. It is in agreement 

with some earlier reports in which it varied from 52-57% 4 . Mean values following cotton incubation were 51.3±1.6, 

37.8±1.7, 27.3±1.4 and 24.5±1.3 at 1, 2, 3 and 4 hr, respectively. It is apparent that there was a progressive fall in 

the live sperm percentage following incubation of straws in cotton. 

Mean post-thaw progressive motility was 48.1±1.9% at 0 hr, immediately after thawing. In majority of other studies it 

has been reported to vary between 30-40% 8, 9 and 40-60% 1, 10 . Mean values following incubation were 29.4±1.2% at 1 

hr, 19.6±1.9% at 2 hr, 13.5±1.8% at 3 hr and 4.3±2.12 and 6.5±1.7% at 4 hr incubation in cotton, respectively. It is 

apparent that there was a progressive fall in motility following incubation of straw in cotton. 

The mean values for post-thaw acrosomal abnormality, immediately after thawing, were 17.6±1.0%. Values following 

incubation of straws in cotton were 21.9±1.2% after 1hr, 28.2±1.7% after 2 hr, 35.8±1.7% after 3 hr and 36.1+1.55 and 

37.7±1.5% after 4 hr, respectively. Expectedly, the incidence of abnormalities increased following post-thaw incubation. 

Since the acrosome is involved in the fertilization process, it has been postulated that sperms with abnormal and 

damaged acrosome are unlikely to be capable of fertilization 5 . During freezing procedure spermatozoa have to pass 

through many stress factors, like temperature variation, which leads to acrosomal damage 3 . Accordingly estimation of 

percent intact acosomes has been used as index for the evaluation of frozen semen 6 . 



The implication of these results are obvious in relation to successful implementation of AI in buffaloes in many parts of 

the country, where frozen-thawed semen has to be carried from AI centre to the farmer’s door for insemination. Our results 

indicate that if buffalo spermatozoa follow a similar pattern in female reproductive tract as observed in vivo then this may 

account for invariably low conception rate recorded following AI with frozen semen in this species, particularly during 

door service. Based on these results, it is reasonable to suggest that this practice is likely to affect the conception rate 

adversely in buffaloes and consequently leads to repeat breeding; the incidence of which depends upon the post-thaw 

interval to insemination. Our results indicate that if buffaloes are inseminated at the farmer’s door, 1 to 2 hr post-thaw, 

it may lead to considerable decline in the conception rate and consequently a higher incidence of repeat breeding. This 

conclusion is substantiated from our results that in the buffalo the semen quality (particularly the progressive motility) 

deteriorated much below the prescribed standards even at 1 hr post-thaw. Accordingly, in buffalo it is advisable to do AI 

immediately after thawing the frozen semen straw. 

REFERENCES 

1.Anwar M, Andrabi SMH, Mehmood A and Ullah N. 2008. Effect of Low Temperature Thawing on the Motility and Fertility of Cryopreserved Water 

Buffalo and Zebu Bull Semen. Turkish. Journal of Veterinary and Animimal Sciences 32: 413-416 

2. Campbell RC, Hancock JL and Rothchild L. 1953. Counting live and dead bull spermatozoa. Journal of Experimental Biology 30: 44 – 49. 

3.Gilbert GR and Almquist JO. 1978. Effect of processing procedure on post-thaw acrosomal retention and motility of bovine spermatozoa 

packaged in 3 ml straws at room temp. Journal of Animal Science 46: 225-231. 

4. Kumar S. 1992. Effect of different thawing temperatures on leakage of buffalo spermatozoan enzymes. M.V.Sc. thesis submitted to Punjab 

Agricultural University Ludhiana, India. 

5. Meyer RA and Barth AD. 2001. Effect of acrosomal defects on fertility of bulls used in artificial insemination and natural breeding. Canadian 

Veterinary Journal 42: 627. 

6. Saacke RG and White JM 1972. Semen quality tests and their relationship to fertility. Proc. 4th tech. Conference on AI and Reproduction National 

(US) Association of Animal Breeders Chicago pp 22-27. 

7. Saacke RG, Amman RP and Marshal CE. 1968. Acrosomal cap abnormalities of sperm from subfertile bulls. Journal of Animal Science 27 : 1391- 

1400. 

8. Sidhu SS. 1994. Effect of some additives and presence of fungi on the quality and preservation of buffalo bull semen. Ph.D. Thesis submitted to 

Punjab Agricultural University Ludhiana, India 

9. Tuli RK, Bhela SL, Sengupta BP and Singal S. 1986. Effect of raffinose and membrane stabilizers suplementation on motility and release of 

glutamic oxaloacetic transaminase from frozen buffalo semen. Indian Journal of Dairy Science. 39 : 323-324. 

10. Verma HK, Singh M, Singh GD and Pant HC. 1994. Effect of thaw rates on survival, post thaw motility and thermal resistance of spermatozoa of 

cattle and buffalo. Indian Journal of Animal Science 64 : 745-747 


876 


Effect of using fixed-time artificial insemination 

protocols (Ovsynch vs. Progestogen) on 

pregnancy of Buffaloes (Bubalus bubalis) 

Crudeli, G. A.; Maldonado Vargas, P.; De La Sota, R. L.; Scarnatto, R. E.; Konrad, J. L.; 

Olazarri, M. J.; Breard, M.; Patiño, E. M. 

1- Cátedra de Teriogenologia. F.C.V. UNNE. Corrientes - 2- Cátedra de Teriogenologia. F.C.V. UNLP. La Plata 

3- Cátedra de Tecnología de los Alimentos. F.C.V. UNNE. Corrientes - 4- Cátedra de Derecho Agrario. F.D.C.S. y P. UNNE. 

Corrientes - Sargento Cabral 2139, Corrientes (3400), Argentina. 

gcrudeli @vet.unne.edu.ar 

Abstract 

Were realized a research with the aim of compare the pregnancy rate obtained with two different protocols in buffaloes 

of Mediterranean breed, in the north of Corrientes, Argentina. The experiment was realized between the monts of May and 

July of year 2009. In totally, were used 120 pluriparous buffaloes, with calves. The buffaloes were divided in two groups; 

Group 1(G1) n = 59, Ovsynch protocol, with GnRH, PGF 2á and GnRH. The resynchronization: day 28, 8 ìg GnRH; day 35 

pregnancy diagnoses through ultrasonography and PGF 2á to the animals diagnosed empty; day 37, GnRH and day 38 FTAI. 

While Group 2 (G2) n = 61 was used intravaginal dispositives with progestagens. Synchronization: day 1 Estradiol 

Benzoate and putting in the dispositives; day 7, PGF 2á and put out the dispositives; day 8, EB and day 9 FTAI. Resynchronization: 

day 28, EB and second use dispositives, during 7 days; day 35, pregnancy diagnoses through ultrasonography 

and PGF 2á to the animals diagnosed empty; day 36, EB and day 37 FTAI. Starting from the day 55 were 75 and 80% and 

83 and 87% for the first insemination, resynchronization and final pregnancy for G1 and G2 respectively. Concluding, 

that were differences in the pregnancy rate at first FTAI for G2. In the other dates were no significant differences. 

INTRODUCTION 

Keys Words: Buffalo, Synchronization, GnRH, Progestogen. 

The use of artificial insemination (AI) in bovines was widely studied, allowing that the genetic improvement of the 

cattles was faster and efficient. While, in buffaloes this biotechnology has earned a minor number of studies and has been 

less used for the breeders because some difficuties in the identification of the heat manifestations and the appropiate 

time to AI. 

A reproductive characteristic that should be considered in buffaloes is the low incidence of homosexual behaviour 

during heat. Unlike bovines, is rare sow this sintomatology in buffaloes, varying according to authors 3, 12 between a 3, 

4 and 16, 7 %. This behaviour decreases the external observation of the heat and shows that it is indispensable the use 

of markers for the heat detection in this specie. This characteristic asociated to variations in the duration of buffaloes 

heats (6 to 48 hours) becomes the heat detection management more laborious and difficult the AI use. The heat 

synchronization and of the ovulation by hormonal methods in bovines has presented encouraging results for the Fixedtime 

artificial insemination use (FTAI), 16, 17 . The existing synchronization protocols allow for fixed-time artificial insemination 

(in predetermined horary), without the need to observe the heat, facilitating the cattle management and 

optimizing the use of this biotechnology in the field. The study of the follicular dynamic during the estrous cycle 



clarifies the phenomena that interfere in the heat and ovulation synchronization. This depend of the control of some 

important factors as preventing the development of persistent follicles that contain aged oocytes, recruitment of a new 

follicular wave, independently of the estrous cycle status, the luteal phase manipulation and the precise synchronization 

of the future ovulatory follicle 13 . Studying the follicular dynamic during the “Ovsynch” treatment was verify that after the 

first application of GnRH, ovulation occurs and/or the beginning of a new wave of follicular growth, that result in the 

presence of a domiinant follicle 7 days after, the day of PGF 2á application, the induced luteolysis for this one does that 

all the treated animals ovulate between 24 to 32 hours after the second dose of GnRH; demonstrating a high efficiency 

of the “Ovsynch” method in the ovulation synchronization in bovines 16 . Others researchs shows that in buffaloes treated 

with this protocol during the breeding season presented a half conception rate of 50,2%, observed a influence of the 

body condition in the pregnancy rates 7 . Others authors also reported, that verified interference of the body condition 

in the conception rate in buffaloes inseminated artificially 4, 9 . To perform the FTAI must synchronize the ovulation of a 

fertile egg at a given time; this is achieved by inducing the formation of a new follicular wave and subsequent ovulation. 

There are currently many protocols established for this purpose, for example, are those that use the GnRH to induce the 

formation of a new wave, as well, the ovularion at the end of the protocol, this protocol has become popular as Ovsynch 

16 . Has proven effective in buffaloes, with a 56, 5% of pregnancy using the Ovsynch and a 64,2% using LH, instead of the 

second GnRH, in central Brazil 1 . Others authors in north of Argentina, reported that using buffaloes of Mediterranean 

breed, with Ovsynch protocol, in two consecutive years obtained a 50 and 44% of pregnancy respectively for the year 

2002 and 2003 14 . 

There are other FTAI protocols, which used estrogen in combination with progestgens to achieve these objectives. The 

estrogen in the presence of progesterone inhibits the release of GnRH and hence FSH inducing thus involution of the 

follicles once metabolized the estrogen occurs the release of accumulated FSH that induces the formation of a new 

follicular wave around the fourth day of estrogen application. At the end of the protocol is synchronized ovulation again 

with estrogen, as this in the absence of progesterone and with a follicle in clear dominance induces by positive feedback 

the release of LH essential for ovulation 13 . the application of intravaginal dispositives (IVD) with P4, are placed for 

7 to 10 days, along with estradiol benzoate (EB), simulating a artificial corpus luteum (CL), while the EB generates a new 

follicular wave, to remove the dispositive, applies PGF 2á and the next day is placed again EB or GnRH for ovulation 

synchronize, inseminated between 50 to 54 hours following the removal of the dispositive. In India 18 , used IVD by eight 

days, obtaining a 80% of heats post- removal of the IVD and a pregnancy to a FTAI between 60 to 84 hours, of 33%. In 

Brazil 8 , used an progestagen implant (Crestar) vs a IVD, during 9 days, find an interval of initiation of treatment and 

emergence of the wave, of 5,5 and 8,7 days respectively and a pregnancy of 47 and 49% for both groups respectively. In 

another experiment carried out in northern Argentina 15 , used different synchronization and resynchronization protocols, 

such as Ovsynch (G1), comparing Ovsynch plus IVD for 7 days (G2), in the breeding season, vs Ovsynch, outside the 

breeding season (G3). The results described were 66, 6, 40, 5 and 33, 3% of pregnancy to cousin insemination for the 

groups G1, G2 and G3 respectively, while the pregnancy achieved with resynchronization were 33, 3, 40, 9 and 0% for the 

same groups respectively. It´s important to note that the G3 obtains at the first and second FTAI had an embryonic death 

of 60%. In a research made in southeast of Brazil 11 , comment that using single-use and second-use dispositives, and 

evaluating the effect over the follicular wave and the achieved pregnancy rate, comparing the effect of the use of hCG or 

GnRH, 48 hours after dispositive removal, find the following results of 1, 39; 1, 21; 1, 39 and 1,11mm for the dominant 

follicle (DF), for hCG, GnRH in single-use dispositives and hCG and GnRH in second-use. While for the same items obtained 

a pregnancy rate of 49, 3; 54, 4; 55, 6 and 49, 1% for cousin insemination respectively. Researchers from Italy 2 , working 

with pluriparous dairy buffaloes of Mediterranean breed, compared a dispositive with Prid (dispositive with P4), for 10 

days, placing in a group (A), the day 7 PMSG plus PGF 2á and in the (B) the day 10, inseminate all 72 hours after 

dispositive removal. The results obtained were 56, 2 and 57, 4% of pregnancy for the groups A and B respectively. Some 

suggest that high progesterone levels in buffaloes conditions their reproductive performance 6 . By the above, the aim of 

this research was to compare the pregnancy in synchronized buffaloes with an Ovsynch protocol and a progestagen. 



878 


The experiment were realized during the breeding season of the year 2009, between the monts of may and july in a ranch 

located at 70km south of the Corrientes city in the Empedrado department, being located to 27º 59´ 37´´ south latitude 

and 58º 44´ 06´´ W and at a height of 64 meters on sea level, with a pluvial regime of 1200 mm annuals. 

Were used 120 pluriparous buffaloes, with calves, of Mediterranean breed, of differents ages, all of which are older than 

7 and under 11 years old, with a half body condition (BC) of 3,8 evaluated on a scale of 1 (emaciated) to 5 (obese). 

The buffaloes were randomly divided in two groups; 

Group 1 (G1) n = 59 Ovsynch protocol. Synchronization: day 0, 8ìg of buserelin (GnRH, Receptal ® , Intervet Argentina); 

day 7, 150 mg cloprostenol (PGF 2á , Preloban ® , Intervet Argentina); day 9, 8 ìg GnRH; day 10 fixed-time artificial 

insemination (FTAI). Resynchronization: day 28, 8 ìg GnRH; day 35, pregnancy diagnose through ultrasonography and 

150 ìg PGF 2á to the animals dignosed empty; day 37, 8 ìg GnRH and day 38 FTAI. 

Group 2 (G2) n = 61 Progestagens protocol. Synchonization: day 0, 2 mg of estradiol benzoate (EB, benzoato de 

estradiol ® , Biogénesis Argentina) and a intravaginal dispositive of P4 during 7 days (TRIU-B ® of single-use, Biogénesis 

Argentina); day 7, 150 mg PGF 2á ; day 8, 1 mg EB; day 9, FTAI. Resynchronization: day 28, 1 mg EB and a TRIU-B ® of 

second-use, during 7 days; day 35, pregnancy diagnose through ultrasonography (PD 35) and 150 ìg PGF 2á for the 

animals diagnosed empty; day 36, 1 mg EB; day 37, FTAI. View figure 1. 

From the day 55 were realized re-rides with bulls until day 75, that same day were realized ultrasound to determine which 

were pregnant in the resynchronization (PD 75). 

The day 100 was realized ultrasound to determine the pregnancy by bull and final (final PD). The buffaloes were inseminated 

with frozen-defreeze semen of a buffalo with fertility probated. The inseminator was the same for both groups. 

The pregnancy diagnoses were realized with an ultrasound PIE MEDICAL ® Aquila model with rectal transducer of 8 MHz. The 

pregnancy data obtained for both dates were analyzed through the chi square test, of Statistix program for Windows 

version 1.0, 1999. 

RESULTS 

At next were observed the pregnancy data as treatment, at the 35 days, 75 days and final pregnancy, in table 1. 

Table 1. Reproductive efficiency obtained using two synchronization 

and resynchronization ovulation treatments in buffaloes. 

PD: pregnancy diagnose, a different of b , P

DISCUSSION 


The obtained results here agree with the related by authors of Brazil 1 , under dairy animals conditions with controlled 

nutrition 14 , was similar at realized in a same area that the realized in our area, having averages results of 50% for 

different BC, while that lower at the obtained for 15 , for Ovsynch protocols and higher at the obtained with IVD for the 

authors, in the breeding season, being that out of this, the results are lower. The resynchronization data were good for 

both treatments although animals were few how could had differences. Is important to comment that could visualize, 

after the ultrasound, there were some pregnant at cousin FTAI and later appear empty. This variations had to be more 

studies, because according to data 10 , in the 20 to 40 days of pregnancy, occurs in the inseminated buffalo a 22, 9% of 

embryonic loss. While Pellerano et al, 2005, tells higher values but detected out of breeding season (January) with a 60% 

of embryonic death, were the thermal stress had to be a crucial issue. This issue had to be, for their relevance, a 

researching issue in our area. Were concludes that 

In the conditions how were realized our research, the protocol with IVD, was higher in pregnancy to Ovsynch, in cousin 

insemination, while were similar in resynchronization and final pregnancy for the others dates. 

Acknowledge 

To Mr. Hernán Gomez Danuzzo and the workers of the ranch “Rincon del Madregón”, for providing the animals and 

infrastructure for made this research. 

REFERENCES 

1. Araujo Berber R, Madureira EH, Baruselli PS. 2002. Comparation of two Ovsynch protocols (GnRH versus LH) for fixed timed insemination in 

buffalo. (Bubalus bubalis). Theriogenology 57: 1421-1430. 

2. Barile VL, Terzano GM, Allegrini S, Maschio M, Razzano M, Neglia G, Pacelli C. 2007. Relatinship among preovulatory follicle, corpus luteum and 

progesterone in oestrus synchronized buffaloes. VIII World Buffalo Congress, Caserta, Italia, Ital. J. Anim. Sci, 6: 663-666. 

3. Baruselli PS. 1994. Basic requiriments for artificial insenination and embryo transfer in buffaloes. buffalo j., 2: 53-60. 

4. Baruselli PS, Barnabe VH, Barnabe RC. 1995. Efeito da condição corporal sob a tasa de gestación en búfalos. Anais Reunião Anual da Sociedade 

Brasileira de Transferência de Embriões (Atibada, Brasil), p. 229. 

5. Baruselli OS, Muccelolo RG, Viana WG, Castro-Junior FG, Reichert RH, Alvarez RH. 1996. Involución uterina no período pós-parto en hembras 

bubalinas (Bubalus bubalis). B. Indústr. Anim., N. Odessa, 53: 51-55. 

6. Baruselli OS, Mucciolo RG, Visintin JÁ, Viana WG, Arruda RP, Madureira EH, Oliveiras CA, Molero-Filhof JR. 1997. Ovarian follicular dynamics during 

the estrous cycle in buffalo (Bubalus bubalis). Theriogenology 47:1531-1547. 

7. Baruselli OS, Oliveira JFS, Mattos JCA. 1998. Eficiência reprodutiva de búfalos criados no Vale do Ribeira - SP. Congresso Brasileiro De Reprodução 

Animal, 10. Belo Horizonte. Anais, 2: 285. 

8. Baruselli PS, Carvalho NA, Enriquez CH, Nichi M. 2000. Pre-synchronization with GnRH 7 days before Ovsynch protocol for timed insemination in 

buffalo. Anais I Simpósio de Búfalos de las Américas (Belem, Brasil), p. 414-417. 

9. Bhalaru SS, Tiwana MS, Singh N. 1987. Effect of body condition at calving on subsequent reproductive performance in buffaloes. Indian J Anim 

Sci 57: 33-36. 

10. Campanile G, Di Palo R, Neglia G, Vecchio D, Gasparrini B, Prandi A, Galiero G, D’Occhio MJ. 2007. Corpus luteum function and embryonic 

mortality in buffaloes treated with a GnRH agonist, hCG and progesterone. Theriogenology, 67: 1393–1398. 

11. Carvalho NAT, Nagasaku EM, Vannucci FS, Toledo LM, Baruselli PS. 2007. Ovulation and conception rates according intravaginal progesterone 

device and hCG or GnRH to induce ovulation in buffalo during the off breeding season. VIII World Buffalo Congress. Caserta, Italia, Ital. J. Anim. 

Sci, 6: 646-648. 

12. Crudeli GA, Maldonado Vargas P, Flores Barbarán MS. 1996. Comportamiento reproductivo del búfalo en el Nordeste Argentino. Actas del XV 

Congreso Panamericano de Ciencias Veterinarias, Campo Grande, Brasil, p 388. 

13. Driancourt MA. 2000. Regulation of ovarian folicular dynamics in faro animals. Implications for manipulation of reproduction. Theriogenology 


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14. Kizur A, Pellerano GS, Maldonado Vargas P, Rodriguez S, Crudeli GA. 2003. Eficiencia en el uso del protocolo de sincronización “Ovsynch” con 

resincronización en Búfalos en el NEA Argentino. Facultad de Ciencias Veterinarias, UNNE. Comunicaciones Científicas y Tecnológicas 2003, V-041. 

15. Pellerano, G.; Crudeli, G.; De Sa da Silva, A.; Amuchastegui, F. 2005. Evaluación de diferentes protocolos de sincronización y resincronización 

con inseminación artificial a tiempo fijo en búfalos en el noreste argentino. XXVI Sesión de comunicaciones científicas. Facultad de Ciencias 

Veterinarias. UNNE. Julio. 

16. Pursley JR, Mee MO, Wiltbank MC. 1995. Sinchronization of ovulation in dairy cows using PGF2 and GnRH. Theriogenology 44: 915-923. 

17. Thatcher WW, Drost M, Savio JD. 1993. New clinical uses of GnRH and its analogues in cattle. Anim Reprod Sci 33: 27-49. 

18. Rajamahendran R, Thamotharam M. 1983. Effect of progesterone releasing intravaginal device (PRID) on fertility in the post-partum buffalo 

cow. Anim Reprod Sci 6: 111-118. 


ABSTRACT: 


Effects of Different Hormone Combinations 

on Superovulation in River Buffaloes 

Guang-sheng Qin1, 2, 3 , Ming-tang Chen1 , Xian-wei Liang1* , Xiu-fang Zhang1 , Chun-ying Pang1 , 

Sheng-ju Wei2 , Fen-xiang Huang1 2, 3* 

, He-sheng Jiang 

1Guangxi Buffalo Research Institute, Chinese Academy of Agricultural Science, Nanning 530001, China 

2College of Animal Science & Technology, Guangxi University, Nanning, 530004, China 

3Nanning Ovagene Biotechonology Co., Ltd, Nanning, 530003, China 

First author: Tel: +86 771 3338631 Fax: +86 7713338814 - E-mail: qinguangsheng2004@126.com 

Corresponding author: Tel: +86 771 3338631 Fax: +86 771 3338814 - E-mail:liangbri@126.com - hsjiang@tom.com 

This study was conducted to evaluate the effects of different hormone combinations with FSH, PGC, LHRH-A3 and LH on 

superovulation in river buffaloes. Thirty-five heads of river buffaloes were divided into six groups as follows: Group I, FSH 

(Japan, total doses 26 AU) + PGc (Shanghai, 0.6 mg); Group II, FSH (Japan, total doses 26 AU) + PGc (Shanghai, 0.6 mg) 

+ LHRH-A3 (Made in Ningbo, 50 ìg) ; Group III, FSH (Beijing, total doses 20 mg ) + PGc (Shanghai, 0.6 mg) + LHRH-A3 

(Ningbo, 50 ìg) ; Group IV, FSH (Beijing, 20 mg) + PGc (Shanghai, 0.6 mg) + LH (Ningbo, 50 ìg); Group V, FSH (Canada, 

800 mg) + PGc (Shanghai, 0.6 mg); Group VI, FSH (Canada, 800 mg) + PGc (Shanghai, 0.6 mg) + LHRH-A3 (Ningbo, 50 

ìg). The results showed that superovulation rate was 97.14% (34/35). There were 8.71 mature follicles per head in 

superovulation (296/34). The average number of CL was 5.0 (170/34). The average ovulation rate was 57.3% (170/296). 

The average number of embryo collection was 2.72. Average transferable embryos were 1.33 (24/18). Recovery rate and 

transferable rate were 39.84% and 48.98 %, respectively. The mean number of CL in Group II (6.86±5.96) and the 

ovulation rate in Group VI (76.92 %) were the highest among the six groups. The results showed that the ovulation rate 

in Group VI with LHRH-A3 was higher than those in the other groups without LHRH-A3 (12.12%) and that with LH 

treatment (28.64%), respectively. 

1. INTRODUCTION 

KEYWORDS: River buffalo, Superovulation, FSH, LHRH-A3 

There are more than 172 million heads of buffaloes in the world in 2006 (FAO). More than 95 percent of the world 

population is found in Asia where buffaloes play a leading role in rural livestock production. According to statistical data 

(FAO, 2006), the total number of buffaloes in China in 2003 was 22.28 million. Most of them are swamp buffalo. Buffalo 

is an important livestock resource, which provide milk, meat, and work power. The swamp buffalo has the characters of 

low milk production and slow growth. It is urgent to improve species by using embryo biology technique. One possibility 

for enhancing their reproduction efficiency is through genetic improvement of the existing population. Superovulation 

and embryo transfer are commonly used techniques for improving genetic potential. The use of the super ovulation to 

obtain the transferable embryo and perform the embryo transfer can completely highlight the potentiality of the excellent 

female cattle, reduce the generation’s interval and is one of the ways of rapid reproduction of the female cattle 

bearing a special gene. But in buffalo, because of that the effect is not ideal, that technology has not been applied. This 

trial aimed to optimize the study scheme by the use of the different hormones combination treatment to improve the 

effectiveness of the superovulation of the dairy water buffalo. 


2. MATERIALS AND METHODS 

2.1 Animals and management 

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Water buffaloes reared at Guangxi Buffalo Research Institute with known good reproduction records and in normal 

reproductive function were examined and selected for use in this experiment. At any day of the reproductive cycle, 

uterine infusion of chlorine cloprostenol (PGc, Chlorine cloprostenol, Shanghai Institute of Family Planning) 0.6 mg/ 

head was done to perform the synchronization treatment. Female buffaloes observed in estrus, with ovulation and corpus 

luteum on the ovaries were chosen as donors. The average age and body weight of the donors were 6.31±2.14 years old 

and 607.44±81.33 kg., respectively. 

2.2 Experimental design 

According to the method of the association of the hormones, the experiment is subdivided into 6 groups. There were 35 

hds of animals used in this experiment and were randomly divided into six groups: 

Group I : FSH - Japan (26 AU, Japan Denka Pharmaceutical Co., Ltd) + PGc – Shanghai (0.6 mg); 

Group II: FSH-Japan (26 AU) + PGc - Shanghai (0.6 mg) + Ningbo LHRH-A3 (50 µg,Gonadotropin-releasing hormone, LH, 

Ningbo Hormones Products Co., Ltd.); 

Group III: FSH - Beijing (20 mg, Chinese Science Institute Animal Research Institute) + PGc - Shanghai (0.6 mg) + 

Ningbo LHRH-A3 (50 µg); 

Group IV: FSH - Beijing (20 mg) + PGc - Shanghai (0.6 mg) + LH (50 µg, Ningbo); 

Group V: FSH - Canada (800 mg, Folltropin-V, Canada) + PGc – Shanghai (0.6 mg); 

Group VI: FSH – Canada (800 mg) + PGc – Shanghai (0.6 mg) + LHRH-A3 (50 µg). 

2.3. Superovulation treatment 

On day 9 to 11 after estrus synchronization, FSH was administrated intramuscularly to the donors for four consecutive 

days at a decreasing dose for Groups I, II, III, IV or equal in dose for Groups V, and VI. On the third day, PGc was given 

twice intramuscularly. After standing estrus was observed to animal, first insemination was done. In the corresponding 

group, the intramuscular injection of LHRH-A3 or LH was done at the same time with the first insemination. Second 

insemination was done at an interval of 8~12 h from the first insemination. Recovery of embryo was performed on days 

5.5 ~ 6.5 after the first insemination. 

Superovulation started 10 th days after the occurrence of estrus .With 26 AU of the FSH (Japan) as example, the superovulation 

methods and its dosage of hormones was presented in Table 1. 

Table 1. Superovulation method, its processes and dosage of hormones 


2.4 Statistical analyses 


After embryo recovery, the number of corpus luteum and the unovulated follicles (=10 mm) were counted by palpation of 

the ovaries per rectum and B-mode ultrasound equipped with a transrectal 5.0 MHz linear-array transducer (HS-101V, 

Japan, Honda Co.). The non-parametric ANOVA for a single factor (Kruskal–Wallis one-way ANOVA) of SAS/STAT was used in 

the analysis. 

3. RESULTS 

3.1. Superovulation response 

After superovulation treatment, it was observed that 34 out of the 35 donors carried two or more mature follicles with an 

overall superovulation effectiveness rate of 97.14 % (34/35). It was also found out that all buffaloes in Groups I, III, IV, 

V and VI while only 7 hds of buffaloes in Group II had mature follicles and corpus luteum after superovualtion treatment. 

As indicated in Table 2, overall means obtained for mature follicles was 8.71 (296/34) with 5 (170/34) for corpus luteum 

and 57.43 % (170/296) for ovulation rate. 

Table 2. Comparison of the superovulation responses among the different groups 

Numbers with different letters (a, b) differed significantly (P < 0.05). 

3.2. Effects of different hormonal treatments on the follicles and ovulation of the donors 

The numbers of corpus luteum in Groups I (6.17), and II (6.86) were higher than those obtained from other Groups, but 

found to have no significant differences (P£¾0.05) among the treatment groups. The average number of unovulation per 

head was highest for Group I (5.33±2.94) and the lowest was on Group VI (1.50±1.29). Group IV had the lowest 

ovulation rate which accounted to 35.48 % while other Groups had the rates 50% and above. Based on the results, it 

appears that Group II is the best treatment and superior over the other Groups. 

3.3. Corpus luteum and ovulation rate of donors treated with FSH from different sources 

The average corpus luteum of FSH-Japan(6.54±4.52) treated buffaloes was higher than those in FSH(Canada , 5.37±1.85) 

and FSH-Beijing(3.23±1.83), and was found to be significantly different (P£¾0.05) from each other (Table 3). However, 

almost similar results were obtained on the number of unovulated follicles in each group. On the ovulation rate, FSH- 

Japan and FSH - Canada treated groups were significantly better (P£¾0.05) than FSH – Beijing group. 


884 


Table 3. Superovulation effects from different kinds of FSH 


2.4. Effects of LHRH-A3 or LH on the ovulation rate of the donors 

From the Table 4 the results showed that the ovulation rate (67.91%) in LHRH- A3 Group was higher than one (55.79%) 

in no LHRH-A3 and LH Group. It was 12.12%. Also it was higher than one (39.29%) in LH Group. It was 28.62%. Those 

results could also be obtained from the Table 1, which the use of the LHRH-A3 in the superovulation experiment was 

beneficial in enhancing the ovulation rate. However, in this experiment, the use of the LH contrarily reduced the 

ovulation rate, which could be in relation with the fewer number of the female buffaloes in the experiment. 

Table 4. Effects of LHRH-A3 and LH in superovulation 


2.5. Correlation between the number of luteum corpus numbers and embryo recovery 

The presence of corpus luteum in the ovary reflects strong or weak action of FSH on the ovary of the buffalo. On the 

other hand, one factor for worse or better result of the embryo recovery depends on the embryo flushing technical skill. 

The results presented in Table 5 showed that the embryo recovery and the corpus luteum were highly correlation to each 

other. It was experienced that even if there are many corpus luteum in the ovary, if the embryo flushing manipulation 

is not completely done can have a bad effect on the embryo recovery. The curved structure of the horn of uterus of the 

dairy buffalo made difficult also to collect embryos. As such, the average recovery rate obtained in this experiment was 

only 39.84 % from the Table5. 


Table 5. Embryo recovery in each group 

3. DISCUSSION 

3.1. Superovulation effect with FSH 


In the comparison of results from Tables 2 and 3 revealed that in the series of experiments conducted to know the effect 

of different hormonal treatments to buffaloes, even to whether with mature follicles or corpus luteum, the FSH – Japan 

treatment was found to be most effective. The average head number was respectively 10.46 and 6.54, followed by the 

Canadian group with the mean head number of 8.38 and 5.37 from Table3. The Beijing FSH had a slightly bad effect, the 

average head number was only 7.15 and 3.23. The experiment results were similar with the reference (He et al, 2004). 

3.2. The action of the LHRH-A3 and LH on superovulation 

In the treatment of the superovulation, a lot of follicles rapidly develop and mature due to the stimulation of the ovary 

by the use of the FSH. If the LH secretion is not enough, it can not induce sufficiently to mature follicles and then turn 

into corpus luteum. LH level in the serum can be enhanced by the injection of the LH, or by the injection of the GnRH 

or LHRH-A3 to regulate the body FSH and LH synthesis and releasing. In this experiment LHRH-A3 and LH were used 

respectively to determine the effect of two kinds of hormones to stimulate superovulation. From Table 4, we can find that 

the application of LHRH-A3 has improved the ovulation rate, however, the use of LH compared to other groups without 

the ovulation stimulus, had low ovulation rates (39.29% and 55.79%), which was in close agreement with the reports of 

some researchers (Carvalho, et al., 2002). The reason to consider of the results indicated above might be due to the 

addition of an inappropriate dose of LH thus inhibited ovulation. In addition, the number of treated animals is just few 

to conclude something hence further studies with the increase number of animals is necessary. There was another report 

(M.A. Beg et al., 1996) to confirm that the priming of donor buffaloes prior to superovulation or administration of GnRH 

on the day of estrus had no effect on the onset or duration of estrus, ovulation rate or steroid profile. And progesterone 

levels on the day of initial FSH injections and on the day of palpation (per rectum) were positively correlated with 

ovulation rate. 

3.3. The factors influencing the superovulation, embryo recovery and availability 

Out of 34 female donors, 18 donors with many corpora lutea and with good ovulation effect were chosen. Embryo 

flushing was done by one person with different results obtained in each donor. The average embryo recovery per head was 

2.72. The mean available embryo number per head was 1.33. The embryo recovery rate was 39.84 % and the availability 

was 48.98 %. The highest number recovered in one female was 17 and the lowest was zero. Results showed that under the 


886 


same condition, the number of the embryo obtained could be influenced by the embryo flushing technical skills. The 

skilled manipulation not only can help to obtain more embryos, but also shorten the manipulation duration and lighten 

the stress and injury of the animal. When the three-ways embryo recovery tube was used, it was noted that the embryo 

recovery tube must be placed deeply to obtain good results. Because of the strong flexibility of the uterine horn, 

intubations deeply can involve the curling and tilting of the large ligament. To obtain a high embryo recovery rate, only 

the suitable intubation maintaining the embryo recovery point in the lower level of the uterine horn must be performed. 

Some researchers (M. Anwar et al., 1998) reported that it was hard to recover embryos completely if one buffalo was 

slaughtered to collect embryos from uterine horn. As for the transferable embryos, Guo et al. (1998) considered that in 

cattle superovulation must be performed with two to three times of insemination and in each insemination the effective 

spermatozoon number must not be less than to 50×10 6 . It was also considered that the oocytes of development, 

ovulation and movement were disturbed by the use of the FSH, which affected fertilization and reduced the fertilization 

rate. Moreira et al., (2002) have shown that bST (bovine Somatotropin) increased the available embryo number by 

increasing the fertilization rate and the early embryonic development ability which was opposed in the study of Gray et 

al. (1993) who used bST in superovulation found to have no improvement in superovulation response in cattle. Chen et 

al. (1992) considered that the purity of the FSH was related with embryonic quality. Better embryonic development and 

excellent embryos were obtained with the FSH product. Its mechanism relates that maybe the FSH reagent was mixed 

with excessive LH. Oocytes were activated before it developed maturation thus fertilization rate was reduced and the rate 

of embryonic degeneration was increased. In this experiment, the lower embryonic availability obtained was due to the 

lower vitality and fertilization ability of the frozen spermatozoon and might be also to some other factors such as 

hormone quality. It was suggested that further research studies must be undertaken to elucidate hormonal effects and 

improve superovulation in buffaloes. 

ACKNOWLEDGEMENTS 

This work was supported by the Guangxi Young Scientific Foundation (0731046) and the Guangxi Projects for Young 

Scientific Technological Talents Innovation (05112001-10). 

REFERENCES 

Gray, B.W., String, F.D.A., Riddel, M.G., 1993. The effect of treatment with BST on the superovulatory response of cattle. Theriogenology. 39: 227. 

Chen J.B., Ding H., 1992. The Comparison of Effects for Superovulating Dairy Cattle with Different FSH Products. Journal of China Animal Husbandry. 

Volume, 28(2):14-16. (In Chinese) 

Guo Z.Q., 1998. Editor in chief. Animal Embryo Engineering. Science and Technology of China Press. Beijing. (In Chinese) 

Beg, M. A., Sanwal, P. C., Yadav, M. C., 1996. Steroid hormone profile and superovulatory response following priming and GnRH treatment in 

buffaloes.Animal Reproduction Science. Volume 44, Issue 1, Pages: 33-39. 

Anwar, M., Ullah, N., 1998. Early development and location of embryos in the reproductive tract of Nili Ravi buffalo (Bubalus bubalis): A 

retrospective analysis. Theriogenology, Volume 49, Issue 6, Pages: 1187-1193. 

Moreira F., Bandinga L., Burn Ley C., 2002. Bovine Somatotropin in creases embryonic development in superovulated cows and improves posttransfer 

pregnancy rates w hen given to lactation recipient cows. Theriogenology. 57: 1371-1387£® 

Carvalho, N.A.T., Baruselli, P.S., Zicarelli, L., Madureira, E.H., Visintin, J.A., D , Occhio, M.J., 2002. Control of ovulation with a GnRH agonist after 

superstimulation of follicular growth in buffalo: Fertilization and embryo recovery. Theriogenology. 58:1641-1650£® 

Wang Y.Q., Liu W.H., Chen Y.Z., 2008. Study on superovulation and frozen embryos of cattle. China animal husbandry & veterinary medicine. 

35(2):25-27. (In Chinese) 

www.fao.org/FAO Production Yearbook (2006). 

Z.X., He, C.X., Zhang, B.Z., Yang, X.P. Yuan, Z.Q., Li, M.T., Chen, X.F.,Zhang., 2006. The impact of dairy buffalo follicle development and ovulation 

with different FSH doses. Proceeding of the 5th Asian Congress on water buffalo research process and the industrialization of the status of China, 

Volume ?, Nanning, China. p 140-142. (In Chinese) 

Copyright – Papers accepted for publication become copyright of Editorial Office, 9th World Buffalo Congress. 



Embryonic mortality in artificially inseminated 

buffaloes during the breeding season 

Summary 

Neglia G., Vecchio D., Di Palo R., Rossi P., Di Russo C., Campanile G. 

Dipartimento of Scienze Zootecniche ed Ispezione degli Alimenti, Faculty of Veterinary Medicine, 

Via F. Delpino 1, 80137 Napoli, Italy 

The aim of this study was to evaluate the embryonic mortality (EM) rate in Italian Mediterranean buffaloes artificially 

mated during a period of decreasing daylight length. The study was conducted between October and December on 133 

multiparous Italian Mediterranean Buffaloes. The animals were selected by clinical examination before AI and synchronized 

by the Ovsynch-TAI Program, which consists of administration of a GnRH agonist on day 0, a PGF2a analogue on 

day 7 and GnRH agonist again on day 9. Artificial inseminations were performed by the same operator and each buffalo 

was inseminated twice, 16 and 40 h after the second injection of GnRH agonist. Twenty-five and 45 days after AI, 

buffaloes underwent transrectal ultrasonography to assess embryonic development and buffaloes pregnant on day 25, but 

not on day 45 were considered to have undergone EM. Pregnancy rate on Day 25 after AI was 54.9% (73/133) and 

declined to 48.1% (64/133) by Day 45, which represented an EM rate of 12.3% (9/73). This value is definitely lower than 

those reported during the transition period (passage from decreasing to increasing daylight length) by our research 

group. In conclusion this trial represents a further confirmation of buffalo sensitivity to seasonality. 

Keywords: Buffalo, Artificial insemination, Embryonic mortality, Progesterone. 

Introduction 

The buffalo is a photoperiodic species and females show a decline in reproductive activity from mid-winter to spring in 

response to increasing day length 11. The seasonal decline in reproductive activity is manifested by a reduced incidence 

of estrous behaviour, a decrease proportion of females that undergo regular estrous cycles and generally low conception 

rates. This is partially due to the phenomenon of embryonic mortality, that is considered one of the major causes of 

fertility loss in buffalo species 3. In animals mated by artificial insemination (AI) EM can reach 20-40 % during seasons 

characterized by high number of light hours 4. Also in buffaloes naturally mated the incidence of embryonic mortality is 

about 20% and a higher incidence is observed between 28-60 days of gestation in buffaloes that conceive during 

increasing daylight length 10. In any case, EM in buffaloes mated by AI in mid-winter appears to occur later (between 25 

and 45 days), than in cattle 3. Interestingly, a 7% incidence of EM has been reported during periods characterized by 

decreasing daylight length 1. In contrast to the previous work, an EM rate of 20% was reported for buffaloes close to the 

equator 9. However, no information are available regarding the incidence of EM during periods characterized by decreasing 

daylight length in Mediterranean areas. Therefore, the aim of this study was to evaluate the EM rate in Italian 

Mediterranean buffaloes artificially mated from October to December, that are the months characterized by the lowest 

number of light hours in Italy. 

Materials and Methods 

The study was conducted between October and December on 133 multiparous Italian Mediterranean Buffalo cows at 152 

± 59 days in milk and bred in Southern Italy (between 40.5_N and 41.5_N parallel). The animals were selected by clinical 

examination (rectal palpation of ovaries for follicles and corpora lutea to confirm cyclic status and of the reproductive 

tract for any gross abnormalities such as uterine fluid) before AI and only those in a healthy reproductive status were 


888 


included in the study. They were maintained in open yards that allowed 15 m2 for each animal. A total mixed ration 

consisting of 50–55% forage and 45–50% concentrate, containing 0.90 milk forage units ? kg of dry matter and 15% 

crude protein ?dry matter was fed daily in a group pen situation. 

Only animals with a corpus luteum and ?or follicle ‡1.0 cm were selected for synchronizing the oestrous cycle and for AI. 

The synchronization protocol used, Ovsynch with timed-AI, was similar to that developed for cattle 8 and previously 

applied in buffaloes 6. Briefly, it consists of administration of a GnRH agonist (buserelin acetate, 12 ?g; Receptal?, 

Intervet, Milan, Italy) on day 0, a PGF2a analogue (luprostiol, 15 mg; Prosolvin?, Intervet) on day 7 and GnRH agonist 

(buserelin acetate, 12 ?g; Receptal?, Intervet, Milan, Italy) again on day 9. Artificial inseminations were performed by 

the same operator and each buffalo was inseminated twice, 16 and 40 h after the second injection of GnRH agonist. 

Because of the relatively low intensity of oestrous behaviour in buffaloes 7, animals were palpated per rectum (immediately 

before AI) to assess oestrous status (follicle ‡1.0 cm) and a tonic uterus with the presence or absence of mucous 

vaginal discharge. Twenty-five days after AI, buffaloes underwent transrectal ultrasonography to assess embryonic development. 

Ultrasonography was conducted with an Aloka SSD-500 unit equipped with a 5.0 MHz linear array probe (Aloka 

CO., Tokyo, Japan) by the same operator. Pregnancy diagnosis was confirmed on day 45 and 70 after AI by rectal 

palpation. Buffaloes pregnant on day 25, but not on day 45 were considered to have 

undergone EM. 

Results and Discussion 

Pregnancy rate on Day 25 after AI was 54.9% (73/133) and declined to 48.1% (64/133) by Day 45, which represented 

an EM rate of 12.3% (9/73). The proportion of buffaloes with embryonic development on Day 26 after AI was also similar 

to that reported for buffaloes that underwent estrus synchronisation and AI during a period of decreasing day length 1. 

Embryonic mortality rate is quite higher than that reported by other authors 1 during periods characterized by decreasing 

daylight length, but is definitely lower than those reported during the transition period by our research group 2,4,5. 

It was previously demonstrated that infectious agents as a likely cause of embryonic mortality accounted for only around 

8%of the losses 4, hence it would be confirmed that the seasonality play a fundamental role in buffalo reproductive 

activity. Also in buffalo naturally mated the incidence of embryonic mortality is about 20% and a higher incidence is 

observed between 28-60 days of gestation in buffaloes that conceive during increasing daylight length 10. 

In conclusion this trial represents a further confirmation of buffalo sensitivity to seasonality, although additional 

studies are needed in order to reduce the incidence of the phenomenon also in periods characterized by increasing 

daylight length. 

References 

1. Baruselli PS, Visintin JA, Barnabe VH, Barnabe RC, Amaral R, Souza AC. 1997b. Early pregnancy ultrsonography and embryonic mortality 

occurence in buffalo. Proc. V World Buffalo Congress, Caserta, Italy, October 1997b, pp. 776-778. 

2. Campanile G, Di Palo R, Neglia G, Vecchio D, Gasparrini B, Prandi A, Galiero G, D’Occhio MJ. 2007.Corpus luteum function and embryonic mortality 

in buffaloes treated with a GnRH agonist, hCG and progesterone. Theriogenology 67:1393-1398. 

3. Campanile G, Neglia G. 2007. Embryonic mortality in buffalo cows. Italian Journal of Animal Science 6 (Suppl. 2 – Part 1):119–129. 

4. Campanile G, Neglia G, Gasparrini B, Galiero G, Prandi A, Di Palo R, D’Occhio MJ, Zicarelli L. 2005. Embryonic mortality in buffaloes synchronized 

and mated by AI during the seasonal decline in reproductive function. Theriogenology 63:2334–2340. 

5. Campanile G, Vecchio D, Di Palo R, Neglia G, Gasparrini B, Prandi A, Zicarelli L, D’Occhio MJ. 2008. Delayed treatment with GnRH agonist, hCG and 

progesterone and reduced embryonic mortality in buffaloes. Theriogenology 70:1544–1549. 

6. Neglia G, Gasparrini B, Di Palo R, De Rosa C, Zicarelli L, Campanile G. 2003. Comparison of pregnancy rates with two oestrus synchronization 

protocols in Italian Mediterranean Buffalo cows. Theriogenology 60:125–133. 

7. Ohashi OM. 1994. Estrous detection in buffalo cow. Buffalo J 2:61–64. 

8. Pursley JR, Mee MO, Wiltbank MC. 1995. Synchronization of ovulation in diary cows using PGF2a and GnRH. Theriogenology 44:915–923. 

9. Vale WG, Ohashi OM, Sousa JS, Ribeiro HFL, Silva AOA, Nanba SY. 1989. Morte embrionária e fetal em bufalos, Bubalus bubalis. Lin Revista Brasileira 

de Reprodução Animal 13:157–165. 

10. Vecchio D, Di Palo R, Zicarelli L, Grassi C, Cammarano A, D’Occhio MJ, Campanile G. 2007. Embyonic mortality in buffalo naturally mated. Italian 

Journal of Animal Science, 6 (suppl 2–part 1):677-679. 

11. Zicarelli L. 1997. Reproductive seasonality in buffalo. Proc. Third Course on Biotechnology of Reproduction in Buffaloes, Caserta, Italy, Issue 

II, pp. 29–52. 



Ethno-veterinary practices for the treatment of 

anoestrus and retained fetal membranes in 

buffalos in District Faisalabad, Pakistan 

ABSTRACT 

Arfan Yousaf 1 , Muhamamd Younis 1 , Tanveer Ahmad 1 , Nafees Akhtar 2 

1 Department of Clinical Medicine and Surgery, University of Agriculture, Faisalabad, Pakistan 

2 Department of Theriogenology, University of Agriculture, Faisalabad, Pakis 

arfan_yousaf@yahoo.com 

The present study was carried to document the ethno-veterinary practices currently in use for the treatment of two most 

common reproductive disorders i.e. anoestrus and retained fetal membranes (retained placenta) in buffaloes in the five 

villages of district Faisalabad (Pakistan) using rapid and participatory appraisal (RRA & PRA) techniques over a period of 

6 months. The data was collected from 100 respondents including traditional veterinary healers and farmers using the 

structured interview technique. As a result of this study 46 medicinal plants belonging to 30 families, 11 materials other 

than plants and 14 food materials were identified being used for the treatment of subject reproductive problems of the 

buffaloes. Most frequently reported (= 10 times) plants represented family Apiaceae, Brassicaceae, Compositae, Fabaceae, 

Liliaceae, Linaceae, Malvaceae, Musaceae, Pedaliacee, Piperaceae, Poaceae, Rosaceae, Solanaceae, Theaceae and 

Zingiberaceae. The four most frequently reported plants were Trachyspermum ammi, Linum usitatissimum, Brassica campestris 

and Gossypium hirsutum. Materials other than plants included ammonium chloride, sodium chloride, sodium bicarbonate 

, pigeon’ s faeces, and cloth-washing soap. Food materials included jaggary, curd, sugar (cheeni or shaker), 

diluted curd (called lassi in vernacular), water, canal water, milk, candied roses, honey of small bee, camel milk, goat milk, 

filth from Jalebi (a type of sweet), knead flour, bread, vegetable ghee , refined butter (called desi ghee in vernacular) and 

eggs from back-yard poultry. Mostly the route of administration was oral. Different parts of plants were reported to be 

used in different formulations and there was a great variation in the dose range. 

Key Words: anestrous, dairy, ghee, oestrus cycle, reproduction 


890 


Evaluation of a different time of the second 

GnRH injection during Ovsynch protocol 

in buffaloes 

Barile, V.L. 1 ; Pacelli, 2 C.; Barbato, O. 3 ; Maschio 1 , M.; Baldassi, D. 1 ; Terzano, G.M. 1 ; Borghese A. 1 

1 Animal Production Research Center (CRA-PCM), Monterotondo (Rome), Italy; 2 Department of Animal Production Science, 

University of Basilicata, Potenza, Italy; 3 Department of Biopathological Veterinary Science, Faculty of Veterinary Medicine, 

University of Perugia, Italy E-mail: vittorialucia.barile@entecra.it 

Abstract 

The aim of the work was to verify the effect on fertility after altering the timing of the second GnRH injection in the 

Ovsynch protocol for the application of fixed time artificial insemination (AI) in buffalo. Forty-nine Italian Mediterranean 

buffalo cows were submitted to this trial. All buffaloes received GnRH, followed 7 days later by PGF2á, and then 

received one of the following: 1) GnRH 48h after PGF2á (Ovsynh-48; n=24); 2) GnRH 56h after PGF2á (Ovsynh-56; n=25). 

At the time of AI (16h after the second GnRH) a further GnRH dose was done. Starting 24 h before the AI, the ovaries 

were ultrasonografically examined to study the preovulatory follicle and the time of ovulation. The conception rate 

observed 40 days after AI was 45,83% in the Ovsynch-56 and 20% in the Ovsynch-48 (P=0.05). The efficiency of the two 

protocols, taking into account the conception rate, the size of the preovulatory follicle and the ovulation rate, is 

discussed. 

INTRODUCTION 

Key words: Buffalo, Oestrus synchronization; Ovsynch, Artificial insemination 

Management schemes that do not require oestrus identification so, contributing to the increase in the use of artificial 

insemination (AI) in buffalo, are easy to perform. Different hormonal treatment schedules for a timed artificial insemination 

(TAI), such as Ovsynch (GnRH+ PGF2á+GnRH), have been proposed. Efficiency of this protocol is variable depending 

moreover on the season in which it is employed (1). Several studies have been performed in cows to provide information 

regarding the optimal time of AI in relation to the ovulation and the optimal time to deliver the final GnRH, but no 

information is reported for buffalo. The aim of this work was to verify the effect on fertility after altering the timing of 

the second GnRH injection in the Ovsynch protocol in buffalo cows. 


The trial was carried out on 49 lactating Italian Mediterranean buffaloes cows of different ages and parity, starting from 

February until May (low breeding season for Italian buffalo). Animals were subjected to a treatment of oestrus syncrhonization 

and fixed time artificial insemination (AI) using Ovsynch protocol. All buffaloes received GnRH (20 ug 

buserelin), followed 7 days later by PGF2á (0.15 mg cloprostenol), and then received one of the following: 1) GnRH (20 

ug buserelin) 48h after PGF2á (Ovsynh-48; n=24); 2) GnRH (20 ug buserelin) 56h after PGF2á (Ovsynh-56; n=25). At the 

time of AI (16h after the second GnRH) a further GnRH at the same dose was done. Pregnancy was diagnosed by rectal 

palpation 40 days after AI. Ovarian ultrasound examination was performed by a 7.5 MHz linear rectal probe, 24h before 

AI, at AI and 24h post-AI, to measure the preovulatory follicle and verify the ovulation. Data were analyzed by ANOVA and 

c 2 test. 




Overall conception rate (CR) at AI was 30.18%. A lower CR was found in buffaloes synchronized with the standard Ovsynch 

protocol (GnRH - 7days PGF2á - 48h GnRH – 16h AI) compared with those in which the second GnRH injection was 

delayed at 56h respect to PGF2á injection (20% in Ovsynch-48 vs 45,83% in Ovsynch-56 group; P=0,05). This result 

supports the hypothesis proposed, that the differences in time from PGF2á until GnRH could affect the fertility, although 

a higher overall CR was expected. In fact, in previous works aimed to study the efficiency of Ovsynch+TAI in buffaloes, we 

found a CR of 42.5% (2) with the standard protocol and double AI at 16h and 40h, and a lower CR of 27.14% (3) when 

a single AI delayed at 40h was done. There are not references regarding the use of Ovsynch-56 in buffaloes, but in cows 

there is evidence that Ovsynch-56 provides an increase of fertility (38.6%) compared with Cosynch-48 (29.2%) and 

Cosynch-72 (25.4%) (4), although the study did not provide information on whether Ovsynch-56 is superior to the 

standard Ovsynch protocol. The results of our study provide evidence that Ovsynch-56 in buffalo affect positively the CR. 

We have supposed that the GnRH injection 56 h after PGF2á could have increased the size of ovulatory follicle compared 

with GnRH done at 48 h, but analysis of follicular size data does not support this hypothesis. In fact, no significant 

differences were found in the size of ovulatory follicle between the two treatments, even though a larger follicle was 

recorded when the GnRH was delayed (14.5±0.6 vs 13.1±0.6 mm respectively in Ovsynch-56 and Ovsynch-48 groups). 

Peters and Pursely (5) reported, in cow, that increasing the time between decline of P4 until the LH surge increased 

fertility, probably due to a slightly longer time with lower circulating progesterone and higher circulating oestrogen that 

allow to a better oviduct environment for the survival and transport of spermatozoa and ovum. The ovulation rate, also, 

was not significant different in the two treatments (83.33% in Ovsynch-56 and 80% in Ovsynch-48. In conclusion, from 

this study seems that postpone the final GnRH treatment 56 h after PGF2á in the Ovsynch+TAI protocol, improve fertility 

rate at AI. Nevertheless, further study involving more farms and animals are needed to verify the validity of the protocol 

proposed. 

Table 1. Effect of the administration of final GnRH of Ovsynch at two different times from PGF2á (48 vs 56 h) in 

buffalo cows on conception rate, ovulatory rate and ovulatory follicle size 

a,b=P=0.05 

REFERENCES 

1. Barile, V. L., 2005. Reproductive efficiency in female buffaloes. In: “Buffalo Production and Research”, A. Borghese (Ed). FAO, Rome - REU 

Technical Series; 67, 77-107. 

2. Barile V.L., Pacelli C., De Santis G., Penna L., Veloccia C., Allegrini S., Lomolino R., Barbato O., Borghese A., 2004. Fixed time artificial 

insemination in buffalo using two different hormonal schedale for oestrus synchronization. Preliminary results. Proc. of 7th World Buffalo Congress, 

Manila (Philippines), 20-23 October 2004, vol.II, 585-587. 

3. Barile V.L., Pacelli C., Terzano G.M., Allegrini S., Penna L., Veloccia C., Coletta A., Borghese A., 2005. Conception rate at artificial 

insemination using two different oestus synchronization schedule in buffaloes. Atti 3° Congresso Nazionale sull’Allevamento del Bufalo and 1st Buffalo Symposium of Europe and Americas, Capaccio-Paestum (Italy), 12-15 Ottobre 2005, 226-227. 

4. Brusveen, D. J.; Cunha, A. P.; Silva, C. D.; Cunha, P. M.; Sterry, R. A.; Silva, E. P. B.; Guenther, J. N.; Wiltbank, M. C., 2008. Altering the time 

of the second gonadotropin-releasing hormone injection and artificial insemination (AI) during Ovsynch affects pregnancies per AI in lactating 

dairy cows. Journal of Dairy Science, 91(3), 1044-1052. 

5. Peters, M. W.; Pursley, J. R., 2003. Timing of final GnRH of the Ovsynch protocol affects ovulatory follicle size, subsequent luteal function, and 

fertility in dairy cows. Theriogenology 60(6), 1197-1204. 


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Evaluation of Recovery, Quality and In Vitro 

Nuclear Maturation of Oocytes Obtained 

from Buffalo and Bovine Ovaries 

Leal, L. S. 1 ; Moya-Araújo, C. F. 1 ; Fernandes, C. B. 1 ; Martins, L. R. 1 ; 

Landim-Alvarenga, F. C. 1 ; Oba, E. 1 

1 Department of Animal Reproduction and Veterinary Radiology, College of Veterinary Medicine and Animal Science, São Paulo 

State University (UNESP) - 18618-000 -Botucatu, São Paulo State, Brazil E-mail: lu_s_leal@yahoo.com.br 

Abstract 

Ovaries of 86 buffaloes and 95 cows were collected from slaughterhouses and transported to the laboratory in saline 

solution at 36 o C. The Cumulus-oocyte complexes (COCs) were recovered by follicular aspiration and only grades I and II 

COCs were selected and matured in TCM 199 supplemented with 10% fetal calf serum, sodium pyruvate, LH, FSH, estradiol, 

gentamicin and cysteamin, for 22-24 hours. A total of 714 and 1983 COCs were recovered from buffaloes and cows, 

respectively. In the buffaloes, the recovery rates of each COCs categories (grade I: 25.9%; grade II: 30.7%; grade III: 

10.2%; denuded 18.6% and expanded: 14.6%) were lower than cows (31.8; 30.6; 15.4; 4.5; 17.7%, respectively) 

according to Mann-Whitney Test (p< 0.05). The percentage of bubaline oocytes that reached metaphase II (63.4% - 

242/ 396) was lower than bovine oocytes (67.8% - 696/ 1234) under the same laboratory conditions . These differences 

observed in all analysis realized indicate that each species has peculiar phisiological characteristics. 

INTRODUCTION 

Keywords: buffaloes, cows, in vitro maturation, oocytes. 

Researchers around the world have investigated the function of biological factors and events during folliculogenisis and 

in the process of in vitro maturation of oocytes in several species in order to improve technologies of embryo production 

and manipulation. 


The ovaries were obtained from 86 Murrah x Mediterranean buffaloes and 95 Zebu cows in slaughterhouses located in the 

cities Lençóis Paulista (Frigol ® , distance 55 Km), Rancharia (Better Beef ® , distance 318 Km) and Cajati (Frivale ® , distance 

460 Km), São Paulo State, Brazil. Weight and age were not determined but all evaluated females were adult. The time of 

ovary transportation to the laboratory was from 4 to 6 hours for buffaloes and 45 minutes for cows. 

The ovaries from each female were separately collected, kept in labeled plastic bags containing heated saline solution 

(36ºC) added of 100 units/mL penicillin and 100 µg/mL streptomycin, and transported to the laboratory in an isothermal 

box. 



The Cumulus-oocyte complexes (COCs) were recovered by follicular aspiration and only grade I (COCs compact and uniform, 

with 3 or more layers of cells and homogeneous cytoplasm) and grade II (COCs slightly less compact, with less than 

3 layers of cell or cytoplasm less homogeneous) were selected and matured in TCM 199 supplemented with 10% fetal calf 

serum, sodium pyruvate, LH, FSH, estradiol, gentamicin and cysteamin. In vitro maturation was carried out at 38,5 o C 

under a controlled gas atmosphere of 5% CO 2 in humidified air for 22-24 hours. For the evaluation of nuclear maturation 

the oocytes were removed from medium after the period of IVM and placed in TCM 199 medium added with type v 

hialuronidase where the granulosa cells were extracted by a glass micropipette. The denuded oocytes were trasferred to 

10 µL of Hoescht 33342 in glass slide and the chromosomic configuration was evaluated by on inverted fluorescent 

microscope (Leica DMIRB). 

Statistical analysis was carried out in the Department of Biostatistics, Institute of Biosciences, São Paulo State University 

– UNESP, Botucatu, São Paulo State. Results were subjected to statistical treatments of mean, standard deviation, 

median, first and third quartiles, and percentage. In all analysis, differences were considered significant when p< 0.05. 

RESULTS AND CONCLUSION 

A total of 714 and 1983 COCs were recovered from buffaloes and cows, respectively. In the buffaloes, the recovery rates 

of each COCs categories (grade I: 25.9%; grade II: 30.7%; grade III: 10.2%; denuded 18.6% and expanded: 14.6%) were 

lower than cows (31.8; 30.6; 15.4; 4.5; 17.7%, respectively) according to Mann-Whitney Test (p< 0.05, Table 1). 

Table 1. Median [1 0 and 3 0 quartiles] of the variables GI, GII, GIII, DEN, EXP e TOT RECOV according to animal species. 

GI (grade I), GII (grade II), GIII (grade III), DEN (denuded), EXP (expanded) and TOT RECOV (total number of oocytes 

recovered). 

The percentage of bubaline oocytes that reached metaphase II (63.4% - 242/ 396) was lower than bovine oocytes 

(67.8% - 696/ 1234) under the same laboratory conditions (Table 2 and Figure 1). 

Table 2. Median [1 0 and 3 0 quartiles] of variables GV, GVB, MI, MII, DEG e TOT STAIN according to animal species. 

GV (germinal vesicle), GVB (germinal vesicle breakdown), MI (metaphase I), MII (metaphase II), DEG (degenerated) e TOT 

STAIN (total number of oocytes stained). 


894 


Figure 1. Photography (increase of 400x) of buffalo (1a) and cow (1b) oocytes that reached metaphase 

II stage stained with Hoechst 33342 visualized by on inverted fluorescent microscope (Leica DMIRB). 

These differences observed in all analysis realized indicate that each species has peculiar phisiological 

characteristics. 

Acknowledgements. Frigol, Better Beef and Frivale slaughterhouses. This study was supported by CAPES, FAPESP 

(05/51151-2) and FUNDUNESP (00181/07 – DFP) – Brazil. 


INTRODUCTION 


Normal spermatogenesis in scrotal mammals depends upon maintenance of optimum testicular temperature. In bulls, 

testicular temperature is maintained 4 - 5° C below body temperature. The effect of increased testicular temperature has 

detrimental effects on semen quality and sperm motility 3 . However, since blood flow in the testis does not include at all 

or not enough to match the increased metabolic rate of the heated testis tissue 5,6 . 

The objective of the present study was to evaluate the effects of heat on sperm production in buffaloes submitted to 

experimental testicular insulation. 


The experiment was conducted at the northeast region of Pará State. Were used six murrah buffaloes with mean age of 50 

± 2 months. After a period of adaptation and conditioning, the animals were submitted to semen collection by artificial 

vagina, for evaluation of physical and morphologic characteristics before, during and after insulation. 

In six animals were used bag, made of plastic, Arabic gum to fix the cotton to scrotal skin, tape and adhesive tape. The 

bag was set around the cord and scrotum, for seven days and checked the rectal‘s temperature, the cord‘s temperature and 

the temperature between the skin of the scrotum and the plastic bag, at different times. 

The experimental period was divided into five phases: pre-insulating (PI), insulation (I) after insulation up to thirty 

days (P30), within sixty (P60) and up to ninety (P90). 

The significatic different were calculated by Statistical Analisys System (SAS, 1997) using SNK (Student-Newman-Keuls) 

to evaluate the data, with a probability of 1%. 


Experimental Study of Testicular 

Insulation In Buffalo 

2 Garcia, O. S.; 1 Vale, W. G.; 3 Garcia, A. R.; 1 Ribeiro, H. F. L.; 4 Ferro, R. S.; 1 Rolim Filho, S. T.; 5 Sousa, E. M. 

1 Division of Animal Reproduction. ISPA/UFRA. 2 Master Students – Animal Science – UFRA/UFPA/EMBRAPA. 

3 Empresa Brasileira de Pesquisa Agropecuaria – EMBRAPA. 4 Laboratório de Genética e Biotecnologia da Reprodução – 

UEMA 5 Graduate Student -UFRA E-mail: onelsolano@yahoo.com.br 

The results showed that increased testicular temperature for a short period of time has altered the physical and morphological 

characteristics of semen in all animals, ranging in intensity between individuals (Table 1 e 2). 

The color, appearance and volume of semen collected, have not changed the insulation testicular (P>0,01) and do not 

represent reliable parameter to evaluate the effects of thermal challenge testicular 2, 3 . 

Key-Words: Andrology, buffaloes, testicular degeneration, semen 


896 


Table 1 – Means ± SEM of volume, turbulence, progressive motility individual and sperm vigor of buffaloes submitted 

to testicular insulation. 

PI – Pre-insulation; I – Insulation; P30 – Post-insulation (day 1-30); P60 - Post-insulation (day 31-60); P90 – Postinsulation 

(day 61-90). 

*Means in the same column followed by same letter do not differ by test of SNK (Student-Newman-Keuls) (P>0,01). 

Turbulence was significantly reduced (P


Table 3 - Means ± SEM of major defects, minor defects and total defects of buffaloes submitted to testicular insulation. 

PI – Pre-insulation; I – Insulation; P30 – Post-insulation (day 1-30); P60 – Post-insulation (day 31-60); P90 – Postinsulation 

(day 61-90). 

* Means in the same column followed by same letter do not differ by test of SNK (Student-Newman-Keuls) (P>0,01). 

Significative variations (P

898 


Expression of Estrogen Receptors β 

in Ovarian Follicles of water buffalo tissues 

(Bubalus bubalis) 

Salvetti, N.R. 2 ; Baroni, E.E. 1 ; Pellerano, B. 1 ; Gasparini, S. 1 ; Montero, R. 1 ; Ortega, H. H. 2 

1 Cátedra de Farmacología-Toxicología. Facultad de Cs Veterinarias. Universidad Nacional del Litoral. Argentina. 

2 Departamento de Ciencias Morfológicas. Facultad de Cs Veterinarias. Universidad Nacional del Litoral. Kreder 2805-(3080) 

Esperanza. Argentina. E-mail: hhortega@fcv.unl.edu.ar 

INTRODUCTION 

The ovarian steroid hormones perform several important functions related to reproduction through endocrine mechanisms 

of action. The genomic effects of these hormones are mediated through interaction with specific intracellular 

receptors that are members of the nuclear receptor families. Hormone binding to their receptors induces structural and 

functional changes in the receptor structure that associates ligand-receptor complexes with specific target genes to 

regulate their transcription 1-4 . 

Two major forms of estrogen receptor (ER) have been identified in mammalians: ERá and ERβ. These receptors have a 

differential distribution in the different organs both in the male as female. The existence of subtypes may partly explain 

the selective action of estrogen in different target tissues and in the same tissue during different physiological states. 

The two receptors bind 17β-estradiol with high affinity and specificity. Although ERβ shares many functional characteristics 

with ERα, the molecular mechanisms that regulate its transcriptional activity and its tissue location are different 

from those of ERα 5-9 . Several studies have demonstrated the cellular distribution of ERβ in the female reproductive 

organs of various species including cow 3-7,9-10 . 

The purpose of this study was to determine the expression of Estrogen Receptor Beta (ERβ) in ovarian follicular structures 

from adult buffalo cows by immunohistochemistry. 


Ovaries were collected at abattoir and fixed in 10% buffered formalin and then washed in phosphate buffer saline (PBS). 

Tixed tissues were dehydrated in an ascending series of ethanol, cleared in xylene, and embedded in paraffin. Serial 

sections (5 µm in thickness) were mounted on 3-aminopropyl triethoxysilane (Sigma, USA)-coated slides, and dried for 

24 h at 37°C 4,10 . 

A streptavidin-biotin immunoperoxidase method was done as previously described 4,10 . In brief, sections were deparaffinized, 

hydrated and microwave pre-treatment (antigen retrieval) was performed. The endogen peroxidase activity was 

inhibited with 1% H2O2 and nonspecific binding was blocked with 10% normal goat serum. All sections were incubated 

with the primary antibody (Polyclonal anti - Estrogen Receptor β; BioGenex, San Ramón, CA, USA) 18 h at 4ºC and then, 

after having been washed in PBS, the samples were incubated for 30 min at room temperature with preabsorbed biotinylated 

secondary antibodies. 

Keywords: ovary, estrogen receptor, steroid, buffalo 



The visualization of antigens was achieved by the streptavidin-peroxidase method (BioGenex, San Ramon, CA, USA) and 

3.3-diaminobenzidine (Liquid DAB-Plus Substrate Kit - Zymed, San Francisco, CA, USA) was used as chromogen. Finally, 

the slides were washed in distilled water and counterstained with Mayer’s hematoxylin, dehydrated and mounted. 

RESULTS 

A summary of the immunohistochemical expression is given in figure 1. 

ERβ protein was detected in nuclei of granulosa and theca interna of all follicles studied, although the intensity of ERβ 

in granulosa cell layer was stronger in secondary follicles than in tertiary and atretic follicles. A weak immunostaining was 

evident in follicular cells of primary follicles. 

DISCUSSION 

Figure 1: Expression of estrogen receptor â in the ovary of Buffaloes as revealed 

by immunohistochemistry in secondary (left) and tertiary (right) follicles. 

In conclussion, ovaries from buffalos exhibited a ERβ expression pattern similar to Bos Taurus cows 3,9,10 , an these could 

play an important role in the ovarian physiology and pathology. On the other hand, changes in the concentration of the 

different types of receptors in granulosa and theca cells of follicles could alter the ratio ERα/ERβ causing modifications 

in the way of action of the estrogen on its target cells. Recent researches on ER knockout animals revealed that the 

presence of both ERs is a prerequisite for the proper functioning of the hypothalamic-pituitary-ovarian axis and successful 

ovulation 1,2 . Further studies are necessary to fully understand and appreciate the implications of these observations. 

REFERENCES 

1. Beato M & Klug J. 2000. Steroid hormone receptors: an update. Human Reproduction Update 6:225-236. 

2. Drummond AE, Britt KL, Dyson M, Jones ME, Kerr JB, O’donnell L, Simpson ER & Findlay JK. 2002. Ovarian steroid receptors and their role in 

ovarian function. Molecular and Cellular Endocrinology 191:27-33. 

3. Berisha B, Pfaffl M & Schams D. 2002. Expression of estrogen and progesterone receptors in the bovine ovary during estrous cycle and pregnancy. 

Endocrine 17:207-214. 

4. Ortega HH, Salvetti NR & Padmanabhan V. 2009. Developmental programming: prenatal androgen excess disrupts ovarian steroid receptor 

balance. Reproduction 137:865-877. 

5. Byers M, Kuiper GG, Gustafsson JA & Park-Sarge OK. 1997. Estrogen receptor-â mRNA expression in rat ovary: down-regulation by gonadotropins. 

Molecular Endocrinology 11:172-182. 

6. Pelletier G, Labrie C & Labrie F. 2000. Localization of oestrogen receptor á, oestrogen receptor â and androgen receptors in the rat reproductive 

organs. Journal of Endocrinology. 165:359-370. 

7. Wang H, Eriksson H & Sahlin L. 2000. Estrogen receptors á and â in the female reproductive tract of the rat during the estrous cycle. Biology of 

Reproduction 63:1331-1340. 

8. Couse JF, Yates MM, Sanford R, Nyska A, Nilson JH & Korach KS. 2004. Formation of cystic ovarian follicles associated with elevated luteinizing 

hormone requires estrogen receptor-â. Endocrinology 145:4693-4702. 

9. D’Haeseleer M, Van Poucke M, & Van den Broeck W. 2005. Cell-specific localization of oestrogen receptor â (ESR2) mRNA within various bovine 

ovarian cell types using in situ hybridization. Anatomia Histologia Embryologia 34:265-272. 

10. Salvetti NR, Acosta JC, Gimeno EJ, Muller LA, Mazzini RA, Taboada AF & Ortega HH. 2007. Estrogen receptors alpha and beta and progesterone 

receptors in normal bovine ovarian follicles and cystic ovarian disease. Veterinary Pathology 44:373-378. 


900 


Finding relation between crossbred cattle sperm 

binding to buffalo hemizonae and cattle bull 

fertility by heterologous hemizona assay 

Panda S*, Chauhan MS**, Manik RS**, Palta P** and Singla SK**. 

* PhD scholar, Animal Biotechnology Centre, National Dairy Research Institute, Karnal, Haryana-132001, India. ** Principal Scientists, 

Animal Biotechnology Centre, NDRI, Haryana-1332001, India. E-email: sanabtc@gmail.com 

Abstract 

For prediction of cattle bull fertility using buffalo hemizonae by hemizona assay present study was done in homologous 

{cattle sperms binding to cattle hemizonae (group 1)} and heterologous {cattle sperms binding to buffalo hemizonae 

(group 2)} system. Known fertility frozen semen of four cattle bulls (36.7 to 51.8%) were grouped into control (two 

higher fertility bulls) and test (two lower fertility bulls) semen. Significant difference was found by students’t test when 

matching hemizonae were incubated with different semen samples but not in same semen sample. Between homologous 

and heterologous groups hemizona indexes for each bull were not significantly different. When hemizona indexes were 

correlated with respective pregnancies of semen sample in each group significant positive correlation was found in 

homologous group (for group 1 and r=0.89, P


nipulation system. Then ooplasm inside each hemizona was dislodged and placed in 50ml droplet of Ham’s F-10 medium. 

Semen samples were processed with DPBS and resuspended in F-10 medium and final concentration of 10 6 sperms/ml was 

adjusted. A 50 ml droplet of sperm suspension of individual buffalo and cattle bull was added to each hemizona droplet 

and co-incubated at 38.5°C for four hours. After incubation, the sperm hemizona complexes were rinsed with DPBS and 

stained with Hoechst 33342. The total numbers of bound spermatozoa were counted with fluorescence illumination. 

Experimental Design 

Homologous {cattle sperms binding to cattle hemizonae (group 1)} and heterologous {cattle sperms binding to buffalo 

hemizonae (group 2)} systems were made. Semen of four cattle bulls of which two were having superior known fertility 

rate of 51.8% and 51.4% respectively (those were used as control) and other two having inferior fertility rate of 43.2% 

and 36.7% (used as test semen samples) were taken in five combinations in each group to find out HZA indexes. The HZA 

index was defined as percent of sperm bound to test hemizona compare to control hemizona. Comparison of hemizona 

indexes was done between the groups and correlation was made between hemizona indexes of test semen sample and its 

pregnancy rate for each group. 

Table 1. 

Bull codes were A: KF6465, 

B: KF6469, C: KF6422, D: KF 6442 

Table 2. Bull codes were 

A: KF6465, B: KF6469, 

C: KF6422, D: KF 6442 

*Non Significant at 5% level 

*Non Significant at 5% level 


Table 3 

Bull CombinationHemizona 

Index (Mean ± S.E.) 

RESULTS 

902 


Results for validation of HZA showed the equivalent number of bound spermatozoa to matching hemizonae while incubating 

with same bull semen for group 1 and 2 (Table 1 and 2). Results for bull wise comparison of hemizona indexes between 

different groups showed there was no significant variation of sperm binding in two groups (Table 3). Correlation between 

HZAI and in vivo fertility rates showed significant positive correlation coefficient for group 1 is r= 0.89, P



This project was supported by Embryo Biotechnology Lab., National Dairy Research Institute, Karnal, Haryana-132001, 

India. 

REFERENCES 

1. Amann RP. 1989. Can the fertility potential of a seminal sample be predicted accurately. J Androl 10:89-98. 

2. Burkman LJ, Coddington CC, Franken DR, Kruger TT, Rosenwaks Z and Hodgen GD. 1988. The hemizona assay (HZA): development of a 

diagnostic test for the binding of human spermatozoa to the human hemizona pellucida to predict fertilization potential. Fertil Steril 49(4): 688- 

697. 

3. Fazeli AR, Zhang BR, Steenweg W, Larsson B, Bevers MM, van den Broek J, Rodriguez-Martinez H and Colenbrander B. 1997. Relationship 

between sperm-zona pellucida binding assays and the 56-day nonreturn rate of cattle inseminated with frozen-thawed bull semen. Theriogenology 

48(5):853-863. 

4. Fazeli AR, Holt C, Steenweg W, Bevers MM, Holt WV and Colenbrander B. 1995. Development of a sperm hemizona binding assay for boar 

semen. Theriogenology 44 : 17-27. 

5. Fazeli AR, Steenweg W, Bevers MM, van den Broek J, Bracher V, Parlevliet J and Colenbrander B. 1995. Relation between stallion sperm 

binding to homologous hemizonae and fertility. Theriogenology 44: 751-760. 

6. Franken DR, Kruger TF, Oehninger S, Coddington CC, Lombard C, Smith K and Hodgen GD. 1993. The ability of the hemizona assay to predict 

human fertilization in different and consecutive in-vitro fertilization cycles. Human Reproduction 8:1240-1244. 

7. Franken DR, Coddington CC, Burkman LJ, Oosthuizen WT, Oehninger SC, Kruger TF and Hodgen GD. 1991. Defining the valid hemizona assay: 

accounting for binding variability within zonae pellucidae and within semen samples from fertile males. Fertil Steril 56(6): 1156-1161. 

8. Oehninger S, Coddington CC, Scott R, Franken DA, Burkman LJ, Acosta AA and Hodgen GD. 1989. Hemizona assay: assessment of sperm 

dysfunction and prediction of in vitro fertilization outcome. Fertil Steril 51(4): 665-670. 


ABSTRACT 

904 


Frequency of estrous in Nili-Ravi buffaloes 

during different seasons of the year 

Makhdoom A. Jabbar, A. A. Channa, S. Raffat and S. Naveed 

University of Veterinary and Animal Sciences, Lahore, Pakistan 

Eight adult non-lactating and normally cycling Nili-Ravi buffaloes were procured to study the estrous frequency during 

different months of the year. These were fed total mixed ration with protein 12 % and ME 2.5 Mcak/kg through out the 

year. They were maintained in a semi opened shed under similar management conditions. Animals were not bred during 

the experimental period. Heat detection was done with the help of teaser bull daily in the morning and eve3ning. The 

blood samples from each animal were collected at weekly interval for progesterone estimation for detection of heat. 

Comparatively high frequency of heat (44.7%) was noticed during September to January (Winter) whereas it was lowest 

(18.89 %) during May to August (Summer). Both the met5hods of heat detection through teaser bull and blood progesterone 

concentration showed almost similar results. Nutrition did not seem to have any effect on the seasonality breeding 

response of buffaloes as it was same during the year. 

Key Words: Buffalo, Estrous frequency, season, Total mixed ration 


INTRODUCTION 


Histopathology of Buffalo Testis Submitted 

To Experimental Testicular Insulation 

2 Garcia, O. S.; 1 Vale, W. G.; 3 Garcia, A. R.; 1 Ribeiro, H. F. L.; 1 Assunção, W. L. P.; 4 Ferro, R. S.; 1 Rolim Filho, S. T.; 

5 Sales, M. S.; 2 Barbosa, E. M.; 2 Ayala, H. D. M.; 2 Simões, A. R.; 6 Vidal, S. C.; 2 Miyasaki, M. Y. A. 

1 Division Animal of Reproduction. ISPA / UFRA. 2 Posgraduate Students. Animal Science. UFRA / UFPA / EMBRAPA. 3 Empresa 

Brasileira de Pesquisa Agropecuária – EMBRAPA. 4 Laboratory of Genetics and Biotechnology of the Reproduction – UEMA 

5 Universidade Federal do Piauí – UFPI. 6 Graduate Student –UFRA - E-mail: onelsolano@yahoo.com.br 

The buffalo has higher performance in relation to other domestic species, demonstrating good adaptation skills, especially 

in areas considered unlikely to conventional calving, like in Amazon lowlands, with seasonal parcial or complete 

flooding 7 . 

Elevation of normal testicle temperature has deleterious effects in spermatogenesis, reducing it completely, like in 

cryptorchidic or experimentally induced animals 5 . 

Through thermal insulation method, evolving all scrotum surface, archived efficient induction of prolonged and transitory 

testicular degeneration, declining spermatic motility, vigor and concentration and increasing spermatic morphological 

alterations 2 . 


keys words: histopathology - testicular - insulation - buffalo 

To realize the histophatological exam, unilateral orchiectomy was performed twenty, forty and sixty days after testicular 

insulation. 

Fragments of 10 mm by 20 mm from proximal, medial and distal parts of the testicles and epididymis were collected. The 

fragments were fixed in ALFAC solution then cut, dehydrated, cleaned in xylene, embedded in paraffin, microtomyzed 5 

mm thick and stained with hematoxylin-eosin solution 6,9 . 

In histophatological examination, using binocular microscope with 20x and 40x objective lenses, seminiferous epithelium, 

various epididymis segments and cellular content in the lumen of seminiferous tubules and epididymis ducts were 

evaluated. 


The histophatological examination demonstrated spermiogenesis and testicles with distinct cicles in buffalo A (Figure 

1). The presence of some normal seminiferous tubules and many others with celular desquamation in spheroid configuration 

(spermatogonium), and more widely, enlogated cells (spermatids), which indicates the presence of spermatic cells 


906 


on ejaculate, in the most critical phase of the experiment (P30). The tubular basal membrane did not showed any 

alteration, the same was observed to interstitial cells. The epididymis presented vacuolized and degenerated epithelium. 

In the tubule lumen, collision of sperm and many desquamated germ cells and nuclear morphology with eosinophilia 

(necrosis) (Figure 2) 4,10 . 

Figure 1 - Tubules presenting germ cells desquamation, 

with typical karyolysis and nuclear pyknosis, 

and cytoplasmic acidophilia (necrosis) 

(1). Presence of vacuolized germ cells (2) H.E, 

40X. 

Figure 2 - Epididymal tubule showing normal 

sperm filling. Full spermatogenic cells in necrosis, 

spermatocytes (1) and spermatids (2) are 

observed. Epididymal cells vacuolization (3). H.E, 

40X. 

In case of animal “B”, 40 days after insulation, vacuolization of germ cells in seminifeous tubules in general, were 

observed. These considerably reduced cells were spermatogonium and spermatocytes, with rare round spermatids (Figure 

3). Similarly, in tubule lumen, desquamation cells mostly living were observed. Additionaly, tubules contening almost 

exclusively Sertoli cells, many with citoplasmatic vacuolization was observed. There was no vacuolation of epididymal 

epithelial cells and there was no sperm in the lumen, while some epididymal tubules showed desquamated cells and 

eosinophilic substance with spheroid droplets (Figure 4) with a diagnosis indicating a severe testicular degeneration 1,3,4 . 

Figura 3 – Seminal tubules in general, with vacuolization 

of predominantly Sertoli cells (1). 

Germ cells considerably reduced in number, and 

the ones present are the spermatogonium (2) 

and spermatocytes types(3). H.E., 40X. 

Figure 4 - Absence of sperm cells in the lumen 

(1). Vacuolization of epididymal epithelium cells 

(2), presence of apoptotic bodies (3). H.E., 40X. 



Sixty days after the insulation of animal “C”, the seminiferous tubules showed spermatogenesis with the absence of 

vacuolized cells, and in the tubular lumen, numerous necrotic desquamated cells, as well as the absence of changes in 

interstitial cells and basement membrane of seminiferous tubules (Figure 5). 

In the epididymis there was vacuolization and few epithelial cells desquamated and necrotic with pyknosis and cytoplasmic 

hypereosinophilia, besides the presence of spermatozoa in the epididymal tubules (Figure 6). 

This analysis demonstrates a slow recovery, corroborating the findings with the morphology examinations 4,8 . 

Figure 5 - Seminiferous tubules with spermatogenic 

activity (1). Desquamation of tubular lumen 

cells is observed (2). H.E., 40X. 

Figure 6 – Epididymal tubules showing normal 

filling by spermatozoids (1). H.E., 20X. 

In this study, the artificial insulation of scrotum, testis and epididymis, although caused an accentuated degenerative 

condition, it was possible to regenerate spermatogenesis in most studied animals. 

Acknowledgements: UFPA, EMBRAPA, CEBRAN, FAPEMA, CAPES. 

REFERENCES 

1. Ahmad, M. N. et al. Post mortem studies on infertile buffalo bulls: Testicular histology. Veterinary Record, n. 122, p. 229-231. 1988. 

2. Barth, A. D.; Bowman, P. A. The sequential appearance of sperm abnormalities after scrotal insulation or dexametasone treatment in bulls. Can. 

Vet. J., 35:93-102, 1994. 

3. Blanchard, T. L. et al. Testicular degeneration in large animals: Identification and treatment. Veterinary Medicine, May, 1991. 

4. Fonseca, V. O. 1976. Efeito da elevação térmica experimental sobre a espermatogênese no zebu: aspectos físicos e morfológicos do sêmen, 

anátomo - patológicos do testículo e epidídimo e alguns processos endócrinos relacionados à afecção. 148p. Dissertação de Mestrado – Curso de 

pós-graduação, Universidade Federal de Minas Gerais, Belo Horizonte, 1976. 

5. Gabaldi, S. H; Wolf, A. 2002. A importância da termorregulação testicular na qualidade do sêmen em touros. Ciên. Agr. Saúde. FEA, Andradina, 

v. 2, n. 2, jul-dez, p. 66-70. 

6. Luna, L. G. 1968. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. Mc Graw-Hill, 3a Ed. New York, p. 258. 

7. Perera. O. et al. 2005. Buffalo. Cameo-Projects to help resource-poor buffalo keepers in Sri Lanka and Brazil. Livestock and Wealth Creation, Cap. 

23, p. 451- 471. 

8. Rao, V. D. N.; Rao, A. R. 1977. Influence of heat induced testicular degeneration on semen characteristic and testicular histology in rams. Indian 

Vet, n. 5, v. 54, p. 719-726. September. 

9. Sartori, R et al. 2002. Avaliações ultra-sonográfica, macroscópica e histológica da biopsia testicular em ovinos. Arq. Bras. Med. Vet. 

Zootec. vol.54 n°.3, Belo Horizonte, June. 

10. Vale Filho, V. R., et al. 1980. Fertility of the bull in Brazil study on 1.088 bulls and 17.954 ejaculations of Bos indicus and Bos taurus, raised 

in tropical conditions, comparatively, 9th Intern. Congr. Reprod., Madrid, v.1, p.545 – 548. 


Abstract 

908 


The low efficiency of estrus detection leads to inappropriate time for AI insemination in swamp buffalo. The ovu lation 

synchronization (O vsynch ) protocol has successfully applied in bovine and riverine buffalo but not in swamp buffalo . 

Our previous studies demonstrated that Ovsynch can be applied with a 34.6% p regnancy rate (Chaik hun et al., 2009). 

The aim of this study was to observed ovulation time and changes in hormonal profiles during ovsynch of Thai swamp 

buffaloes . The experiments were ca rried out in twelve buffaloes, estrus induction was performed by PGF2? (D-13) - PGF2? (D- 

2) – - PGF (D7) – D9) GnRH(D0 ) 2? GnRH ( (Presynch - Ovsynch) treatment. Blood samples were collected from jugular vein on days 

0, 4, 7, 9 and every 4 h a fter second GnRH injection. Plasma progesterone and estradiol concentrations were dete 

rminated by radioimmunoassay technique. The ovarian changes observed by ultrasonography and behavior of estrus 

signs investigated by rectal palpation and visual ob servation. Ten buffaloes (83.3%) ovulated with the average ovula 

ted dominant follicles was 1. 24 ± 0.2 mm. and the me an + SD o f ovulation time was 30 + 2.8 h . A low level of 

progesterone (0.36 ± 0.15 ng/ml.) was maintained during the period from day 9 to day 12. During the estrus , estradiol 

17- level fluctuated in a pulse fashion with the averag e level was 3.84 ± 1.0 pg/ml. recorded at about 24 h after the 

onset of heat. From the rectal palpation show that 12 h after the highest (+3) tone of uterus was the basic criteria for 

indicated ovulation time in swamp buffalo. The results show high efficiency of ovsynch program for induce ovulation and 

indicate the interval between the last GnRH administration of the ovsynch protocol and AI should be 28-32 h in Thai 

swamp buffalo. 

INTRODUCTION 

Hormonal Profiles and Ovulation Time 

in Thai Swamp Buffaloes after Ovulation 

Synchronization Program 

Thuchadaporn Chaikhun 1 , Akachart Promdireg 2 , Wanvipa Suthikrai 3 , Ratree Jintana 3 , 

Jatuporn Kajaysri 1 , Mongko l Techakumphu 4 * 

1 Clinic for Obstetrics Gynaecology Andrology and Artificial Insemination in 

Domestic Animals, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok 10530, Thailand. 2 Department 

of Veterinary Te chnology, Faculty of Veterinary Technology, Kasetsart University, Bangkok 10900, Thailand 

3 Research and Development Center for Livestock Production Technology Faculty of Veterinary Science, Chulalongkorn University, 

Bangkok 10330, Thailand. 4 Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn 

University, Bangkok 10330, Thailand - E-mail: tmongkol@chula.ac.th 

Keywords: swamp buffalo, ovulation synchronization, ovulation time 

Decreasing of swamp buffalo population in Thailand (more than 60% for 10 years) is the main problem which leading to 

loss the swamp buffalo genetic diversity 1 . 

Therefore, because of high meat consumption and a castrated bulls traditional for wo rking were induced inbreeding 

proplem, including their productivity is limited by poor reproductive efficiency such as inherent late maturity, a p 

rolonged intercalving interval, decreasing of ovarian function especially in summer, especially, poor estrus expression 

and detecting estrus that cause difficulty in predicting the time of ovulation and artificial insemination 2-10 . 



The Ovsynch protocol, a sequence of GnRH, PGF2alpha, and GnRH treatments, has successfully synchronized 

ovulation in lactating dairy cows, resulting in fertility to timed artificial insemination (TAI) that was similar to that of 

cows inseminated after detection of estrus 11–12 . However, there are only recent reports using the Ovsynch protocol in 

dairy buffalo: half -bred (Murrah x M editerranean) buffaloes 13 , Mediterranean buffaloes 5 , Murrah buffaloes 9 and Egyptian 

buffaloes 14 . It is noteworthy that the use of the Ovsynch protocol in swamp buffalo in its native tropical environment 

in Thailand has been reported that showed 34.6% pregn ancy rate 15 The aim of this study was to observed ovulation 

time and changes in hormonal profiles during ovsynch of Thai swamp buffaloes. 


Animals 

Twelve female buffaloes were selected with the inclusion criterias; the cows had more than 60 days of postpartum, normal 

reproductive organs and cyclicity of estrus cycle were examined by ultrasounography, base on the presence of a functional 

corpus luteum or follicle before treated at least 7 days. 

Treatment 

All buffaloes were performed to Presynch -Ovsynch protocol (figure1) characterized by the administration of PGF2 

(Cloprostenol 500 g, Estrumate ® , Intervet Shering -Plough Animal Health, The netherlands ) at day -14 and -2, then 

injected GnRH (Buserelin 10 g, Receptal®, Intervet Shering -Plough Animal Health, The netherlands ) at days 0 and 9, 

and PGF2 at days 7 (PP-GPG). 

On days 0, 4, 7, 9 and every 4 h until 72 h after second GnRH injection, blood samples were collected from jugular vein, 

estrous signs such as standing heat, vu lva edema, vaginal discharge, uterine tone of buffaloes were recorded, including 

follicle size and ovulation time were mornitored by ultrasound using a B-mode scanner equipped with a 7.5 MHz lineararray 

transducer . Plasma was harvested and store at - 70 C until th e analysis of progesterone and estradiol 17concentration 

by radioimmunoassay (RIA) 16 . The estradiol 17- intra -assay coefficients of variation (%CV) of low, 

medium and high control were 11.15, 10.1 and 2.43, respectively and inter-assay (%CV) of low, medium and high control 

we re 15.12, 8.12 and 4.97, respectively. The recovery rate was 84.64 and s ensitivity of the estrogen antibody was 2.59 

pg/ml. The progesterone intra -and inter-assay coefficients of variation were 7.7 and 13.9, respective ly and sensitivity 

was 0.05 ng/ml. 

RESULTS AND DISCUSSIONS 

Table 1 Mean (± S.D.) characteristics of dominant follicle, ovulation time, progesterone and estradiol 17- concentrationin 

Presynch- vsynchbuffaloes. 

Ovulated Animals Anovulated Animals 

Number (%) 10 (83.3 ) 2 (16.7) 

Mean Ovulatory/ anovulation dominant 

Follicular s ize (mm.) 

Mean of ovulation time 

(hour after second GnRH injection) 

Mean of Progesterone level on D9 -D12 (ng/ml.) 

Mean of Estradiol 17- level on estrus (pg/ml.) 

1.24 ± 0.2 2.37 ± 0.3 

30 ± 2.8 - 

0.36 ± 0.15 0.35±0.15 

3.84 ± 1.0 3.91±2.54 


910 


The ovulation rate in this study was 83.3% (10/12) which was lower tan previous study in riverine buffaloes (90% in 

Murrah 9 and 93.3% in Murrah x Mediterranean4). The average ovula ted dominant follicles was 1.24 ± 0.2 mm. The ovulation time was 30 

+ 2.8 h, it showed higher than river ine buffaloes (23±1.3 h in Murrah 9 and 26.5±9.6 h in Murrah x Mediterranean 4 ). From 

the estrous signs, only uterine tone at 12 h after 2 nd GnRH was the highest (+3) that was the basic criteria for indicated 

ovulation time in swamp buffalo. In the present study, there was a typical pattern of progesterone decline near the time 

of ovulation. Estradiol 17- level fluctuated in a pulse fashion during estrous period, it maybe elevated estradiol concentrations 

during estrus are not necessarily accompanied by overt signs of estrous behavior in buffalo 6 . 

The results show high efficiency of ovsynch program for induce ovulation and indicate the interval between the last 

GnRH administration of the ovsynch protocol and AI should be 28-32 h in Thai swamp buffalo. 

Acknowledgements. This research was supported by grants from Mahanakorn University of Technology and The 

Thailand Research Fund - Senior Research Scholar 2007.Intervet-Schering-Plough Animal Health (Thailand) Co., 

Ltd. for product supported. 

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REPRODUCTION - Facultad de Ciencias Veterinarias

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