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BUFFALO BULLETIN 

ISSN : 0125-6726 

Buffalo Bulletin is published quarterly in March, 

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any aspect of research or development, progress 

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http://ibic.lib.ku.ac.th/e-Bulletin 

BUFFALO BULLEITN 

IBIC, KASETSART UNIVERSITY, P.O. BOX 1084 

BANGKOK 10903, THAILAND 

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Buffalo Bulletin (December 2007) Vol.26 No.4 

EFFECT OF GROWTH HORMONE ON ONSET OF CYCLICITY, MILK YIELD AND BLOOD 

METABOLITES IN POST-PARTUM LACTATING RIVERINE 

BUFFALOES (BUBALUS BUBALIS) 

A. Mishra, P.K. Pankaj, B. Roy, I.J.Sharma and B.S. Prakash 

ABSTRACT 

Twenty-two post-partum riverine buffaloes 

(Murrah breed) of first or second lactation were 

selected from the National Dairy Research Institute 

(NDRI) herd to determine the length of post-partum 

period for onset of cyclicity by growth hormone 

(GH). Blood samples (5-10 ml) were collected twice 

a week until commencement of cyclicity as 

determined by progesterone analysis or for period 

of 3 months (whichever was earlier). Out of these 

22 animals, ten were found to be cyclic as 

determined through progesterone analysis. Blood 

samples were collected twice a week at 3-4 day 

intervals from all animals by means of jugular vein 

puncture 5 days post-partum. To fulfill the objectives 

of the present study, plasma GH was assayed by 

enzyme immunoassay (EIA) techniques, and plasma 

progesterone was analyzed by a direct 

radioimmunoassay (RIA) method. The plasma GH 

profiles in these two groups of animals were not 

significantly different (P>0.05). Further, no 

significant correlation was found between GH 

concentration and days to commencement of 

cyclicity. In high yielders, a significant (P0.05). 

Keywords: growth hormone, ycyclicity 

commencement, milk yield, blood metabolite, buffalo 

INTRODUCTION 

Early resumption of ovarian activity in postpartum 

buffaloes is required to achieve calving-tofirst-service 

and to-conception intervals of 55 and 

85 days, respectively, which are necessary targets 

if a 365 day calving interval is to be attained. After 

regression of the CL of pregnancy, there is a variable 

anovulatory period before first ovulation takes place. 

The length of this anovulatory period can be affected 

by the level of nutrition, body condition, suckling, 

lactation, dystocia, breed, age, month of calving, 

uterine pathology and chronic debilitating disease 

(Chauhan et.al., 1984). The initiation of follicular 

growth is not clearly understood in this early postpartum 

period. 

Riverine buffaloes are the important milkproducing 

animals along and who contribute to 

draught animal power (DAP) and meat production 

in many Asian countries. Malven (1984) defined the 

problem of the post-partum period as requiring 

recovery of function after pregnancy and parturition 

of both the brain-pituitary-ovarian system and the 

genital tract. Functional recovery of these major 

components of the reproductive system occurs 

simultaneously and there are obvious interactions. 

Hence, the principal hormones, which could 

be playing a major role in determining the length of 

post-partum period for cyclicity commencement, are 

GH and prolactin. The former is also a known 

anabolic hormone and has been seen to play a role 

in enhancing growth and early commencement of 

Department of Veterinary Physiology, College of Veterinary Sciences & A.H., 

Jabalpur, M.P-482 001 (India) 

106


puberty in cattle and buffaloes (Simpson et al., 1991 

and Haldar, 2004) and therefore could be playing an 

important role in the postpartum period. Very little 

information is available on post-partum 

endocrinology in buffaloes, especially in relation to 

milk production, blood metabolites (non-esterified 

fatty acids (NEFA), glucose, and α-amino nitrogen 

(AAN)) and cyclicity commencement. In the present 

investigation, the endocrinology of the riverine 

buffaloes (Murrah breed) has been studied with 

emphasis on the endocrine causes for the postpartum 

delay in cyclicity commencement in lactating 

buffaloes. 

MATERIALS AND METHODS 

Hormone analysis 

Plasma GH was assayed by enzymeimmunoassay 

(EIA) techniques, and plasma 

progesterone was analyzed by a direct RIA method 

developed in our laboratory. 

Radioimmunoassay (RIA) for progesterone 

quantification 

Progesterone was estimated by a direct RIA 

procedure used routinely in our laboratory (Kamboj 

and Prakash, 1993) with some modifications. Plasma 

samples and progesterone standards (containing 

progesterone concentrations ranging from 0 to 250 

pg/20 µl) prepared in plasma (20 µl each) were 

pipetted out in duplicate in 12x75 mm tubes. RIA 

assay buffer (300 µl) was then added to each tube. 

Subsequently, 100 µl of anti-progesterone serum 

(diluted 1:16,000 in the RIA assay buffer) and 100 l 

of tracer (~ 10,000 dpm/100 µl) were added to each 

tube. The resulting mixture was vortexed and 

incubated at 4 o C in a refrigerator overnight. The 

free and antiserum-bound hormone were separated 

by the addition of 0.5 ml of freshly prepared cold 

(4 o C) charcoal-dextran suspension (0.625% 

activated charcoal + 0.0625% dextran in RIA assay 

buffer without-gelatin) under constant stirring at 4 o C. 

The tubes were then stirred, incubated at 0 o C in an 

ice-water bath for 10 minutes and then centrifuged 

at 3,000 rpm at 4 o C for 15 minutes. The supernatant 

containing the bound progesterone was decanted into 

scintillation vials. Scintillation fluid (5ml) was then 

added to each vial after which the vials were counted 

after being kept overnight at room temperature. In 

addition to the above, three sets of tubes were also 

run. Blank tubes, in duplicate, containing 400 µl RIA 

assay buffer, 20 µl progesterone-free buffalo plasma 

and 100 µl tracer, for the calculation of non-specific 

binding for the charcoal separation procedure. Four 

tubes containing 300 l RIA assay buffer, 20 µl 

progesterone-free buffalo plasma, 100 µl antiprogesterone 

serum and 100 µl tracer for the 

calculation of the maximum binding of tracer by the 

antibody. Total count tubes were prepared, in 

duplicate, containing 100 µl tracer and 920 µl RIA 

assay buffer, for obtaining total counts of tracer 

added. This mixture was decanted directly into 

scintillation vials. 

Assessment of RIA 

In the process of estimation of hormones 

and evaluation of the assay system, quality control 

in terms of sensitivity, specificity and precision was 

carried out for each hormone. The sensitivity of the 

assay for progesterone by direct plasma estimation 

was 4 pg/tube, which corresponds to 0.2 ng/ml, the 

50 percent binding limit being 70 pg/tube. The intraand 

inter-assay coefficients of variation for 

progesterone were 8.4 and 12.0 percent, 

respectively. The cross-reactivity of the antiprogesterone 

serum against different compounds 

related to progesterone was worked out to quantify 

the specificity of the progesterone antisera (Table1) 

(Prakash and Madan, 2001). 

Enzyme Immunoassay (EIA) procedure for 

plasma GH 

Preparation of affinity purified goat lgG 

antirabbit lgG 

Affinity purified goat lgG antirabbit lgG was 

prepared as described by Anandlaxmi and Prakash 

(2001). About 40 ml plasma from a goat immunized 

against rabbit lgG was mixed with rabbit lgG agarose 

and loaded onto a small column (column size: 1.5 x 

5.0 cm). The non-specific proteins were eluted with 

‘PBS’; pH 7.2. The bound proteins were then eluted 

with 15 ml of 0.1 M glycine-HCl, pH 2.0. All the 

steps were performed at room temperature. The 

eluted fractions (3 ml each) were collected in vials 

107


containing 0.2 ml of 1 M Tris-HCl, pH 8.0. The 

eluted lgG was dialyzed overnight against ‘PBS’ and 

the protein content determined by measuring the 

absorbance spectrophotometrically at 260 and 280 

nm, and extrapolated from a normograph. 

EIA for GH in plasma 

A highly sensitive enzyme immunoassay 

procedure using the second antibody coating 

technique developed in the laboratory (Prakash et 

al., 2003) was carried out to estimate plasma GH. 

Preparation of GH-biotin conjugate 

To 40 µg bovine GH (USDA-bGH-B-1) 

dissolved in 200 µl of 0.1 M carbonate buffer (50 

mM, pH 9.6), 20 µl biotinamidocaproate-Nhydroxysuccinimide 

ester (Biotin, Sigma, Germany) 

dissolved in dimethyl sulfoxide (5 mg/ml, Sigma, 

Germany) was added and the mixture was 

immediately vortexed and incubated for 3 hours at 

room temperature under constant agitation. The 

coupling reaction was stopped by the addition of 20 

µl (1 M) NH 4 

Cl and the reaction mixture was further 

incubated for 30 minutes before addition of 2 ml of 

a solution of 0.1% BSA (Sigma, Germany) in PBS, 

pH 7.4 (50 mM NaPO 4 

, 0.15 M NaCl, pH adjusted 

with 5 N HCI). Biotin-GH conjugate was isolated 

by dialysis of the mixture in dialysis sacks (250-7U, 

Sigma, USA) overnight at 40 0 C with three changes 

in PBS. After dialysis, the conjugate was mixed with 

an equal volume of glycerol (Hi Media, India) to 

prevent freezing and preserved at -20 0 C in 1 ml 

aliquots. 

bGH antibody 

The bovine GH antibody (Rabbit 3-antibGH, 

Pool 7-12) used in the present investigation 

was specific for estimation of bovine GH. The details 

of the specificity of the antiserum were given by 

Hennies and Holtz (1993). 

EIA procedure 

First coating 

The first coating was performed by adding 

0.63 µg of goat lgG anti -rabbit lgG dissolved in 100 

µl of coating buffer (15 mM Na 2 

C0 3 

, 35 mM 

NaHCO 3 

, pH 9.6) per well of the microtitre plate, 

(Linbro, Flow Laboratories, Scotland). These plates 

were subsequently incubated overnight under 

refrigerated conditions. 

Second coating 

For saturating the remaining binding sites, 

300 µl of PBS containing 1 percent BSA was added 

to all the wells and incubated for 40 to 50 minutes at 

room temperature under constant shaking on 

microtiter plate shaker (Titertek, Flow Laboratories, 

Germany). 

Washing 

The coated plates were washed twice with 

350 µl of washing solution (0.05% Tween-20, 10% 

PBS in distilled water, Sigma, Germany) per well 

using an automated microtitre plate washer (Model: 

EL 50 x 8MS, USA). 

Assay protocol 

Duplicates of 100 µl of unknown plasma or 

bovine GH standards prepared in assay buffer 

ranging from 40 pg/100 µl/well to 10,000 pg/100 µl/ 

well were simultaneously pipetted into respective 

wells along with 100 µl of charcoal treated plasma 

(C.T.P). GH antibody diluted 1: 40,000 in assay buffer 

(50 mM NaPO 4 

, 0.15 M NaCI, 0.02% Thiomersal, 

pH 7.4) were added with the aid of a dilutor 

dispenser (Microlab 500 series, Hamilton, 

Switzerland). Thereafter, the plates were incubated 

overnight at room temperature after 30 minutes 

constant agitation. The next day, plates were 

decanted and washed two times with washing 

solution before addition of 100 µl of biotinyl-GH 

conjugate diluted 1:3,000 in assay buffer. The plates 

were further incubated for 30 minutes with constant 

agitation, decanted and washed four times with 

washing solution. Then 20 ng streptavidinperoxidase 

(Sigma, Germany) in 100 µl of assay 

buffer was added to all the wells using digital multichannel 

pipette (Flow Titertek, Finland) and the 

plates were wrapped in aluminum foil and incubated 

further for 30 min under constant agitation. All steps 

were performed at room temperature. 

Substrate reaction 

The plates were then washed four times 

with washing solution and incubated further in the 

dark for 40 minutes after addition of 150 µl of 

108


substrate solution per well [Substrate buffer: 0.05 

M citric acid, 0.11 M Na 2 

HPO 4 

, 0.05% ureum 

peroxide, pH 4.0 with 5 N HCl, substrate solution: 

17 ml of substrate buffer plus 340 µl 3,3',5,5'– 

tetramethyl benzidine; 12.5 mg/ml dimethylsulfoxide 

(Sigma, Germany)]. The reaction was stopped by 

the addition of 50 µl 4 N H 2 

S0 4 

and the colour 

produced was measured at 450 nm with a 12-channel 

microtitre plate reader (ECIL, Microscan, India). 

GH concentration in buffalo plasma samples was 

calculated from the standard curve plotted against 

absorbance at 450 nm by using graph pad 

OR 

PRISM 3.0, software package. The quality control 

of the assay was carried out by performing the 

following: 

Assay sensitivity 

The lowest GH detection limit significantly 

different from zero concentration, was 40 pg/100 µl 

plasma/well, which corresponds to 0.4 ng/ml in 

plasma, and the 50 percent relative binding was seen 

at 900 pg/100 µl/well. Intra- and inter-assay 

coefficient of variations were determined using 

pooled plasma containing 2.0 ng/ml and were found 

to be 3.68 and 6.89 percent, respectively, from eight 

assays. The specificity of the antiserum used was 

determined by Hennies and Holtz (1993). The 

antiserum showed high specificity for GH. 

Blood metabolites estimation 

The blood metabolites viz., non-esterified 

fatty acids (NEFA), glucose and α-amino nitrogen 

(AAN), were estimated fortnightly throughout the 

experimental period in plasma samples from 

buffaloes as described below: 

Non-esterified fatty acids (NEFA) deter - 

mination: 

The copper soap solvent extraction method 

modified by Shipe et al. (1980) was adopted for the 

estimation of plasma NEFA. The standard curve 

was prepared as per Koops and Klomp (1977). 

Alpha amino nitrogen estimation 

Blood α.-amino nitrogen is an indicator of 

protein status of the animal and was estimated by 

the method given by Goodwin (1970). 

Plasma glucose determination 

Plasma glucose was estimated by GOD/ 

POD method using commercial kits (Span 

Diagnostics Ltd., India; Code No # 25940). 

Statistical analysis 

The data obtained were analyzed by using 

Microsoft Excel 2000 and Graphpad Prism software 

package, 1995. Paired t-test was employed to test 

the difference between plasma GH amongst high 

yielders and low yielders. To test the metabolic and 

hormonal parameters, analysis of variance technique 

was used in the following statistical model: 

Yijk = µ+ αI + βj + eijk where, 

Yijk = the dependent variable 

µ = Overall populations mean 

αI = the effect of i th treatment, 

βj = the effect of j th week of experimental period 

and 

eijk = Random error associated with k th individual, 

normally and independently distributed with mean 

zero, and variance σ 2 . 

The correlations among these traits were 

calculated within each group, and the significance 

was tested using standard statistical methods as 

described by Snedecor and Cochran (1968). 

RESULTS AND DISCUSSION 

Cyclicity commencement 

Commencement of cyclicity was 

determined by progesterone analysis at regular 3-4 

day intervals. Continuous monitoring of progesterone 

levels (from 5 days post–partum up to 3 months) 

was carried out on 12 animals, which incidentally 

had also calved recently. On the basis of 

progesterone profiles, six commenced cyclicity 

during the course of 3 months (Table 2), while six 

animals had not commenced cyclicity even up to 90 

days post-partum (Table 3). 

109


Milk production performance 

On the basis of milk yield, data obtain from 

the animals were also divided into two groups viz. 

low yielders (n=6) and high yielders (n=6). The high 

yielders were giving 9.0 to 13.0 litres milk/day 

whereas the low yielders produced between 5.0 to 

8.5 litres milk/day (Figure 1). The overall milk yield 

+SEM for the low yielders was 6.58+0.469 kg/day 

as against 10.92+ 0.608 kg/day for the high yielders, 

which was significantly (P0.05) 

difference between mean growth hormone 

concentrations (Figure 2) post-partum (n=6), which 

was 14.91+2.315 ng/ml and 10.51+1.646 ng/ml for 

the high and the low yielders, respectively. 

While no reports are available for comparing 

our data on growth hormone in high and low yielding 

buffaloes (or even cows), the magnitude of growth 

hormone levels were similar to those reported by 

Sartin et al. (1988), who recorded hormone levels 

of 19.3, 16.4 and 13.6 ng/ml at 1 to 20, 21 to 40 and 

41 to 56 days lactation in cows, respectively. In 

lactating Murrah buffaloes, the level of growth 

hormone has been reported as 2.48 ng/ml (Jindal 

and Ludri, 1990) but in contrast to this, according to 

Patel (2001), this level is 14 to 20 ng/ml and 

decreases in advanced lactation. Normal 

concentration of growth hormone in plasma of cattle 

ranges between 3 and 30 ng/ml, depending upon age, 

sex and stage of lactation (Schams et al., 1989). 

The variation in growth hormone level in 

different studies can also be attributed to the 

different sources of purified growth hormone used 

in different studies as well as the differences in 

antisera specificities. No significant trend in growth 

hormone profile was recorded for either the high or 

low yielders (Figure 2) over 90 days post-partum. 

Vasilatos and Wangsness (1981) reported in cattle 

that during early lactation (30 days post-partum) the 

plasma growth hormone concentration was elevated 

(13.2 ng/ml) compared to that in later lactation (90 

days post-partum) 9.8 ng/ml. However, they stated 

that the increased growth hormone status in early 

lactation was due to greater magnitude of individual 

secretory spikes rather than a difference in frequency 

of spikes or baseline plasma levels of GH. 

GH and milk yield: In the high yielders (Figure 2) 

a significant (P


plasma GH profiles in the two groups of animals 

were not significantly different (P>0.05). Further 

no significant correlation was found between GH 

concentration and days to commencement of 

cyclicity (Table 4). We had not come across any 

reports either in cattle or in buffaloes which provide 

information of relationship of plasma GH to 

commencement of cyclicity. 

Plasma profiles of blood metabolites 

Plasma glucose in high and low yielders: The 

mean plasma glucose concentration in the high 

yielders (n=6) was 62.74+2.522 mg/dl and ranged 

from 53.796+3.626 to 69.455+2.637 mg/dl (Table 

5) whereas in the low yielders (n=6) plasma glucose 

was 67.08+3.786 mg/dl and ranged from 

58.733+5.631 to 72.462+3.132 (Table 5). The 

statistical difference for plasma glucose 

concentrations between the two groups was nonsignificant 

(P>0.05). Glucose is a commonly used 

indicator of energy status but is very tightly 

controlled, and changes in blood glucose are usually 

very small, as reported by Domecq et al. (1997). 

Plasma glucose and commencement of 

Cyclicity: The relationship of plasma glucose to 

cyclicity commencement is provided in (Table 4). 

Plasma glucose was non-significantly correlated 

(P>0.05) with post-partum commencement of 

cyclicity (days). 

Plasma NEFA in high and low yielders: The 

mean plasma NEFA concentration in the high 

yielders (n=6) was 302.30+2.301 µmol/litre and 

ranged from 292.061+7.488 to 309.9+3.896 µmol/ 

litre (Table 5) whereas in the low yielders (n=6) 

plasma NEFA was 301.00+1.320 µmol/litre and 


litre (Table 5). The statistical difference for plasma 

NEFA concentrations between the two groups was 

non-significant (P>0.05). 

The non-significant changes in relation to 

milk yield may indicate that the animals were in a 

similar state of negative energy balance. However, 

the observations have to be viewed with caution 

since the sampling data was limited to only a few 

observations. It is also possible that the magnitudes 

in the differences in the milk yields in the two groups 

were not large enough to bring about a substantial 

change in the NEFA concentrations. 

NEFA and commencement of cyclicity: Plasma 

NEFA was non-significantly correlated (P>0.05) 

with post-partum commencement of cyclicity (days) 

shown in Table IV. Plasma NEFA concentrations in 

animals executing early commencement of cyclicity 


litre without showing any significant trend over the 

period of investigation. The plasma NEFA 

concentration in animals executing delayed 

commencement of cyclicity (more than 90 days) 

were non-significant (P>0.05) and ranged from 

295.936+4.721 to 305.364+3.113 µmol/litre without 

exhibiting any definite trend during the period of 

investigation up to 90 days. The result suggests that 

plasma NEFA concentrations remain unchanged 

irrespective of the commencement of cyclicity in 

buffaloes. No studies have been carried out earlier 

in either buffaloes or cattle in relation to NEFA 

concentrations and cyclicity commencement. 

Alpha Amino Nitrogen (AAN) in high and low 

yielders: The mean plasma AAN concentration in 

the high yielders (n=6) was 2.494+0.0426 mmol/litre 

and ranged from 2.169+0.35 to 2.585+0.05 mmol/ 

litre (Table 5) whereas in the low yielders (n=6) 

plasma AAN was 2.538+0.0346 mmol/litre and 

ranged from 2.362+0.088 to 2.592+0.039 mmol/litre 

(Table 5). The statistical difference for plasma AAN 

concentrations between the two groups was nonsignificant 

(P>0.05). We have not come across any 

studies correlating the AAN profiles with milk yield 

during early post-partum period in buffaloes. The 

preliminary investigations suggest that the AAN 

concentrations were not affected by milk yield. 

However, the number of observations in the present 

investigations being limited, detailed study involving 

a greater number of animals and greater degree of 

variations in the milk yield is wanted. 

AAN and commencement of cyclicity: The 

relationship of plasma AAN to cyclicity 

commencement is provided in Table 4. Plasma AAN 

was not-significantly correlated (P>0.05) with post- 

111


partum commencement of cyclicity (days). Plasma 

AAN concentrations in animals executing early 

commencement of cyclicity ranged from 2.377+0.11 

to 2.591+0.057 mmol/litre without showing any 

significant trend over the period of investigation. The 

plasma AAN concentration in animals executing 

delayed commencement of cyclicity (more than 90 

days) were non-significant (P>0.05) and ranged 

from 2.373+0.084 to 2.606+0.036 mmol/litre without 

exhibiting any definite trend during the period of 

investigation up to 90 days. 

The result indicates that the commencement 

of cyclicity is independent of the concentrations of 

the AAN in blood plasma of buffaloes. No other 

studies have been carried out with which we can 

compare our results with reference to changes in 

AAN in relation to cyclicity commencement in dairy 

cattle and buffaloes. 

SUMMARY AND CONCLUSIONS 

The endocrine profiles associated with 

interrelationship of the principal hormone were 

studied with respect to plasma GH and progesterone 

using sensitive EIA and RIA procedures standardized 

in our laboratory. In the high yielders, a significant 

positive correlation of was found between GH and 

milk yield. In the low yielders, positive correlation 

of was recorded between plasma GH and milk yield. 

The plasma GH profiles in the two groups of animals 

were not significantly different. Further no significant 

correlation was found between GH concentration 

and days to commencement of cyclicity. The positive 

correlation of GH with milk yield indicates that the 

hormone can be used as an index of production 

performance in riverine buffaloes. Although growth 

hormone did not show any significant correlation with 

plasma glucose and NEFA concentrations we did 

observe a negative correlation of growth hormone 

with AAN in low milk yielding buffaloes, which 

indicates that the hormone is responsible for 

mobilization of amino acids for milk synthesis. The 

statistical difference for plasma glucose 

concentrations between the two groups was nonsignificant. 

The statistical difference for plasma 

NEFA concentrations between the two groups was 

non-significant. Plasma NEFA was non-significantly 

correlated with post-partum commencement of 

cyclicity (days). The result suggests that plasma 

NEFA concentrations remain unchanged irrespective 

of the commencement of cyclicity in buffaloes. The 

statistical difference for plasma AAN concentrations 

between the two groups was non-significant. The 

result indicates that the commencement of cyclicity 

is independent of the concentrations of the AAN in 

blood plasma of buffaloes. No correlation of growth 

hormone was found with NEFA, AAN and glucose 

in the high yielders. The GH was found out to be 

negatively correlated with AAN in the low yielders. 

ACKNOWLEDGEMENTS 

The authors would like to thank the Director 

of National Dairy Research Institute for providing 

financial support during the period of research work. 

Table 1. Specificity of the progesterone antiserum. 

Steroids 

Percent cross reaction 

4-pregnane-3, 20 dione 100 

11α-hydroxy progesterone 90 

Corticosterone


Table 2. Mean ± SEM milk yield and days of commencement of cyclicity in the early-commencing group. 

Animal No. Milk Yield/ Day Days to first Estrus 

3543 12.67±0.152 29 

5057 6.447±0.19 21 

4950 5.101±0.28 25 

4658 10.756±0.25 31 

4045 9.10±0.280 59 

5071 8.289±0.198 71 

Mean 8.727±1.131 - 

Table 3. Mean ± SEM milk yield and days to commencement of cyclicity in late commencing group. 

Animal No. Milk Yield/ Day Days to first Estrus 

5059 7.053±0.200 >90 

4700 6.579±0.254 >90 

4461 12.58±0.190 >90 

5098 7.763±0.310 >90 

4181 10.83±0.367 >90 

5036 9.667±0.321 >90 

Mean 9.07±0.916 

Table 4. Correlation coefficients (r) of different hormones and commencement of cyclicity in high and low 

yielders. 

High yielders 

GH 

MILK YIELD 

Low yielders 

GH 1.0000 0.1832* 

NEFA 0.2656 -0.286 

AAN 0.0853 -0.1626 

Glucose 0.1614 -0.0087 

Commencement of Cyclicity (days) -0.1781 -0.2370 

*, P


Table 5. Mean ± SEM concentrations of blood metabolites in high and low yielders. 

High 

Yielder 

Low 

Yielder 

ANIMAL 

NO. 

GLUCOSE 

(mg/dL) 

NEFA 

(µmol/L) 

AAN 

(mmol/L) 

4461 69.455± 2.637 309.900± 3.896 2.307±.0.087 

3543 66.524± 2.622 292.061± 7.488 2.412± 0.123 

4045 63.832± 2.346 299.481± 3.442 2.585± 0.050 

4658 53.796± 3.626 305.780± 2.882 2.169± 0.035 

4181 62.265± 7.716 299.130± 4.808 2.450± 0.034 

5036 56.052± 5.244 301.687± 4.539 2.592± 0.039 

Mean+SE 62.74±2.522 302.30± 2.301 2.494±0.0426 

4700 72.462± 3.132 297.099± 2.512 2.612± 0.030 

5057 72.034± 4.681 303.535± 3.133 2.575± 0.049 

4950 66.846± 4.315 295.716± 2.895 2.362± 0.088 

5098 58.733± 5.631 308.941± 3.791 2.522± 0.113 

5059 62.244± 5.168 304.103± 3.043 2.512± 0.045 

5071 65.181± 7.091 297.777± 2.759 2.592± 0.039 

Mean+SEM 67.08±3.876 301. 00± 1.320 2.538±0.0346 

Figure 1. Comparative milk yield of high yielders and low yielders. 

114


40 

HY 

LY 

GH Conc(ng/ml) 

30 

20 

10 

0 

0 2 4 6 8 10 12 14 16 18 20 22 

Weeks 

Figure 2. Comparative plasma GH of high yielders and low yielders. 

20 late commencement early commencement 

GH Conc(ng/ml) 

15 

10 

5 

0 

15 30 45 60 75 90 

Postpartum days 

Figure 3. Plasma GH (Mean± SEM) profile in postpartum lactating buffaloes. 

115


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and growth hormone fragments. Br. J. Nutr., 

43: 179. 

Bines, J.A. and I.C. Hart. 1982. Metabolic limits to 

milk production, especially roles of growth 

hormone and insulin. J. Dairy Sci., 65: 1375. 

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117


MOLECULAR CHARACTERIZATION OF β-CASEIN EXON 7 GENE IN BUFFALOES 

G. Darshan Raj, Swathishetty, M.G. Govindaiah, C.S. Nagaraja, 

S.M. Byregowda and M.R. Jayashankar 

ABSTRACT 

Caseins are the major part of the milk 

proteins and exist in several molecular forms (α S1 

, 

α S2 

, β and κ) with variant alleles of each. The 

present investigation was aimed at studying the 

genetic variation in β -casein gene locus exon VII 

between the three buffalo breeds.The β -casein 

gene was amplified by PCR using oligonucleotide 

primers standardized for Bos taurus species. The 

sizes of the amplification products were similar in 

all the three breeds, and no variation was found 

when the same PCR amplicons of β -casein gene 

subjected to SSCP and nucleotide sequence 

analysis. The BLAST alignment of 329 bp 

nucleotides of buffalo β -casein gene showed 

homogeneity with the Bubalus bubalis β-casein 

gene. Amino acid variations were observed at β- 

casein gene between Bubalus bubalis and Bos 

taurus species .The milk components and milk yield 

parameters could not be associated with buffalo β - 

casein genotypes due to their monomorphic 

haplotype. 

Keywords : genetic polymorphism , BLAST, 

haplotype, oligonucleotide primers, amplification. 

INTRODUCTION 

Caseins are the major part of the milk 

proteins and exist in several molecular forms (α S1 

, 

α S2 

, β and κ) with variant alleles of each. The milk 

casein is a raw material for the cheese making 

industry. The bovine casein genes (α S1 

, α S2 

, β and 

κ) are distributed in less than 250 kb. The four casein 

genes have been mapped on chromosome 6 in cattle 

and goats (Hayes et al., 1993, Threadgill et al., 

1990) and chromosome 7 in buffaloes (Rijnkels et 

al., 1997). Polymorphisms of all caseins are known 

(Eigel et al., 1984). Polymorphism of β-casein is 

quite complex due to its high genetic variability and 

to the presence of a large number of cases not 

characterized or not well clarified variants. The first 

evidence of polymorphism in β-casein dates back 

to 1961, when Aschaffenburg (1961) discovered, by 

paper electrophoresis at alkaline pH, three different 

β-casein bands, named A, B and C, in order of their 

decreasing mobility. Many nucleotide substitutions 

have occurred during the divergence of these caseins, 

but no major insertions, deletions, or other sequence 

rearrangements are evident in the α S2 

- and α S1 

-like 

caseins (Stewart et al., 1987). 

β-casein is important in determining the 

surface properties of casein micelles and essential 

for curd formation when milk is clotted by the 

proteolytic enzyme chymosin (Pearse et al., 1986). 

Curd formation is physiologically important since it 

ensures the retention of milk protein in the stomach 

of the infant and allows further digestion to occur. 

Bovenhuis et al. (1992) showed in a 

population of 6803 Holstein Friesian cows that the 

β-casein phenotypes could be ranked in the order 

of decreasing milk production as A2B > A1A3, 

A1A2, A1A1> A1B > BB. Among the Ayrshires, 

Kim et al. (1996), reported milk yield of 6077 kg for 

A2 variant of β-casein compared to 5838 kg for the 

A1 variant. The results of Bovenhuis et al. (1992) 

and Kim et al. (1996) were in agreement with earlier 

Department of Animal Breeding Genetics and Biostatistics, Veterinary College, Karanataka Veterinary 

Animal and Fisheries Sciences University, Hebbal, Bangalore- 560024, Karanataka, India. 

118


reports of Ng-Kwai-Hang et al. (1984;1986;1990) 

and Chung et al. (1991) in that the A2 variant of β- 

casein is associated with higher milk production than 

either the A1 or B variant. 

Studies by McLlean et al. (1984), Ng- 

Kwai-Hang et al. (1986), Bovenhuis et al. (1992) 

and Chung et al. (1996) have revealed the existence 

of differences in fat content among the different 

phenotypes of β-casein. 

Malik et al. (2000) studied the frequencies 

of the alleles of CASK and CASB in crossbreds 

and Sahiwal breed of cattle using polymerase chain 

reaction (PCR) and sequence specific oligonucleotide 

probes (SSOP). Their results showed 

predominance of CASK-A allele in Sahiwal, while 

CASK-B allele was predominant in crossbreds. At 

CASB level, CASB-A2 allele showed predominance 

in both Sahiwal and crossbreds and an increased 

frequency of CASB-B allele in crossbreds. 

Ikonen et al. (2001) concluded that β-CN 

locus had a strong effect on milk and protein 

production and fat percentage, and that the κ-CN 

locus had a strong effect on protein percentage. In 

addition, there may be interaction effects between 

the β-CN and κ-CN loci or other quantitative trait 

loci that were associated with certain casein 

combinations affecting milk production. 

The buffalo contributes about 54 percent 

of the total milk produced in India. Although the 

economic importance of buffaloes has always been 

known, yet very little work has been carried out to 

exploit the genetic potentials of this animal. 

Though extensive studies have been carried out on 

characterization of milk protein genes in cattle, 

similar studies in buffaloes are scarce. The studies 

on genetic potentiality of South Indian non-descript 

buffaloes like South Kanara has been limited in 

comparison to northern and western buffalo breeds. 

Hence a study was undertaken to identify 

polymorphism with in the β-casein gene and to 

associate such polymorphic pattern with the milk 

components and milk yield parameters. 


A total of 150 lactating buffaloes comprising 

50 South Kanara, 50 Surti, and 50 Murrah buffaloes 

from different villages and farms of Karnataka State, 

India were chosen for the present study. 

DNA isolation was carried out adopting the 

high salt method as described by Millers et al. 

(1988). 

The purity and concentration of DNA 

samples were estimated by spectrophotometer. The 

ratio of 260/280 nm OD was calculated. A ratio of 

1.7 to 1.9 was considered as high purity of DNA. 

PCR amplification: 

The primers CB1 and CB2 (Sigma-aldrich, 

Bangalore) are the flanking regions of the 329 bp 

fragment of beta casein gene locus in the exon 7 

between the location +8059 to +8387 and were 

designed based on the bovine nucleotide information 

as described by Medrano and Sharrow(1991). The 

nucleotide sequence information used for the PCR 

amplification of β-casein gene in buffalo was as 

follows. 

Primer Primer sequence 

CB1 5' CCCAGACACAGTCTCTAGTC 3' 

CB2 5' CACGGACTHGAGGAGGAAACA 3' 

All the reactions were carried out in 200 µl 

reaction tubes. Just before setting of the reaction, a 

master mix was prepared combining 10X PCR buffer 

(500 mM KCl, 100 mM Tris.HCl, pH 8.3)(MBI 

Fermentas) , 2.0 mM MgCl 2 

(MBI Fermentas), 100 

mM dNTP’s)(MBI Fermentas), 1.0 unit of Taq 

DNA polymerase(MBI Fermentas), 2.5 M.mol of 

each primer and filtered Milli Quartz (FMQ) water. 

Each reaction mix consisting of 19 µl of master mix 

and one µl (100 ng) of template DNA was then 

placed in the thermal cycler block. 

119


An initial denaturation at 94 o C for two 

minutes was done and subsequently denaturation and 

primer extension were carried out at 94 o C for one 

minute and 72 o C for one minute, respectively. The 

annealing temperature and time were determined 

empirically and standardized at 55.5 o C for 30 

seconds. The number of cycles was kept constant 

at 35. After the last cycle, a final primer extension 

was carried out at 72 o C for ten minutes and the 

samples were then cooled to 15 o C until retrieved. 

SSCP analysis: 

Five µl of sample was aliquoted into 

separate tubes and an equal volume of 2x SSCP gel 

loading dye (0.05% bromophenol blue, 0.05% xylene 

cyanol, 95% formamide, 20 mM EDTA) was added 

and mixed. The mixture was denatured at 94 0 C for 

4 minutes in a heating block (Bangalore genei). The 

samples were snap cooled on ice to prevent 

heteroduplex formation and then subjected to 

electrophoresis in an acrylamide:bis acrylamide 

(37.5:1)gel, using Bio-Rad mutation detection unit. 

Acrylamide/bis-acrylamide (37.5:1 acrylamide to 

bisacrylamide), 10x TBE-buffer, TEMED and water 

were mixed in a clean, fat-free beaker and stirred 

well, carefully avoiding air-bubbles, a final 

concentration of 0.09% (v/v) each of ammonium 

persulfate and TEMED solutions were used. The 

running time and polyacrylamide concentrations 

were standardized to observe clear fragment 

separations. DNA visualization in the polyacrylamide 

gel after electrophoresis was done by ethidium 

bromide staining. 

Nucleotide sequencing: 

The PCR products were concentrated to 

50 ng/µl by pooling several tubes to precipitate by 

the isopropanol procedure. In order to obtain clean 

fragment for sequencing, the PCR products were 

separated by electrophoresis in a TAE agarose gel 

containing ethidium bromide using standard 

protocols. The desired PCR product band was 

excised using a clean, sterile razor blade or scalpel 

(band was visualized in a medium or long wavelength 

(e.g., ≥300 nm) UV light, and excised quickly to 

minimize exposure of the DNA to UV light). The 

minimum agarose slice was transferred to a 1.5 ml 

microcentrifuge or screw cap tube and then purified 

by using commercially available gel extraction kits 

(Qiagen). Quantification was done by loading one 

µl of eluted sample in 1% Agarose gel and comparing 

with standard molecular marker (Phi X 174 DNA 

ladder or 100 bp DNA ladder). Only samples with 

good concentration (>50 ng/µl) were selected. 

Samples were labeled and sent for sequencing. 

Sequencing was done by Avesthagen, Bangalore and 

Macrogen, South Korea. 

Sequence data analysis: 

Sequences were edited and initially aligned 

using the Sequencher demo version and then 

optimally aligned visually. Multiple sequences were 

aligned by Clustal format for T-COFFEE 

Version_1.41 on line application. Polymorphic sites 

were analyzed. Polymorphic sites were again 

confirmed by electropherogram results. Coding 

sequences were translated to amino acids by online 

EBI (European Bioinformatics Institute tools, 

Translation tool, www.ebi.ac.uk). 

Database search: 

The database search of sequences for a 

possible match to the DNA sequence of casein gene 

was conducted using the BLAST algorithm available 

at the National Center for Biotechnology 

Information (NCBI, Bethesda, MD). Translated 

protein sequences of different casein genes were 

also subjected to BLAST algorithm. 

Milk sample analysis: 

Milk samples were collected from the same 

target animals and preserved by potassium 

dichromate powder (0.2%). Protein, fat, solid-notfat, 

total solids and lactose of milk samples were 

analyzed by Milko Scan 1338, (Auto Analyzer, 

Denmark) at the Quality Control Unit, Mother Dairy, 

Bangalore. 

120



The present investigation was aimed to study 

the genetic variation at β-casein gene exon VII 

region between the three buffalo breeds viz, South 

Kanara, Surti and Murrah, and to assess the 

genotypic frequency of different haplotypes at 

different regions of the gene. Attempts were also 

made to associate the haplotypes obtained with milk 

composition. There were few or no such studies in 

buffaloes, however, comparison has been made to 

similar work carried out in different cattle breeds 

by other workers. 

PCR technique: 

The high salt method yielded good quality 

of DNA as measured by the OD, which ranged from 

1.7 to 1.9. The quality and quantity of DNA extracted 

was further confirmed by electrophoresis. A 329 

bp fragment of exon 7 of β-casein gene was 

amplified by PCR using oligonucleotide primers 

designed by Medrano and Sharrow (1991). The 

sizes of the amplification products were same for 

all the animals studied, suggesting that this region 

was conserved in buffalo breeds (Figure 1). Malik 

et al. (2000) used the same set of primers for 

studying β-casein alleles in crossbred and zebu cattle. 

The results of the present study are in accordance 

with the amplified product obtained by these workers, 

suggesting the conservation of β-casein gene 

amongst Bos taurus, Bos indicus and Bubalus 

bubalis species. 

SSCP analysis: 

The SSCP protocol was optimized by 

varying gel percentage, quantum of PCR product, 

denaturing solution, time of exposure to denaturation 

and duration of running the gel. The bis ratio and 

running temperature were the most critical factors 

affecting the resolution power. Since no reference 

samples were available, all the samples were loaded 

randomly to record the difference if any in mobility 

of denatured strands. Best results were obtained 

using 8 percent PAGE gels with a ratio of acrylamide 

to NN’ methylene-bisacrylamide of 37.5 :1, 7 per 

cent glycerol, 0.1 percent TEMED and one percent 

APS. The electrophoresis was run for 4-5 hours at 

300 V in 1x TBE at 12 o C. The 150 amplified samples 

of β-casein genes were subjected to SSCP, which 

did not show any significant variation in mobility of 

fragments, thus suggesting homozygous banding 

pattern (Figure 2). No discrepancies between PCR– 

RFLP and PCR-SSCP patterns were detected for 

any of the buffalo breeds.The polymorphic base at 

+8267 bp of β-casein gene recommended by 

Medrano and Sharrow (1991) did not possess any 

restriction enzyme site. Hence, PCR-RFLP analysis 

was not performed. However, Medrano and 

Sharrow (1991) performed PCR- RFLP for the site 

by using MspI enzyme by altering the first base in 

reverse primer, which lies close to the polymorphic 

site. In the present study, samples were directly 

subjected to the SSCP studies to identify different 

haplotypes. Only one SSCP pattern was observed 

in all the buffaloes studied. Since no reference 

sample was available to compare, it was not possible 

to assign the SSCP pattern into any type. Further, 

random samples were sequenced to identify the 

genotype or haplotype. 

Sequencing: 

In the present study, a 329 bp fragment of 

β-casein gene was sequenced in South Kanara, Surti 

and Murrah breeds using the bovine primers. 

Purified PCR products with good concentrations 

(>50 ng/µl) from each breed (South Kanara, Surti 

and Murrah) were selected for sequencing. Initially, 

for confirmation of the PCR product for casein 

genes, one random sample for each gene was chosen 

and sequenced; forward and reverse reactions were 

performed using both the primers and the reactions 

were compared and overlapping regions from one 

of the reaction was removed and whole sequence 

was aligned. 

Sequencing of the 329 bp fragment of exon 

VII β-casein gene showed cytosine (C) at the 

+8267 nucleotide position; hence, a representative 

sequence report is given (Figure 3), which was 

121


Figure 1. PCR amplification of Bubalus bubalis β- casein gene. 

Lane 1 and 2 - South Kanara breed. 

Lane 3 and 4 - Surti breed. 

Lane 5 and 6 - Murrah breed. 

M1 – 1 kb DNA ladder in bp. 

M2-φX174 ladder in bp. 

122


Figure 2. PCR-SSCP pattern of the Bubalus bubalis β-casein gene. 

Lane 1 to 3 - South Kanara breed. 

Lane 4 and 5 - Surti breed 

Lane 6 and 7 - Murrah breed 

M1 – 1 kb DNA ladder in bp. 

NT – Not treated with denaturing dye (Direct PCR product). 

123


1 Q T Q S L V Y P F P G P I P K S L P Q N 

3 CAGACACAGTCTCTAGTCTATCCCTTCCCTGGGCCCATCCCTAAGAGCCTCCCACAAAAC 

21 I P P L T Q T P V V V P P F L Q P E I M 

63 ATCCCGCCTCTTACTCAAACCCCTGTGGTGGTGCCGCCTTTCCTTCAGCCTGAAATAATG 

41 G V S K V K E A M A P K H K E M P F P K 

123 GGAGTCTCCAAAGTGAAGGAGGCTATGGCTCCTAAGCACAAAGAAATGCCCTTCCCTAAA 

61 Y P V E P F T E S Q S L T L T D V E N L 

183 TATCCAGTTGAGCCCTTTACTGAAAGCCAGAGCCTGACTCTCACTGATGTTGAAAATCTG 

81 H L P L P L L Q S W M H Q P P Q P L P P 

243 CACCTTCCTCTGCCTCTGCTCCAGTCTTGGATGCACCAGCCTCCCCAGCCTCTGCCTCCA 

101 T V M F P P Q S V 109 

303 ACTGTCATGTTTCCTCCTCAGTCCGTG 

Figure 3. Sequence report :Amplified 329 bp fragment of β -casein gene exon VII in Bubalus bubalis 

breeds ((NCBI gene bank accession no. EF066481). 

EMBOSS_001 1 QTQSLVYPFPGPIPKSLPQNIPPLTQTPVVVPPFLQPEIMGVSKVKEAMA 50 

||||||||||||||.|||||||||||||||||||||||:||||||||||| 

EMBOSS_002 1 QTQSLVYPFPGPIPNSLPQNIPPLTQTPVVVPPFLQPEVMGVSKVKEAMA 50 

EMBOSS_001 51 PKHKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPPQPLPP 100 

||.|||||||||||||||||||||||||||||||||||||||||.||||| 

EMBOSS_002 51 PKQKEMPFPKYPVEPFTESQSLTLTDVENLHLPLPLLQSWMHQPHQPLPP 100 

EMBOSS_001 101 TVMFPPQSV 109 

||||||||| 

EMBOSS_002 101 TVMFPPQSV 

Figure 4. Blast report: Comparison of β-casein amino acid sequences between Bubalus bubalis and 

Bos taurus species. 

EMBOSS_001 – Amplified 329 bp fragment’s protein sequence. 

EMBOSS_002 – Bovine mammary gland m-RNA for β-casein gene as given by Stewart et al. 

(1987). 

124


observed in genetic variant “A” only. This feature 

affirmatively confirmed that all the animals studied 

were of AA genotype. 

The presence of the amino acid serine 

(AGC) was observed at 76 th position of β-casein 

gene in all the buffalo samples studied. It was 

considered as AA genotype as recommended by 

Medrano and Sharrow (1991). A different genotype 

BB was observed by the same authors in cattle with 

amino acid arginine (AGG) which was not observed 

in present study. 

β-casein B allele was totally absent in all 

the three buffalo breeds studied. However, earlier 

studies in three zebu breeds (Sahiwal, Tharparkar 

and Red Sindhi) revealed the presence of the β- 

casein B allele, but at very low frequencies 

(Aschaffenberg, 1961; Jairam and Nair, 1983). A 

higher frequency of this allele was recorded in the 

crossbred dairy cattle (H.F x Jersey x Hariana) in 

comparison to Hariana breed by Malik et al. (2000). 

BLAST search: 

The BLAST result for the 329 bp β-casein 

gene matched 98 percent with the Bubalis bubalus 

gene sequence reported by Singh et al. (2005) 

(Figure 4). The second highest match was with 

Bubalus bubalis mRNA gene sequence for β- 

casein reported by Preenu et al. (2004). Based on 

these observations, it can be concluded that the 

Bubalus bubalis gene is very much conserved 

within the different buffalo subspecies. Dissimilarity 

in four amino acids at different positions was 

recorded between the Bubalus bubalis and Bos 

taurus species amino acid sequence reported by 

Stewart et al .(1987) (Table 1). The exact effect of 

this mutation has to be investigated further in the 

case of Bubalus bubalis. 

Correlation with milk constituents: 

The overall mean percentage of fat, protein, 

lactose and total solids were recorded as 7.5+0.68, 

4.25+0.23, 5.23+0.14 and 17.45+0.79 percent, 

respectively.All the 150 animals in the current 

investigation were of homozygous (monomorphic) 

genotype for the β-casein gene and hence, it was 

not possible to assess the relationship between 

genetic variant and milk constituents. 

CONCLUSION 

The primers CB1 and CB2 which were 

designed based on the bovine nucleotide information 

successfully amplified 329 bp Bubalus bubalis β- 

casein gene locus in the exon 7.The results of SSCP 

and sequencing showed that all the 150 animals 

belonging to South Kanara, Murrah and Surti were 

homozygous (monomorphic) for the β-casein gene. 

There was a significant difference between the 

nucleotide sequence of β-casein gene of Bubalus 

bubalis and Bos taurus animals, which needs to be 

further studied to assess their effect on milk 

constituents of the two species. 

Table 1. Amino acid variations recorded at β-casein gene of Bubalus bubalis by comparing with Bos taurus 

species. 

Sl 

no. 

Amino acid 

number 

Amino acid observed in 

Bubalus bubalis 

Amino acid observed in 

Bos taurus (Stewart et al ., 1987) 

1 15 Lysine (K) Asparagine (N) 

2 38 Isoleucine (I) Valine (V) 

3 53 Histidine (H) Glutamine (G) 

4 95 Proline (P) Histidine (H) 

125


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Medrano, J.F. and L. Sharrow. 1991. Genotyping 

beta casein loci by restriction site modification 

of polymerase chain reaction (PCR) amplified 

genome DNA. J. Dairy Sci., 74(1): 282. 

Millers, S.A., D. Dykes and H.F. Polesky. 1988. A 

simple salting out procedure for extraction of 

DNA from human nucleated cells. Nucleic 

acid Res., 16:1215. 

Ng-Kwai-Hang, K.F., J.F. Hayes, J.E. Moxley and 

H.G. Monardes. 1984. Association of genetic 

variants of casein and milk serum proteins with 

milk, fat, and protein production by dairy cattle. 

J. Dairy Sci., 67: 835-840. 

Ng-Kwai-Hang, K.F., J.F. Hayes, J.E. Moxley and 

H.G. Monardes. 1986. Relationships between 

milk protein polymorphisms and major milk 

constituents in Holstein-Friesian cows. 

J. Dairy Sci., 69: 22-26. 

Ng-Kwai-Hang, K.F., H.G. Monardes and J.F. 

Hayes. 1990. Association between genetic 

polymorphism of milk proteins and production 

traits during three lactations. J. Dairy Sci., 

73: 3414-3420. 

Pearse, M. J., P. M. Linklater, R. J. Hall, and A. G. 

Maciunlay. 1986. The effect of casein 

composition and casein dephosphorylation on 

the coagulation and syneresis of artificial 

micelle milk. J. Dairy Res., 53: 381-390. 

Preenu, J., P. Muraleedharan, V. Roch and S. 

Kumar. 2004. A new genetic variant of buffalo 

beta casein in exon 7. Accession AY599833. 

Cited in: www.ncbi.com. 

Rijnkels, M., P.M. Kooiman, H.A. deBoer and F.R. 

Pieper. 1997. Organization of the bovine casein 

gene locus. Mamma. Genome, 8: 148-152. 

Singh, S., Pushpendra Kumar and T.K. 

Bhattacharya. 2005. DNA polymorphism of 

kappa and beta casein genes and it’s 

association with milk production and quality 

traits in buffalo (Bubalus bubalis), p.200- 

201. In Proceedings of the National Symposium 

on Domestic Animal Biodiversity: 

Status Opportunities and Challenges. 

Stewart, A. F., I. M. Willis, and A. G. Maciunlay. 

1987. Nucleotide sequences of bovine α SI 

- and 

β-casein cDNAs. Nucleic Acids Res., 12: 

3895-3907. 

Threadgill, D.W. and J.E.Womack. 1990. Genomic 

analysis of the major bovine milk protein genes. 

Nucleic Acids Res., 18: 6935-6942. 

126


SEASONAL VARIATIONS IN BREEDING AND CALVING PATTERNS OF NILI-RAVI 

BUFFALOES IN AZAD KASHMIR, PAKISTAN 

Zulfiqar Hussain 

ABSTRACT 

A total of 10,254 artificial insemination 

records of 5,467 Nili-Ravi buffaloes from (i) the 

Artificial Insemination Section, Directorate of 

Animal Husbandry and (ii) the Livestock 

Development Research Centre, Raroo, Muzaffarabad, 

Azad Kashmir, Pakistan from 1989 to 

2002, were utilized to study the seasonal variation 

and effect of season and month on breeding and 

calving patterns. The highest (56.57) breeding 

percentage was recorded in the autumn (October- 

November) season, while it was the lowest (0.48 

percent) in the dry hot (May-June) season. Seventyone 

(71) percent of Nili-Ravi buffaloes calved in 

the humid hot (July-September) season, while calving 

was the lowest (0.91percent) in the spring (February- 

April) season. The highest percentages (33.75) of 

buffaloes were observed in oestrus in the month of 

October, while the lowest percentage of buffaloes 

(0.13) showed oestrus in the month of June. The 

highest percentage of calvings took place in the 

month of August (34.46), while the lowest 

percentage of calving (0.14) in took place in the 

month of April. Analysis of variance showed highly 

significant effects (P


Directorate of Research and Extension and ii) the 

Livestock Development Research Centre, Raroo, 

Muzaffarad (Azad Kashmir) from 1989 to 2002 was 

thus planned with the following objectives: 

1. To study the seasonality of breeding and 

reproduction in Nili-Ravi buffaloes. 

2. To determine the influence of month and season 

of calving on breeding and calving 

It is envisaged that the information thus 

generated would help in formulating a breeding plan 

most suitable to the farming community under the 

local conditions of Muzaffarabad in particular and 

the whole of Azad Kashmir in general. 

Statistical analysis 

Effect of the month and season on breeding 

and calving was evaluated. Keeping in view the 

climatological data, the year was divided into 

following five seasons: 

Winter December to January 

Spring February to April 

Dry hot May to June 

Humid hot July to September 

Autumn October to December. 

For analysis, the Mixed Model Least Square 

Maximum Likelihood (LSMLMW) computer 

programme (Harvey, 1990) was used. 


Collection of data 

A total of 10,254 artificial insemination 

records of 5,467 Nili-Ravi buffaloes from 1989 to 

2002 from the Artificial Insemination Section and 

from the Livestock Development Research Centre, 

Raroo, Directorate of Animal Husbandry, Azad 

Kashmir, Pakistan from 1989 to 2002, were utilized 

to study the seasonal variation and effect of season 

and month on breeding and calving patterns in these 

animals. 

Classification of data 

The data consisted of, 

a) Identity of the animal/owner. 

b) Species/Breed of the animal (Buffalo/nondescript 

cow/Cross-bred cow). 

c) Date of Artificial Insemination/mating. 

d) Non-return-rate (60-90 days). 

e) Date of calving. 

f) Sex of the calf. 

Based on this data, seasonality of breeding 

and reproduction in these animals was studied and 

the results depicted graphically. 

A spreadsheet, MS Excel RO was used for 

data entry and manipulation. 


1) Breeding 

The highest percentages of buffaloes (33.75 

percent) were observed in oestrus in the month of 

October, followed by November, when 22.86 percent 

of the buffaloes were observed in oestrus. As much 

as 14.17 percent of the buffaloes were observed in 

oestrus in the month of September. The percentage 

of buffaloes in oestrus in the month of August was 

10.96. The lowest percentage of buffaloes (0.13 

percent) showed oestrus in the month of June, 

followed by May, April and March, when only 0.35, 

0.52 and 0.85 percent of buffaloes were observed 

in oestrus, respectively. The percentage of oestrus 

in Nili-Ravi buffaloes was 2.05, 1.41 and 5.59 in the 

months of January, February and December, 

respectively. 

When the data were grouped according to 

the various seasons, the highest percentage of 

buffaloes (56.57 percent), were observed in oestrus 

in the autumn (October-November) followed by the 

humid hot (July-September) season, in which 32.48 

percent buffaloes were bred, while the percentage 

of buffaloes in oestrus was 7.65 in the winter 

(December-January) season. The lowest percentage 

of oestrus in buffaloes was observed in the dry hot 

(May-June) season, when only 0.48 per cent of 

buffaloes were observed in oestrus, followed by 2.82 

per cent in the spring (February-March) season. 

128


Analysis of variance showed highly significant effect 

(P


by modifying management. This will go a long way 

to help solve the problem of spreading out the 

breeding season of the buffalo to ensure a steady 

supply of milk throughout the year. 

REFERENCES 

Afiefy, M. M. 1967. Seasonal variations in thyroxin 

and iodine-contents in relation to fertility in 

buffaloes and cattle. Vet. Med. J. Cairo., 13: 

73-100. 

Agarwal, K. P. 2003. Augmentation of reproduction 

in buffaloes, p. 121. In Proceeding of 4 th 

Asian Buffalo Congress Lead Papers. 

Ashfaq, M. and I. L. Mason. 1954. Environmental 

and genetical effects on milk yield in Pakistani 

buffalo. Empire J. Exper. Agric., 22(87): 161- 

175. 

Harvey, W. R. 1990. Users’ Guide for LSMLMW 

and MIXMDL, Mixed model least squares 

and maximum likelihood computer 

program. PC-2 version, the Ohio State 

University, Columbus, USA. 

Jalatge, E. F. A. and V. Buvanendran. 1971. 

Statistical studies on characters associated 

with reproduction in the Murrah buffalo in 

Ceylon. Trop. Anim. Hlth. Prod. J., 3: 114- 

124. 

Khan, A. 1996. Seasonal variation in breeding 

patterns of buffalo in Punjab. M. Sc. thesis, 

University of Agriculture, Faisalabad, Pakistan. 

Khan, M. A. 1986. Genetic analysis of a purebred 

herd of Nili-Ravi buffaloes. Ph.D thesis, 

University of Agriculture, Faisalabad, Pakistan. 

99p. 

Shalash, M. R. and A. Salma. 1962. Seasonal 

variation in the ovarian activity of the buffalocow. 

Proc. 4 th . Int. Congr. Anim. Reprod., 

2: l90-191. 

Thevamanoharan, K. 2002. Genetic analysis of 

performance traits of swamp and riverine 

buffalo. Ph. D. thesis, Katholieke University 

Leuven, Belgium. 

Zicarelli, L., R. Boui, G. Campanile, S. Roviello, B. 

Gasparrini and Di Palor. 1997. Response to 

super ovulation according to parity, OPU 

priming, persena/abscent of dominant follicle 

and season, p. 748. In Proceedings of 5 th 

World Buffalo Congress, Oct 13-16, 1997 

at Caserta, Italy. 

*Continued from page 133 

REFERENCES 

Detweiler, D.K. 1996. Control mechanism of the 

circulatory system, p. 170-183. In Swenson 

M.J. and W.O Reece (eds.) Duke’s 

Physiology of Domestic Animals, 7 th ed. 

Panima Publishing Corporation, New Delhi. 

Kaneko, J.J., J.W. Harvey and M.L. Bruss. 1999. 

Appendixes, p. 885-905. In Kaneko, J. J., 

J. W. Harvey and M. L. Bruss (eds.) Clinical 

Biochemistry of Domestic Animals, 5 th ed. 

Harcourt Brace and Company, Asia PTE Ltd., 

Singapore. 

Sharma, I.J. 2004. Physiological Basis of 

Veterinary Practice, p. 56-58. Kalyani 

Publishers, New Delhi. 

Snedecor, G.W. and W.G. Chochran. 1989. Statistical 

Method, 8 th ed. The lowa State University 

Press, Ames, lowa, USA. 

130


CARDIO – RESPIRATORY RESPONSES OF NEONATAL BOVINES AS AFFECTED BY 

ACETYLCHOLINESTERASE DURING THEIR DEVELOPMENT 

A.K. Jain, R.K. Tripathi, I.J. Sharma, R.G. Agrawal and M.A. Quadri 

ABSTRACT 

The cardio–respiratory responses are 

associated with the neuronal development of the 

animals. In order to assess the extent of this neuronal 

dependency, an experiment was conducted in 

periparturient buffaloes, cows, buffalo calves and 

cow calves. A separate set of calves was given neem 

oil @ 10 ml per day orally to monitor its potentiating 

effect on the special development of the neonates. 

It was recorded that the activities of acetyl 

cholinesterase was low in early neonatal periods, 

indicating the poor development of cholinergic 

system. It increased with age whereas, the pulse 

rate as well as rate of respiration were higher during 

this period. 

Keywords: acetylcholinesterase activity, cardiorespiratory 

responses, neonatal bovines 

INTRODUCTION 

Intracellular enzymes are involved in the 

process of metabolism of their respective substrates. 

Thus, the secretion of these enzymes depends upon 

their requirement at particular times by the body. 

Development of the nervous system, particularly, the 

autonomic nervous system, is age dependent. As 

per the regulatory function of autonomic nervous 

system, the secretion of acetylcholinesterase (AchE) 

is meagre early in neonatal life as the tonus of the 

parasympathetic (cholinergic) system is low. The 

effect of low cholinergic activity is depicted by higher 

cardio respiratory responses. In order to further 

elucidate this phenomenon of dependency, study on 

the cardio– respiratory response of neonatal buffalo 

calves as influenced by the levels of actylcholinesterase 

was taken up. The effect of neem 

oil on the system under question as a whole it also 

discussed in this paper. 


This study was conducted from 1 st Dec., 

04 to 30 th August, 05 in neonates of bovines as 

indicated in Table 1. The pulse rate and rate of 

respiration of neonates were recorded as per 

standard procedure (Sharma, 2004). Blood samples 

of the calves were collected before colostrum 

feeding, 6 h post colostrum feeding, daily for a week, 

and thereafter at weekly intervals up to 91 days 

postnatally. Each calf was given 10 ml of neem oil 

orally daily from postcolostral feeding till 91days. 

The red cells separated after centrifugations were 

haemolysed. The estimation of acetylcholinesterase 

(AchE) in red cell haemolysate and red cell walls 

was done using ready-made kits supplied by 

Spinreact, SA. The statistical analysis of the data 

was done as per the procedure given by Snedecor 

and Cochran (1989). 


The status of acetylcholinesterase (AchE) 

used as an index of the development of autonomic 

nervous system with that of the age of the developing 

animals and their cardio–respiratory responses have 

been presented in Tables 2 and 3. The activity of 

AchE in the red cell wall of periparturient dams was 

quite a bit higher than in their plasma (pseudocholinesterase), 

and this is indicative of a fully 

Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, Madhya Pradesh - 482001, India 

131


functional parasympathetic system. However the 

activity of this enzyme was found to be only onethird 

as great in newly born calves in comparison to 

their dams. This is indicative of the underdevelopment 

of the cholinergic system, meaning 

simply lower levels of acetylcholine and, thus, lower 

activity of its hydrolyzing enzyme as per its 

requirement. However, the activity of this enzyme 

gradually increased with the age in calves of both 

the species, indicating the gradual development of 

the cholinergic system. The cardio–respiratory 

responses were altered accordingly, as these 

activities are under control of the autonomic nervous 

system. Lower parasympathetic tone is cardioacceleratory, 

leading into rapid increase in the pulse 

rate, which thereafter fell with the increased activity 

of the vagus nerve (Detweiler, 1996). However, no 

such age-wise difference was recorded between 

the neonatal buffalo calves and cow calves. Again, 

the rate of respiration was significantly (p


Table 3. Profile of acetylcholinesterase (P) and (R), pulse rate and rate of respiration of neonatal bovines 

as affected by neem oil. 

Animals AChE (P) (U / L.) ACh E(R) (U / L.)) Pulse rate Respiration rate 

Time 

(per minutes) (per minutes) 

intervals 

Calves Control Neemoil Control Neemoil Control Neemoil Control Neemoil 

treated 

treated 

treated 

treated 

Precolostral Bc 303±45 ……….. 416±16 …….. 115±2.0 …….. 32±0.5* …….. 

feeding Cc 268±23 

402±25 

118±4.0 

42±1.7 

6 h post Bc 283±35* 326±36 425±23 435±25 118±2* 107±1.0* 33±0.5* 32±0.7* 

colost.feed. Cc 464±97 326±39 414±21 396±23 111±3 114±4 38±1.5 39±1.6 

1 st Day Bc 304±66 412±44 438±14 452±32 118±1.7 111±1.6 33±0.7 31±0.6 

Cc 366±35 239±25 439±14 365±29 109±5.2 105±3.5 37±2.5 38±1.0 

2 nd Day Bc 362±41 407±49 510±32 515±29 114±1.6 109±1.1 31±0.5 30±0.6 

Cc 413±74 393±60 512±459 402±12 113±3.9 93.4±3 39±2.5 37±1.4 

3 rd Day Bc 306±52 397±52 525±34 530±35 113±1.7 109±1.6 31±0.7 30±0.7 

Cc 359±59 310±30 568±62 514±32 102±5.4 92±3.2 35±2.1 35±1.1 

4 th Day Bc 255±30 403±36 536±28 542±29 109±1.5 103±2 28±0.6 29±0.6 

Cc 371±51 472±56 529±49 531±23 106±5.5 89±4 34±2.4 34±1.6 

5 th Day Bc 241±35 535±45 557±26 560±34 108±1.3 107±1.1 28±0.3 27±0.4 

Cc 263±32 415±48 589±63 536±34 112±3.5 88±3.7 32±2.5 36±1.8 

6 th Day Bc 305±44 562±95 567±31 574±32 107±1.5 105±1.7 28±0.8 28±0.8 

Cc 216±37 420±25 540±53 546±26 107±3.2 88±3.8 32.5±2 37±1.7 

14 th Day Bc 235±56* 490±72 587±24 614±34 104±2.2 105±2.5 27±0.5 27±0.8 

Cc 410±59 427±43 610±23 582±28 92.4±3.4 78±1.6 29±1.8 37±1.8 

21 st Day Bc 457±62* 388±30 632±26 664±26 111±1.7 102±2.1 28±0.8 28±1.4 

Cc 261±31 277±44 622±30 599±31 88±4.4 82±2 27±2.1 38±1.2 

28 th Day Bc 476±64 474±52 652±30 690±30 96±2.9 86±2.4 27±0.8 28±1.5 

Cc 312±30 406±55 635±34 615±24 87±4.4 82±2.3 24±1.8 37±1.2 

35 th Day Bc 398±43 386±26 666±21 718±29 100±4.2 95±3.0 27±1.1 28±1.1 

Cc 312±30 418±61 638±26 622±34 83±4.7 79±2.3 33±2.2 37±1.4 

42 nd Day Bc 470±39 336±40 732±34 740±38 95±2.9 84±5.0 27±0.5 30±1.3 

Cc 303±51 278±31 635±21 615±34 80±3.5 77±1.6 33±1.2 34±1.8 

49 th Day Bc 347±53 380±29 752±32 768±39 91±2.2 80±4.8 27.3±0.8 28±0.8 

Cc 301±23 307±48 625±26 612±31 82±3.5 76±1.2 31±2.2 36±0.9 

56 th Day Bc 240±31 301±20 775±36 779±35 92±4.1 72±2.5 27±0.9 28±1.4 

Cc 270±53 264±45 628±34 623±42 76±5.4 77±0.8 31±1.8 36±1.3 

63 rd Day Bc 220±32* 30427 735±22 756±33 80±3.9 67±1.1 30±1.2 28±1.2 

Cc 401±57 287±42 634±34 608±35 79±5.0 78±0.9 32±3.0 37±0.9 

70 th Day Bc 185±15* 284±31 699±32 687±28 79±3.3 75±3.7 30±0.6 28±1.1 

Cc 312±47 375±55 625±27 620±34 81±5.5 78±0.9 34±3.5 37±1.0 

77 th Day Bc 223±15 271±25 685±25 659±31 82±3.4 72±3.0 29±1.1 27±0.7 

Cc 312±41 276±36 605±34 599±47 75±3.6 77±1.0 31±1.9 37±0.9 

84 th Day Bc 213±33* 234±23 613±29 615±31 79±4.3 69±3.3 29±0.4 25.9±1 

Cc 422±50 280±56 612±26 590±28 72±2.7 76±0.5 31±2.6 36.4±1 

91 st Day Bc 260±32 234±23 604±45 583±26 69±1.2 70±3.2 28±1.1 26±0.9 

postpatum Cc 312±41 222±53 582±44 572±30 70±4.3 75±1.6 30±2.8 35±1.2 

(*P


ENERGY REQUIREMENTS OF LACTATING MURRAH BUFFALOES 

(BUBALUS BUBALIS) 

Pramod Sharma, R. S. Gupta and R. P. S. Baghel 

ABSTRACT 

A study was conducted to determine the 

energy requirement of lactating Murrah buffaloes 

fed different levels of diammonium phosphate. 

Eighteen lactating Murrah buffaloes of nearly the 

same body weight (551.52±14.63 kg), milk yield, 

parity and stage of lactation were divided into three 

groups of six animals in each and were fed 0, 50 

and 100% diammonium phosphate in the mineral 

mixture of concentrates. The chaffed mixed 

roughage (berseem + wheat straw) was available 

ad libitum as was tap water. A metabolism trial of 

seven days was conducted at the end of experiment. 

The energy requirements of lactating Murrah 

buffaloes were estimated by partitioning the ME or 

TDN intake for maintenance and milk production 

by using multiple regression method. It was 

concluded that the daily maintenance requirement 

of energy for lactating Murrah buffalo was 49.5 g 

TDN and 179.4 kcal ME/w 0.75 kg and for per kg 4% 

FCM was 346 g TDN and 1,252 kcal ME. From the 

partitioning of energy, it appeared that about 64.49% 

TDN was utilized for maintenance of body and 

35.51% diverted for milk production. Similarly 

64.55% ME was utilized for maintenance and 

35.45% ME for milk production. 

Keywords: diammonium phosphate, energy 

requirement, lactating Murrah, milk yield, 

partitioning of energy 

INTRODUCTION 

In India feeding and animal husbandry 

practices are different in different agro-climate 

conditions. Though lot of work has been done on 

nutrient requirements in cattle, little work has been 

done on buffaloes. Hence, the studies were, planned 

to learn the energy requirements of lactating 

buffaloes fed different levels of diammonium 

phosphate in the mineral mixture of their ration. 


Eighteen lactating Murrah buffaloes of the 

same body weight, milk yield, parity and stage of 

lactation were divided into three groups of six 

animals in each. Tap water was available ad libitum. 

A digestion trial of seven days was conducted at 

the end of experiment. The animals were fed mixed 

roughage (green berseem+wheat straw) and 

concentrate mixture to supply their nutrient 

requirements as per ICAR (1998). The concentrate 

mixture consisted of yellow maize 40, wheat bran 

22, mustard cake 35.5 , mineral mixture 2, and salt 

0.5 parts. In the mineral mixture of the control diet 

(T 1 

), dicalcium phosphate was used; this was 

replaced by diammonium phosphate at 50% in T 2 

and 100% levels in T 3 

(Table 1). 

A calculated amount of urea was added to 

the mineral mixture of the T 1 

and T 2 

diets and 

limestone powder in increasing amounts in T 1, 

T 2 

and T 3 

diets, to make all the diets identical in nitrogen 

and calcium content (Table 1).The phosphorus 

Department of Animal Nutrition, College of Veterinary Science and Animal Husbandry, 

J. N. Krishi Vishwa Vidyalaya , Jabalpur- 482001 (M.P.) India. 

134


content was the same(21.8%) in the dicalcium 

phosphate and diammonium phosphate. 

Samples of feed, faeces and urine were 

analyzed for proximate composition using A.O.A.C. 

(1990). The fat percentage in the milk was estimated 

by the Gerber’s method (Agarwala and Sharma, 

1961).The data obtained during experiment were 

analyzed by using randomized block design method 

as described by Snedecor and Cochran (1980). 

The chemical composition of the 

experimental diets (concentrate mixtures) and mixed 

roughage offered to the experimental animals are 

presented in Table 2. Due to replacement of DCP 

at 50% in T 2 

and 100% in T 3 

diet the chemical 

composition in respect of CP, EE, CF, ash, Ca and P 

content did not vary as compared to the control (T 1 

). 

The energy requirements of lactating 

Murrah buffaloes were estimated by partitioning the 

ME or TDN intake for maintenance and milk 

production using the following model described by 

Moe et al. (1970). 

Y = a + b 1 

X 1 

+ b 2 

X 2 

where Y is TDN intake (kg) or ME intake 

(Mcal), X 1 

is metabolic body size (w 0.75 kg) and X 2 

is 

4% FCM production (k/d). In the above model, b 1 

represents the energy requirement for maintenance 

(kcal/ w 0.75 kg) and b 2 

represents the energy required 

or spared for 1 kg 4% FCM production (k/d). The 

constant ‘a’ represent an amount of energy which 

is not attributable to any specific variable in this 

model. This amount of energy was assigned to the 

maintenance term (Moe et al., 1970). This was done 

by dividing ‘a’ by the average metabolic body size 

and adding it to the coefficient b 1 

. 


Intake of all the nutrients (Table 3) was 

similar in the control (T 1 

) and the experimental feed 

(T 2 

and T 3 

) group. During the experimental period 

the animals showed very little body weight changes, 

which were negligible and not considered in 

partitioning of energy. 

All the three diets (T 1, 

T 2 

and T 3 

) were 

comparable in dry matter intake, digestibility of 

organic nutrient and balances of nutrients (nitrogen, 

calcium and phosphorus), hence the data were 

pooled for eighteen lactating Murrah buffaloes and 

the energy requirement for lactating Murrah 

buffaloes was calculated by using multiple regression 

method. 

The maintenance requirement (Table 4) of 

lactating Murrah buffalo was found to be 49.5 g 

TDN /w 0.75 kg. This was similar to that (49.20 g/ 

Table 1. Ingredient composition of mineral mixtures. 

Ingredients T 1 T 2 T 3 

DCP 31.34 15.67 - 

DAP - 15.67 31.34 

LSP 21.18 33.15 45.12 

Common salt 21.66 21.66 21.66 

TM* 1.87 1.87 1.87 

Urea 14.26 7.13 - 

Filler 9.67 4.84 - 

Ca% 15.34 15.34 15.34 

P% 6.58 6.42 6.26 

*Trace mineral contained cobalt chloride 40 g, copper sulphate 240 g, ferrous sulphate 780 g, manganese 

sulphate 780 g, sodium selenite 8 g and potassium iodide 24 g. 

135


w 0.75 kg) reported by Tiwari and Patle (1983) for 

lactating Murrah buffaloes and 49.85g (Neville and 

McCullough, 1969) for exotic pure breed cows. The 

daily maintenance requirement of ME was 179.4 

kcal /w 0.75 kg, which was very close to 171 kcal ME/ 

w 0.75 kg in milch Murrah buffaloes (Tiwari & Patle, 

1983) and 175 kcal ME/w 0.75 kg in lactating HF cows 

(Ranjhan et al., 1977). 

The energy requirement for production of 

1 kg 4% FCM was 0.346 kg TDN (Table 4). This 

value was very close to 0.323 kg TDN as reported 

by Shtivastava (1970) and 0.340 kg TDN by Kearl 

(1982) in buffaloes. The energy requirement for 

production of 1 kg 4% FCM was 1.252 Mcal ME 

which was similar to the values(1.17 Mcal ME) 

suggested by Shtivastava (1970) in buffaloes, Kearl 

(1982) 1.230 Mcal ME and Sen et al. (1977) as 

1.188 Mcal ME/ Kg 4% FCM in buffaloes. 

Partitioning of energy was also worked out 

and is presented in Table 5. It appeared that about 

64.49% TDN was utilized for maintenance of body 

and 35.51% diverted for milk production. Similarly 

64.55% ME was utilized for maintenance and 

35.45% ME for milk production. 

From the experiment, it was concluded that 

the daily maintenance requirement of energy for 

lactating Murrah buffalo was 49.5 g TDN and 179.4 

kcal ME/w 0.75 kg and for per kg 4% FCM was 346 g 

TDN and 1,252 kcal ME. 

Table 2. Chemical composition of concentrate mixtures and mixed roughage on DM basis (%). 

Particular 

Concentrate mixtures 

T 1 T 2 T 3 

Mixed roughage 

DM 93.34 93.18 93.03 41.37 

CP 19.76 19.69 20.18 5.98 

EE 4.57 4.39 4.81 2.18 

CF 6.30 6.80 6.70 31.18 

Ash 11.50 12.01 12.21 9.14 

NFE 57.87 57.11 56.10 51.52 

Ca 1.17 0.98 1.06 0.30 

P 0.86 0.75 0.83 0.18 

Table 3. Daily nutrient intake, live weight changes and milk production in lactating Murrah buffaloes. 

Particulars T 1 T 2 T 3 Average 

DMI(g/w 0.75 kg) 122.01± 4.96 115.18 ± 5.95 116.16 ± 4.18 117.78 

DCP(g) 784.01±7.01 766.5±8.79 792.00±5.57 780.83 

TDN(kg) 8.70±0.63 9.13±0.48 8.96±0.91 8.93 

ME(Mcal) 31.46±0.75 33.03±0.92 32.41±0.67 32.3 

Gain/loss(g/d) 120.18± 39.91 24.01± 57.98 24.40 ± 85.31 56.19 

4% FCM production 

(kg/d) 

8.43 ± 0.63 8.74 ± 0.48 8.57 ± 0.41 8.58 

136


Table 4. Requirement of energy for maintenance and milk production. 

Energy Maintenance/w 0.75 kg Per kg 4% FCM production 

TDN 49.5 g 346 g 

ME 179.4 kcal 1,252 kcal 

Table. 5 Partitioning of Energy Intake. 

Energy intake 

TDN (kg) 

8.936 

ME (Mcal) 

32.359 

Maintenance 

Milk Production 

Total Percentage Total Percentage 

5.763 64.49% 3.173 35.51% 

20.889 64.55% 11.47 35.45% 

REFERENCES 

A.O.A.C. 1990. Official Methods of Analysis, 15 th 

ed. Association of Official Analytical 

Chemists. Arlington, Va. USA. 

Agarwala, A. C. and R. M. Sharma. 1961. A 

Laboratory Manual of Milk Inspection, 4 th 

ed. Asia Publishing House, Bombay. 

ICAR .1998. Nutrient Requirement of Livestock 

and Poultry, 2 nd Revised ed. Indian Council 

of Agricultural Research. Krishi Anusandhan 

Bhawan, PUSA, New Delhi, India. 

Kearl, L.C. 1982. Nutrient Requirements of 

Ruminants in Developing Countries. 

International Feed Stuffs Institute, Utah 

Agricultural Experiment station, Utah state 

University, Logan Utah, USA. 

Moe, P.W., H.F. Tyrrell and W.P. Flatt. 1970. Energy 

metabolism of farm animals, p. 65. In Schurch, 

A. and C. Wenk. (eds.) Proc. 5 th Symp. 

Energy Metal. Vitzan, Switzerland. EAAP. 

Publ. No. 13. Juris, Verlag Zurich. 

Neville, W. E. (Jr.) and M.E. McCullough. 1969. 

Calculated energy requirement of lactating 

and non-lactating Hereford cows. J. Anim. 

Sci., 29: 823. 

Ranjhan, S.K., D.V. Mohan and R. Singh. 1977. 

Energy and protein requirement of Holstein- 

Friesian and Holstein-Friesian X Hariana 

crosses for maintenance and milk production. 

Indian J. Anim. Sci., 45: 71. 

Sen, K.C., S.N. Ray and S.K. Ranjhan. 1977. 

Nutritive Value of Indian Feeds and Fodder, 

ICAR, New Delhi. 

Snedecor, G.W. and W.G. Chochran. 1989. Statistical 

Method, 8 th ed. The lowa State University 

Press, Ames, lowa, USA. 

Srivastava, J.P. 1970. (Title not available). Ph.D. 

Dissertation, Agra. Univ., Indian Veterinary 

Research Institute, Izzatnagar, India. (cited by 

Ranjhan, personal communication). 

Tiwari, D.P. and B.R. Patle. 1983. Utilization of 

Mahua seed cake by lactating buffaloes. 

Indian J. Dairy Sci., 36: 394. 

137


EFFICIENCY OF UTILIZATION OF DIETARY ENERGY FOR MILK PRODUCTION 

IN LACTATING MURRAH BUFFALOES (BUBALUS BUBALIS) 

Pramod Sharma, R. S. Gupta and R. P. S. Baghel 

ABSTRACT 

Studies were conducted on the efficiency 

of utilization of dietary energy for milk production in 

lactating Murrah buffaloes. Eighteen lactating 

Murrah buffaloes of nearly same body weight 

(551.52±14.63 kg), milk yield, parity and stage of 

lactation were divided into three groups of six 

animals each and were fed 0, 50 and 100% diammonium 

phosphate(DAP) in the mineral mixture 

of concentrates for 120 days. The chaffed mixed 

roughage (berseem + wheat straw) and concentrate 

mixture was fed to supply about 30:70 concentrate 

to roughage ratio on dry matter basis. Tap water 

was available ad lib. A metabolism trial of seven 

days was conducted at the end of experiment to 

study digestibility of organic nutrients and balances 

of energy. Diammonium phosphate did not affect 

the nutrient intake, body weight changes, digestibility 

of DM, CP, EE, NFE and daily milk yield, However 

the digestibility of CF was improved significantly 

(P


was offered to the animals twice daily. The ration 

scheduled was adjusted weekly on the basis of the 

milk production of the buffaloes. All the animals were 

offered weighed amounts of mixed roughage 

(berseem + wheat straw). Concentrate allowance 

was offered in two portions, one at morning 

milking(4.00 AM) and the other at afternoon 

milking(3.30 PM). The concentrate mixture 

consisted of 40 parts crushed maize, 22 parts wheat 

bran, 35.5 parts mustard cake, 2 parts mineral 

mixtures and 0.50 part common salt. Milk records 

were kept for individual cows throughout the 

experimental period. 

Animals were fed experimental rations for 

120 days inclusive of seven days metabolism trial, 

which was conducted at the end of the trial period. 

Faeces and urine were quantitatively collected and 

were preserved for further analysis. Aliquots of milk 

were taken during morning and afternoon for each 

animal. Faeces, urine, feeds, residues and milk were 

analyzed for proximate constituents by AOAC 

(1990) methods. The fat content of milk was 

determined in Soxhlet apparatus (Agarwala and 

Sharma, 1961). The data obtained during experiment 

were analyzed by using randomized block design 

method as described by Snedecor and Cochran 

(1994). 

Table 1. Ingredient composition of mineral mixtures. 

Ingredients T 1 T 2 T 3 

DCP 31.34 15.67 - 

DAP - 15.67 31.34 

LSP 21.18 33.15 45.12 

Common salt 21.66 21.66 21.66 

TM* 1.87 1.87 1.87 

Urea 14.26 7.13 - 

Filler 9.67 4.84 - 

Ca% 15.34 15.34 15.34 

P% 6.58 6.42 6.26 

*Trace mineral contained cobalt chloride 40 g, copper sulphate 240 g, ferrous sulphate 780 g, manganese 

sulphate 780 g, sodium selenite 8 g and potassium iodide 24 g. 

(1989). 

GE of a feed was calculated from its chemical composition as per the formula suggested by Ewan 

GE (kcal/kg) = 4,143+ (56X% EE) + (15X% CP) - (44X% ash) 

139


DE was calculated from the TDN value obtained (1g TDN= 4.4kcal DE). Urine (10%) and methane 

(8%) losses were calculated from DE. 

The gross efficiency of milk production was calculated presuming 1kg 4% FCM contained 750 Kcal 

and 1 kg TDN contained 3600 kcal ME (McDonald et al., 1979) .The gross efficiency of ME of milk production 

was calculated as follows: 

Gross efficiency of milk production = 

750 × FCM ( kg) 

3,600 × TDN 

I 

( kg) 

× 100 

The net efficiency of milk production was calculated by subtracting TDN or ME utilized for the 

maintenance from total energy intake. 

Net efficiency of milk production = 

750 × FCM ( kg) 

ME 

I 

129kcalME 

/ w 

0.75 

− 

kg 

× 100 

Table 2. Chemical composition of concentrate mixtures and mixed roughage on DM basis (%). 

Particular 

Concentrate mixtures 

Mixed roughage 

T 1 T 2 T 3 

DM 93.34 93.18 93.03 41.37 

CP 19.76 19.69 20.18 05.98 

EE 4.57 4.39 4.81 02.18 

CF 6.30 6.80 6.70 31.18 

Ash 11.50 12.01 12.21 09.14 

NFE 57.87 57.11 56.10 51.52 

Ca 1.17 0.98 1.06 0.30 

P 0.86 0.75 0.83 0.18 


The chemical composition of the 

experimental diets (concentrate mixtures) and mixed 

roughage offered to the experimental animals are 

presented in Table 2. Due to replacement of DCP 

at 50% in T 2 

and 100% in T 3 

diet, the chemical 

composition in respect of CP, EE, CF, ash, Ca and P 

content did not vary as compared to the control (T 1 

). 

140


The average body weight, total dry matter 

consumption and dry matter consumption from 

concentrate and roughages did not differ significantly 

among the groups (Table 3). 

The digestibility coefficient of various 

organic nutrients is shown in Table 4. There was no 

significant difference in the digestibility of DM, CP, 

EE and NFE of experimental diets, however the CF 

digestibility of experimental diets T 2 

and T 3 

were 

significantly (P>0.01) higher than control. The 

digestibility of CF of T 2 

and T 3 

did not differ 

significantly from each other. 

Intake of all the nutrients (Table 5) was 

similar in the control (T 1 

) and the experimental 

groups (T 2 

and T 3 

). During the experimental period 

the animals showed very little change in body weight. 

All the three diets (T 1 

, T 2 

and T 3 

) were 

comparable in dry matter intake and digestibility of 

organic nutrient, hence the data were pooled for 

eighteen lactating Murrah buffaloes and the 

distribution of GE and the efficiency of utilization of 

energy for lactating Murrah buffaloes was 

calculated by a factorial method. 

Percentage distribution of gross energy in 

feeds, faeces, urine, methane, milk and heat 

production and tissue deposition is presented in Table 

6. The energy of heat production and tissue 

deposition was calculated as gross energy consumed, 

which was not excreted in faeces, urine, methane 

or milk. 

In the present study out of 54.55 Mcal GE 

intake level, the losses in faeces, urine, methane and 

heat production was 27.92%,7.20%,5.75% and 

47.33%, respectively, leaving behind a net energy 

retention for milk production as 11.79%. 

Krishnamohan et al. (1975b) reported the various 

losses at the same GE intake level (53.1Mcal) as 

37% in faeces, 2.3% in urine, 5.5% in methane, 

14.8% in milk production and 40.4% in heat 

production and tissue deposition in cross bred cows. 

The lower faecal loss in the present study may be 

due to higher DM digestibility. The higher heat 

production in our study may due to higher roughage 

to concentrate ratio (70:30) compared to 50:50 in 

Krishnamohan et al. (1975b) experiment. The net 

energy retention is similar to that reported by 

Krishnamohan et al. (1975b). 

The gross efficiency of ME for milk 

production was 21.32%, which is within the range 

of 19.1 to 30.6% for various types of roughages as 

reported by Krishnamohan et al. (1975b). Kawalkar 

and Patle (1978) reported that the gross efficiency 

of milk production from 18.54 to 20.11% with the 

complete feed compared with conventional type of 

feeding system. 

The net efficiency of ME for milk production 

was 39.56%, which is similar to that(37.57%) 

reported by Nayak and Maitra (1983) during mid 

lactation. They further reported higher (52.24%) 

during early and lower (29.50%) in late stage of 

lactation. Krishnamohan et al. (1975a) reported that 

efficiency of utilization of energy for milk production 

is governed by a variety of factors viz. ration 

composition, environmental temperature and stage 

of lactation. Wayman et al. (1962) showed that high 

environmental temperature caused a significant 

decrease in efficiency of energy utilization for milk 

production. Similarly, low efficiency of energy 

utilization for milk production in our experiment was 

due to the high environmental temperature (average 

38.31 o C) in the month of June during the metabolic 

trial period. 

From the study, it was concluded that out 

of 54.55 Mcal GE intake, the losses in faeces, urine, 

methane and heat production+tissue deposition were 

27.92%, 7.20%, 5.75% and 47.33%, respectively, 

and the net energy retention for milk production was 

11.79%. The gross efficiency of conversion of ME 

for milk production was 21.32%, and the net 

efficiency of conversion of ME for milk production 

was 39.56%. 

141


Table 3. Average consumption of roughage and concentrate on DM basis. 

Particular T 1 T 2 T 3 

Average BW (kg) 538.27 ± 30.39 587.01 ± 28.41 529.30 ± 40.51 

Roughage (kg) 9.51±0.14 9.48±0.30 9.81±0.17 

Concentrate (kg) 4.07±0.08 4.06±0.19 3.81±0.13 

DM intake (kg/d) 13.58 ± 0.22 13.54 ± 0.49 13.62 ± 0.30 

Ratio of concentrate to roughage 

in dry matter consumed 

30:70 30:70 28:72 

Table 4. Digestibility coefficient of organic nutrients. 

Organic nutrients T 1 T 2 T 3 

DM 66.65 ± 1.11 68.81 ± 1.43 68.33 ± 0.89 

CP 63.41 ± 1.07 64.31 ± 2.06 64.36 ± 2.02 

EE 61.87 ± 2.28 62.63 ± 2.77 62.91 ± 2.20 

CF* 64.75 a ± 1.58 71.5 b ± 1.16 72.09 b ± 0.80 

NFE 69.72 ± 1.03 69.73 ± 1.43 70.24 ± 0.95 

a, b : figures with different super scripts in a row differ different significantly (P>0.01) 

Table 5. Daily nutrient intake, live weight changes and milk production in lactating Murrah buffaloes. 

Particulars T 1 T 2 T 3 Average 

DMI(g/w 0.75 kg) 122.01±4.96 115.18 ±5.95 116.16 ±4.18 117.78 

DCP(g) 784.01±7.01 766.5±8.79 792.00±5.57 780.83 

TDN(kg) 8.70±0.63 9.13±0.48 8.96±0.91 8.93 

ME(Mcal) 31.46±0.75 33.03±0.92 32.41±0.67 32.3 

Gain/loss(g/d) +120.18±39.91 +24.01±57.98 +24.40±85.31 56.19 

4% FCM 

production (kg/d) 

8.43 ± 0.63 8.74 ± 0.48 8.57 ± 0.41 8.58 

Table 6. Distribution of gross energy. 

Energy 

GE 

(Mcal) 

FE 

(Mcal) 

DE 

(Mcal) 

UE 

(Mcal) 

Methane 

(Mcal) 

Milk 

(Mcal) 

Heat production 

and tissue deposition 

(Mcal) 

NE Milk 

(Mcal) 

Distribution 54.55 15.24 39.31 3.93 3.14 32.24 25.81 6.43 

% of GE 100 27.92 72.06 7.20 5.75 59.10 47.31 11.78 

142


REFERENCES 

A.O.A.C. 1990. Official Methods of Analysis, 15 th 

ed. Association of Official Analytical Chemists. 

Arlington, Va. USA. 

Agarwala, A. C. and R. M. Sharma. 1961. A 

Laboratory Manual of Milk Inspection, 4 th 

ed. Asia Publishing House, Bombay. 

Ewan, R. C. 1989. Predicting the energy utilization 

of diets and feed ingredients by pigs. p. 271– 

274. In Van Der Honing, Y. and W. H. Close. 

(eds.) Energy Metabolism, European 

Association of Animal Production Bulletin 

No. 43. Pudoc Wageningen, Netherlands. 

ICAR .1998. Nutrient Requirement of Livestock 

and Poultry, 2 nd rev. ed. Indian Council of 

Agricultural Research. Krishi Anusandhan 

Bhawan, PUSA, New Delhi, India. 

Kawalkar, V. N. and B. R. Patle. 1978. Utilization 

of nutrients from complete rations by crossbred 

(Holstein X Tharparkar) lactating cows. 

Indian J. Anim. Sci., 48: 251. 

Krishnamohan, D. V. G., R. C. Katiyar, Q. Z. Hasan 

and S.K. Ranjhan. 1975a. Efficiency of 

utilization of dietary energy for milk production 

in Holstein and Holstein X Hariana crosses. 

Indian J. Anim. Sci., 45: 4-9. 

Krishnamohan, D. V. G., R. C. Katiyar, S. K. Ranjhan 

and P. N. Bhatt. 1975b. Efficiency of utilization 

of metabolizable energy and nitrogen for milk 

production in exotic and cross bred cows fed 

on different roughages. Indian J. Anim. Sci., 

45: 521. 

McDonald, P., R. A. Edwards and T. F. D. 

Greenhalgh . 1979. Animal Nutrition. Oliver 

and Boyd. Edinburgh.1. 

Nayak, J. B. and D. N. Maitra. 1983. Efficiency of 

nutrient utilization for milk production in crossbred 

cows under hot and humid agro climatic 

conditions. Indian J. Dairy Sci., 36: 2. 

Snedecor, G. W. and W. G. Cochran. 1994. 

Statistical Methods, 8 th ed. Oxford and IBH. 

New Delhi. p. 312-317. 

Wayman, O., H. D. Johnson, C. P. Merilan and I. 

L. Berry. 1962. Effect of ad lib. or force 

feeding of two rations on lactating dairy cows 

to temperature stress. J. Dairy Sci., 45:1472. 

143


EFFECT OF DIFFERENT LEVELS OF SUGAR-BEET TAILINGS SILAGE (STS) REPLACING 

MAIZE SILAGE ON MALE BUFFALO CALVES’ PERFORMANCES 

S. Noroozy 

ABSTRACT 

An experiment was conducted as 

completely randomized design on 16 male buffalo 

calves, aged 6 months old and 120+5 kg body weight, 

to determine the effect of different treatments, i.e.; 

0 (T1, control), 25 (T2), 50 (T3) and 75% (T4) of 

STS which are presented annually in large amounts 

(3,000 tons/dry matter bases) in Kuzestan Province 

of Iran substituted with maize silage on buffaloes 

performances in four replications of four animals 

per each replicate. Mean live weight, daily weight 

gain, dry matter intake, and feed conversion 

efficiency were determined, and the results showed 

that there were no significant differences between 

control group, T2 and T3 (P>0.05) for all traits, but 

they showed significant differences with T4 

(P


composition, but still therefore, the objectives of this 

experiment were to determine the chemical 

composition and the proper percentage of treated 

STS replaced with maize silage in fattening buffaloes. 

Studies showed that using urea in sugar beet tops 

silage increased CP and reduced fungal growth, and 

also adding molasses as a source of carbohydrates 

reduced immediately the pH of silage and increased 

its palatability . Therefore, the objectives of this study 

were to determine the chemical composition and the 

proper replacement percentage of maize silage with 

treated STS in fattening of buffalo. 


This experiment was conducted on 16 male 

buffalo calves, aged 6 months old and 120+5 kg body 

live weight for a period of 180 days in a completely 

randomized design with four treatments, 0, 25, 50 

and 75% of STS treated with 1% urea and 3% 

molasses substituted for maize silage. 

In this experiment, different traits such as 

feed consumption (dry matter intake), initial and final 

body live weight, daily body weight gain and feed 

conversion efficiency were studied on male buffalo 

calves. The chemical composition (DM, CP, ME, 

Ca and P) of beet tailings before and after treating 

and ensiling were calculated (AOAC., 1984). In 

vitro digestibility of organic matter of samples (Tilley 

and Terry,1963) were measured to estimate samples’ 

metabolizable energy Using ME(Mj/kgDM) = 

0.016xDOMO (Mc Donald et al., 1995). Tailings 

were collected from a sugar-beet factory and 

transferred to the Safyabad Animal Science 

Research Station. 

They were sun-dried and turned constantly 

for 3-5 days to decrease the moisture content. 

The pH of silage was measured at two 

week intervals. After 45 days, the silo was opened 

and the silage was of good quality. The main 

experiment started and animals adapted to the silage 

for 15 days gradually. 

Different traits such as final live weight, daily 

weight gain, dry matt intake/days and feed 

conversion efficiency were measured during the 

experiment. Finally, all data were analyzed 

statistically using SAS procedure (1985), and the 

means were compared by the Duncan test (1955). 

Table 1. Ingredient constitutes (%) and nutrient composition (as percent in dry matter) of experimental 

diets. 

Experimental diets (%) 

Ingredients 0 25 50 75 

Maize silage 66.66 49.36 33.00 16.48 

STS - 16.32 33.00 49.50 

Alfalfa 1 16.00 16.32 16.32 16.21 

Concentrate 2 17.34 17.68 17.68 17.59 

Total 100 100 100 100 

Calculated nutrient composition 

ME (Mcal/kg) 8.09 8.05 8.00 8.00 

Cp (%) 12.50 12.34 12.15 12.10 

Ca (%) 0.37 0.38 0.37 0.32 

P (%) 0.31 0.30 0.30 0.30 

1- Alfalfa was dehydrated as 95% dry matter 

2- Concentrate includes barley 4%,wheat bran 35%,dry beet-pulp 9%, cotton seed meal 

15% and salt 1%. 

145


RESULT AND DISCUSSION 

The chemical composition and feed values 

of the experimental diets are presented in Table 2. 

Different percentages of maize silage were replaced 

by treated beet tailing silage and the amount of other 

ingredients were almost the same. 

The calculated nutrient composition shows 

that all levels of experimental diets were more or 

less isocaloric and isonitrogenous. The quality of the 

two silages were tested twice a week and after 45 

days both silos were opened and the quality of silages 

such as 65% dry matter, color, lactic acid, smell and 

pH of 4.3 were similar, and both had a very good 

appearance. 

As it is indicated in Table 2, the CP and ME 

of tailings before and after treating and ensiling 

increased from 7.75 to 8.3% and 2.14 to 2.44 Mcal/ 

kg respectively. Calcium did not show any significant 

differences but phosphorous increased from 0.29 to 

0.80 percent. 

The chemical compositions of STS and 

maize silage were almost the same. The only problem 

of silage making was chocking the big pieces and 

the high amount of water in tailings. By pressing 

and adding 10% wheat straw as Reiisian et al. (1996) 

suggested for tops silage, this problem has been 

solved. 

Effects of different treatments on male 

buffalo calves performances are shown in Table 3. 

There were no significant differences (P>0.05) 

between T1,T2 and T3 for final live weight, but the 

differences between these three treatments and T4 

were significant for the same trait (P0.05). There 

was no significant difference between all treatments 

for feed conversion efficiency (P>0.05). 

Table 2. Chemical analyses of the sugar-beet tailings before and after silage, maize silage, alfalfa and 

concentrate. 

Chemical analyzes 

Ingredients DM (%) CP (%) ME (Meal/kg) Ca (%) P (%) 

Sugar-beet tailings 25 7.75 2.14 1.56 0.29 

Sugar-beet tailings silage 68 8.3 2.44 1.85 0.80 

Maize silage 65 8.5 2.53 0.70 0.95 

Alfalfa 92.5 14.5 2.96 1.13 0.39 

Concentrate 95 17 2.60 0.18 0.84 

Table 3. Effects of different treatments on male buffalo calves’ performances. 

Traits 

Treatments (Composition %) 

T1 T2 T3 T4 

Initial live weight (kg) 120.25 a 120.25 a 120.75 a 120.25 a 

Final live weight (kg) 216.25 a 216.00 a 215.50 a 194.50 a 

Daily body weight fain (g) 532.75 a 530.00 a 521.75 a 409.75 a 

Daily dry matter intake (kg) 4.15 a 4.14 a 4.14 a 4.10 a 

Feed conversion efficiency 7.80 a 7.77 a 7.85 a 9.97 a 

a-b Means in each row with different superscripts are significant (P


According to all results the ME was 

increased from 2.14 to 2.44 Mcal, and it seems 

treating tailings increased the amount of lignin, 

cellulose and hemicelluloses of cell walls. Jackmola 

et al. (1993) reported the improvement of organic 

matter of bagasses in sugarcane treated with urea 

and molasses was 24 to 56 percent, and also caused 

protein percentage increase from 7.75 to 8.3%. 

Kardooni et al. (1997) reported that for achieving 

to a good silage with 1% urea the best amount of 

molasses is 3% (DM). Addition of molasses to 

forage silage which has less than 6-8% soluble sugar 

was reported by Church (1988). Reisian Zadeh et 

al. (1996) reported that the moisture of a good silage 

of beet tailings should not be more than 70%, and as 

the moisture of tailings is high, it is better to add 

10% straw to the silage for reduction of silage 

moisture. 

As it is obvious from all results obtained 

from body live weight, daily weight gain, dry matter 

intake and also feed conversion it can be calculated 

that up to 50 percent substitution of maize silage 

with beet tailing silage is recommended. More than 

that as Mc Donald ed al. (1995) reported in his book 

due to its amount of oxalic acid which is bonded 

with calcium and does not pass the body, is not 

suggested, and reduce the growth and feed intake. 

Therefore, it is concluded that 50% of sugar beet 

tailings can replace maize silage for fattening of 

buffalo male calves. 

REFERENCES 

Association of Official Analytical Chemists. 1984, 

AOAC, 14 th ed. Washington, D.C. 

Church. D. C. 1988. The ruminant animal digestive 

physiology and nutrition. p. 527-528. 

Davis, C. L., D. A. Grenawalt and G. C. Mccoy. 

1983. Feeding value of pressed brewers grains 

for lactating cows. J.Dairy sci., 66:153-159. 

Gohi, B. O. 1975. Tropical Feeds. Food and 

Agricultural Organization of the United 

Nations. Rome, Italy. 661p. 

Jakhmola, R. C., R. Weddel and J. F. D. Greenhalp. 

1993. Ensiling grass with straw. Effect of urea 

and enzyme additives on the feeding value of 

grass and straw silage. Animal Feed Sci. and 

Tech. 41: 87-101. 

Kardooni, A and B. Alem Zadeh. 1997. Application 

of different levels of urea and molasses for 

treating pith and bagasses. Publication of 

Agricultural and Natural Resources Research 

Center of Kuzestan, Iran. 

Lardy, G. and V. Anderson. 1999. Alternative feeds 

for ruminants. NDSU (www.ag.nadu.edu) 

McDonald, P., R. A. Edwards, J. F. D. Greenhalgh 

and C. A. Morgan. 1995. Animal Nutrition, 

5 th ed. John Wiley, Inc New York. 

Ranjhan, S. K. 1982. Animal Nutrition in Tropics. 

Vikas Publishing House, New Delhi. 

Reisian Zadeh, M., S. Parsay and J. Pashny. 1996. 

Effect of different methods of ensiling leaf 

and tops of sugar beet for fattening of 

lamb. First Symposium of Animal Nutrition, 

Animal science Research Institute of Iran. p. 

231-239. 

SAS Institute. 1986. SAS, Users Gnide:statistics. 

SAS Inc., Gary, NC. 

Tilley, J. M. and R. A. Terry. 1963. A two stage 

technique for the in vitro digestion of forage 

crops. J.Br.Grassl.Soc. 18: 104-111. 

147

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relationship, point out exception. Show agreement with 

published research work. The significances of work or 

conductions should be presented in the end of discussion. 

Tables. Number each table with Arabic numerals. Place a 

descriptive caption at the top of each table. 

Figures. (graphs, charts, line drawings, photographs) 

Number each figure with Arabic numerals under the 

illustration. Lettering, data lines and symbols must be 

sufficiently large so as to be clearly visible when the figure 

is reduced to a size commonly used in the journal. 

References. List only those references cited in the text. 

Required format of described below. 

Reference cited format 

Manuscripts should follow the name-year reference format. 

Cite only necessary publications. Primary rather than secondary 

references should be cited, when possible. It is acceptable to cite work 

that is “in press” (i.e., accepted but not yet published) with the pertinent 

year and volume number of the reference. 

In text. Cite publications in text with author name and 

year. Three or more authors use “et al.”. In parenthetical citations, 

separate author and year with a comma. Use suffixes a, b and c to 

separate publications in same year by the same author. Semi-colon 

separate citations of different authors. Cite two or more publications 

of different authors in chronological sequence, from earliest to latest. 

For example: 

….used liquid nitrogen vapour freezing technique from Verma et al. 

(1975) 

….liquid nitrogen vapour freezing technique (Verma et al., 1975) 

…and buffaloes (Singh et al., 1983; Shah et al., 1987; Misra, 1996; 

Pant et al., 2002) 

In reference cited. List only those literature cited in the text. 

References should be listed alphabetically by the first author’s last 

name. Single author precedes same author with co-authors. Type 

references flush left as separate paragraphs. Do not indent manually. 

Write the name of book or journal in italic letters. Use the following 

format. 

• Journal articles: Author(s). Year. Article title. Journal title, 

volume number: inclusive pages. 

Example: Citation in text: Chaudhary et al. (1981) 

Choudhary, P.C., B. Prasad and S.K. Misra. 1981. Note on the use of 

rumen liquor in the treatment of chronic alkaline indigestion in 

cows. Indian J. Anim. Sci., 51: 356-360. 

• Books: Author(s) or editor(s). Year. Title. Publishername,Place 

of publication. Number of pages. 

Example: Citation in text: Snedecor and Cochram. (1980) 

Snedecor, G.W. and W.G. Cochram. 1980. Statistical Methods, 7 th ed. 

The Iowa State University Press, Ames, Iowa, USA. 593p. 

Sattar, A. 1995. Studies on the effect of immunopotentiation of 

vaccinated pregnant buffaloes and cows on neonatal antibody 

titre and hematological profile. Ph. D. thesis, University of 

Agriculture, Faisalabad, Pakistan. 208p. 

• Chapter: Author(s) of the chapter. Year. Title of the chapter, 

pages of the chapter. In author(s) or editor(s). Title of the book. 

Publisher name, Place of publication. 

Example: Citation in text: Sloss and Dufty. (1980) 

Sloss, V. and J.H. Dufty. 1980. Disorders during pregnancy, 

p. 88-97. In Sloss, V. and J.H. Dufty (eds.) Handbook of Bovine 

Obstetrics. Williams and Wilkins, Baltimore, U.S.A. 

Sabrani, M., K. Diwyanto and M. Winugroho 1994. A critical 

review of buffalo research and development activities in 

Indonesia. Past performanceand future strategies, p. 78-89. In 

Proceedings of 1 st Asian Buffalo Association Congress, 

Thailand. 

Submission manuscript 

Submit the following items. 

Cover letter. Identify the corresponding author and provide his/her 

full name, address, numbers for telephone and fax, and e-mail address. 

Manuscript. In 12 point Times or Times New Roman. Type on one 

side of A4 paper. Use one inch margins. Number all pages. Send an 

original manuscript and 1 photocopy. 

Disk. Include an IBM-formatted, 3-1/2" disk or 4-3/4" CD-ROM, 

containing the manuscript in Microsoft Word. 

Mail manuscript to: 

By post: International Buffalo Information Center 

Office of University Library 

Kasetsart University, 

50 Pahonyothin Road, Chatuchak, 

Bangkok 10900, Thailand 

Tel. 66-2-942-8616 

By e-mail: libibic@ku.ac.th


CONTENTS 

Page 

Effect of growth hormone on onset of cyclicity, milk yield and blood metabolites 

in post-partum lactating riverine buffalo(Bubalus bubalis). 

A. Mishra, P.K. Pankaj, B. Roy, I.J.Sharma and B.S. Prakash………….…………….….106 

Molecular characterization of β-casein exon 7 gene in buffaloes. 

G. Darshan Raj, Swathishetty, M.G. Govindaiah, C.S. Nagaraja, 

S.M. Byregowda and M.R. Jayashankar……….……..…………...……………..…..…118 

Seasonal variations in breeding and calving patterns of Nili-Ravi buffaloes 

in Azad Kashmir, Pakistan. 

Zulfiqar Hussain ……….……………………………….…………...………..……....……127 

Cardio-respiratory responses of neonatal bovines as affected by 

acetylcholinesterase during their development. 

A.K. Jain, R.K. Tripathi, I.J. Sharma, R.G. Agrawal and M.A. Quadri…………......…….131 

Energy requirements of lactating Murrah buffaloes(Bubalus bubalis). 

Pramod Sharma, R. S. Gupta and R. P. S. Baghel…….……..……………………..…...…134 

Efficiency of utilization of dietary energy for milk production in lactating 

Murrah buffaloes(Bubalus bubalis). 

Pramod Sharma, R. S. Gupta and R. P. S. Baghel………………...……………….….….138 

Effect of different levels of sugar-beet tailings silage(STS) 

replacing maize silage on male buffalo calves’ performances. 

S. Noroozy.........................…………...…………………….………….....................….144 

BUFFALO BULLETIN 

IBIC, KASETSART UNIVERSITY, P.O. BOX 1084 

BANGKOK 10903, THAILAND 

URL : http://ibic.lib.ku.ac.th 

E-mail : libibic@ku.ac.th 

Tel : 66-2-9428616 ext. 344 

Fax : 66-2-9406688

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