2011 (SBTE) 25th Annual Meeting Proceedings - International ...

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Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A231 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” ENDOMETRIAL BIOPSY IN MARES IN HEAT REPEATER: IMMUNOHISTOCHEMIC OCHEMICAL ANALYSIS OF T LYMPHOCY YMPHOCYTES AND MACROPHAGES Franciele Basso Fernandes Silva, , Joana Fernandes Eigenheer Moreira, Juliana da Silva Leite & Ana Maria Reis Ferreira UFF, NITEROI, RJ, BRAZIL. Heat repeaters mares bring large economic losses, so the endometrial biopsy has been a major procedure in the evaluation of uterine health in horses, besides being easy, safe, cheap and with minimum distress to the animal (SNIDER et al. 2011, Theriogenology 75, 1567–1581; EIGENHEER-MOREIRA et al. 2007, Pesquisa Veterinária Brasileira 27, 506-512). The aim of this study was to evaluate the presence of T lymphocytes and macrophages in the endometrium of heat repeaters mares during estrus and diestrus. Endometrial biopsies were performed in ten heat repeaters mares, five in estrus and five in diestrus. For the control group were selected and examined four mares sound. After collection, samples were preserved in 10% buffered formaldehyde and subsequently subjected to routine histologic processing to perform the immunohistochemistry (IHC). The IHC technique for identification of T lymphocytes and macrophages was performed with antibody rabbit polyclonal anti-CD3 (A 0452, DAKO, Denmark) at 1:200 dilution and mouse antibody monoclonal anti-antigen myeloid/ histiocytic IgG1 Clone MAC 387 kappa (M0747, DAKO, Denmark) at dilution 1:400, respectively. For analysis of the results was made count of immunostained cells in five random fields and the average number of cells per field in two groups and control group was compared to significance level α = 0.05, through the Student’s t statistic for independent groups. The control group had at the time of diestrus, an average of 9.1 immunostained cells for antibody anti-CD3 and 8.1 to antibody anti-antigen myeloid/histiocytic at the time of estrus an average of 13.2 and 1.6 respectively. Heat repeaters mares assessed during estrus showed a mean of 20.4 cells immunostained for the antibody anti-CD3 and 10.0 for the antibody anti-antigen myeloid/histiocytic and animals assessed during the diestrus had an average of 11.3 and 8.3, respectively. When compared with the control group, the group of heat repeaters mares in diestro showed no statistically significant difference for T lymphocytes (Student’s t-test: t =0.434; g.l.=6; p-value = 0.680) and macrophages (Student’s t-test: t = 0.061; g.l. = 6; p-value = 0.953), however the group observed on estrus time showed statistically significant difference for macrophages (Student’s t test: t = 2.814; g.l. = 4; value-p = 0.048) which was not repeated for T lymphocytes (Student’s t-test: t = 2.068; g.l. = 4; p-value = 0.107). This way, we can conclude that heat repeaters mares in estrus period have increased infiltration of macrophages than observed in healthy mares. Keywords: endometrial biopsy, cd3, mac 387. A232 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” MICRO-FTIR BIOCHEMICAL CHARACTERIZA CTERIZATION TION OF BULL SPERMATOZ OZOA IRRADIA ADIATED BY LOW POWER LASER Thiago Revers ers Dreyer er 1 , Adr driano Felip elipe e Siqueir iqueira 2 , Taciana Deprá Magr agrini 3 , Mayr yra Elena Assumpção 4 , Her erculano Silv ilva Mar artinho 5 & Mar arcella Pec ecor ora Milazzotto 6 1,3,5,6 UNIVERSIDADE FEDERAL DO ABC, SANTO ANDRÉ, SP, BRAZIL. 2,4 UNIVERSIDADE DE SÃO PAULO, SÃO PAULO, SP, BRAZIL. Low level laser therapy increases ATP production and consequently the energy supply to the cell. In sperm cells it could reflect in increased motility, acrosomal reaction and consequently in the fertilizing potential. The present study aimed the biochemical characterization, by Fourier Transform infrared micro-spectroscopy, of bovine sperm cells irradiated by low power laser under different fluences. Semen straws were thawed in a water bath at 37°C for 30 s, washed and diluted in TALP medium to a concentration of 7x10 6 spermatozoa/mL. Samples were placed in Petri dishes for irradiation and during incubation times. Irradiation were done with a diode laser in the red region (ë =633nm) with fluences of 30, 150 and 300mJ.cm -2 (obtained due to sample irradiation with laser power of 5mW for 1, 5 and 10 min, respectively); 45, 230 and 450mJ.cm -2 (7.5mW for 1, 5 and 10 min); and of 60, 300 and 600mJ.cm -2 (10mW for 1, 5 and 10 min). Micro-FTIR analyses were held with samples air dried in a platinum slide after irradiation and 30 min after irradiation. Spectra were acquired in a Varian 620- IR FTIR Spectrochemical Imaging Microscope system, in the range of frequencies 400-4000 cm -1 . For statistical analysis, acquired spectra were divided in three different regions, in the range 900-1300cm -1 (nucleic acids region), 1300-1800cm -1 (proteins and phospholipids) and 2820-3000 cm -1 (lipids). The spectra were baseline corrected and normalized. Statistical analysis were held by principal component analysis (PCA) and by t-student test (P < 0.05) from intensities of the spectral frequencies. Treatments with higher fluencies (300 and 600mJ.cm -2 ) resulted in low intensity DNA bands (1083 cm -1 ), which could be due to the negative effect of higher fluences in the molecule integrity. For the fluence of 300 mJ.cm -2 , spectral intensities showed no statistically significant deviation between the used laser powers. Phospholipids (1740cm -1 ), proteins (1655cm -1 ) and lipids (2852cm -1 ) bands were higher for 600mJ.cm -2 fluence compared to 30, 45 and 60mJ.cm -2 , which showed no statistically significant deviation by the applied methodology. Based on these data, we can conclude that irradiation with fluence of 600mJ.cm -2 lead to alterations of the structure of cellular molecules which may be reflected in sperm metabolism. [Acknowledgment: FAPESP, UFABC, Capes, Departamento de Reprodução Animal da Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo]. Keywords: micro-ftir, low power laser irradiation, bovine. s452
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A233 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” COMP OMPARISON OF MRNA EXPRESSION OF STAT3 T3 AND AKR2 IN BOVINE EMBRYOS WITH 7 AND 11 DAYS AFTER IN VITRO FERTILIZA TILIZATION TION Tatiana da Silv ilva Rasc ascado ado, Mid idyan Dar aroz Guastali, Luis Eduar duardo do Ver ergar gara, a, Rosiar osiara a Rosár osária Dias Mazier aziero, Mateus José Sudano, Daniela Mar artins Paschoal, Bianca Andriolo Monteiro & Fernanda da Cruz Landim Alvarenga UNESP- BOTUCATU, BOTUCATU, SP, BRAZIL. The aim of this study was to evaluate the expression of Stat 3 and Akp2, markers of pluripotency in blastocysts (D7) and hatched blastocysts (D11) with seven and eleven days after in vitro fertilization. The oocytes were matured for 24 h in 5% CO2 in air, after they were fertilized and cultured in SOFaaci for seven and eleven days. The RNA of polls of seven embryos of each developmental stage was extracted with Rneasy Micro Kit (Qiagen) and reverse transcriptase IMPROM II (Promega, MA, USA) was used for the synthesis of complementary DNA (cDNA). The polymerase chain reaction (PCR) was performed with the qPCR Master Mix Gotaq (Promega, MA, USA) using as endogenous control genes SDHA and YWHAZ (Goosens et al., 2005) with 130 and 180 base pairs respectively. For each gene was performed negative control in which cDNA was replaced by nuclease-free water. Were performed 10 repetitions with RNAs from different groups of embryos. For statistical analysis of data from 10 replicates was used the analysis of variance with a significance level of 5%. Stat 3 and Akp2 were expressed in blastocysts (D7) and hatched blastocysts (D11) exhibiting 130 and 80 bp respectively. From the results we concluded that Akp2 and Stat 3 are present in embryos indifferenced, but also in the hatched blastocysts in which MCI has already become a fully differentiated epithelium, the hypoblast and epiblast (VEJLSTED et al., 2006). Keywords: pluripotency, gene expression, PCR. A234 SUPPORTIVE BIOTECHNOLOGIES: CRYOPRESERVATION AND CRYOBIOLOGY, IMAGE ANALYSIS AND DIAGNOSIS, MOLECULAR BIOLOGY AND “OMICS” CORREL ORRELATION BETWEEN STALLION FERTILIT TILITY AND DNA FRAGMENT GMENTATION TION OF FROZEN SPERM ANALYSED BY THE ACRIDINA ORANGE TEST Camila de Paula Freitas Dell´Aqua 1 , José Antonio Dell ´Aqua Junior 1 Marco Antonio Alvarenga 2 & Frederico Ozanam Papa 1,2 1 FACULDADE DE MEDICINA VETERINÁRIA E ZOOTECNIA-UNESP, SÃO PAULO, SP, BRAZIL. 2 FMVZ-UNESP BOTUCATU, BOTUCATU, SP, BRAZIL. Analysis of DNA fragmentation from sperm has helped to identify men with infertility but showing spermiogram apparently normal. Regarding that some stallions do not show normal conception rate when considering frozen sperm, but have normal semen evalution. Thus, the objective of this work was to evaluate the relathionship betweens stallion fertility and the DNA fragmentation index or the tests routinely used in frozen semen analysis, such as: computer analysis of sperm movement (CASA), plasma membrane integrity and morphology N sperm. Thirteen animals of different breeds, nine animals Quarter horses, two Lusitano and two Brazilian Equestrian, were considered. From each animal, a pool of straws was thawed and used for the following analyses: sperm movement assessed by CASA, plasma membrane integrity assessed using the association of two fluorescent probes - carboxyfluorescein and propidium iodide (Harrison and Vickers, 1990, Journal of Reproduction and Fertility, 88: 343-52), morphology by Karras staining, and rate of DNA fragmentation assessed by the Acridine Orange test (Naves et al., 2004. Bioscience Journal, 20:117-124). Statistical analyses were performed by Pearson’s test for analysing the correlation between sperm parameters and fertility rates, with significance level P < 0.05. No correlation was found between sperm movement or morphology and fertility. On the other hand, plasma membrane integrity correlated positively(r = 0.8047, P = 0.0016) with fertility whereas the rate of DNA fragmentation correlated negatively (r = -0.78, P = 0.0025). In conclusion, the acridine orange test for evaluation of DNA fragmentation and the plasma membrane integrity using fluorescent probes showed strong relationship with fertility, and thus are useful for assessing quality of sperm frozen. [The authors thank FAPESP for financial support]. Keywords: DNA fragmentation, frozen semen, fertility. s453
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