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. A011 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY ECHOGENICITY EVAL ALUATION OF TESTICULAR PARENCHYMA IN PREPUBERTAL OVINES Pedr edro Paulo Maia Teix eixeir eira, Dio iogo José Car ardilli, Luciana Cristina Padilha, Car arla Cristina D’ama ’amato, Mar aria Emilia Franc anco Oliv liveir eira, Felip elipe e Far arias Per ereir eira Da Câmara Barros, Leandro Nassar Coutinho & Wilter Ricardo Russiano Vicente FCAV/UNESP, JABOTICABAL, SP, BRAZIL. The aim of this study was to establish an ultrasonographic standard of echogenicity of testicular parenchyma of prepubertal ovine correlationated to testicular biometry. Eight Santa Ines ovines, 6 months old, were evaluated in Jaboticabal, SP, Brazil. An Aquila Vet ultrasound with a 6 MHz linear transducer were used. Images of sagittal, transverse and frontal plans were analyzed of left and right testicles. Using echogenitcity image function (grey scale) of this ultrasound, predeterminated areas of testicular parenchyma were selected. The biometrics evaluations were done in according to CBRA (1998). Data were expressed in mean and standard deviation by one-way test ANOVA (non-parametric) and linear regression for correlationing, using the statistical package Grafped Prisma 4. The testicular parenchymas, in a general way, were observed as homogeneous and with an echogenicity of 53,95±6,5%, 55,70±6,4% and 55,68± 6,4% to right, left and the mean of testicles, respectively. Also, a high correlation between echogenicity and scrotal circumference (P = 0,0027, r2= 0,80), was verified. The study showed accurate and practical way a way of measuring the echogenicity of testicular parenchyma of prepubertal sheep, could be utilized as a complementary method of diagnosis, usually early, and great value for andrological evaluation. Keywords: ultrasonography, ovine, testicle. A012 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY EVAL ALUATION OF PROTAMINE AND TRANSITION PROTEIN GENE EXPRESSION IN BOVINE SPERM CELLS AND TESTICLES Renata Simões 1 , Marcella Pecora Milazzotto 2 , Flavia Regina Oliveira De Barros 3 , Marcia Almeida Monteiro Melo Ferraz 4 , Marcilio Nichi 5 , José Antonio Visintin 6 & Mayra Elena Assumpção 7 1,2 UFABC, SANTO ANDRÉ, SP, BRAZIL. 3,4,5,6,7 FMVZ-USP, SÃO PAULO, SP, BRAZIL. Protamines are proteins found in the nucleus of the sperm that are synthesized and deposited in the DNA of the cell at the end of spermiogenesis. These proteins have the function to compact and protect the chromatin, exerting great influence on human and mice fertility. Several studies have demonstrated the positive relationship between unregulated production of protamines and infertility. In humans and mice protamine 1 and 2 (P1/P2) ratio is important to predict fertility and that 1:1 ratio is ideal. According to Aoki et al. (2005, Human Reproduction 20, 1298-1306) and Suganuma et al. (2005, Human Reproduction 20, 3101-08) when the 1:1 ratio is disrupted, the sperm DNA integrity and embryo development are altered. The inactivation of transition protein 2 (TP2) in mice leads to failure in the protamine 2 transduction and a subfertile phenotype, showing less condensed sperm nuclei, and elevated level of breaks in the DNA. However, in the bovine species, the expression of these proteins is not completely understood. Literature data suggest that protamina 2, in humans, is absent in this species. Thus, this study aimed to determine the expression of protaminas 1 and 2 (P1 and P2) and transition proteins 1 and 2 (TP1 and TP2) in bovine testis and sperm cells. Testis and epididymal sperm cells from post-pubertal bulls (n = 9) were obtained from slaughterhouse. The RNA extraction and cDNA synthesis were performed using commercial kits. The gene expression (P1, P2, TP1 and TP2) was determined by real time RT-PCR, using bovine specific primers and β-actin and GAPDH as endogenous controls. A relative expression software tool (REST) was used to compare all samples of each group. All transcripts were present in all animals and tissues, except P2 and TP2 that were not found in one semen sample. The quantification of mRNA relative expression, in testicles, showed that P1 was 4.5 fold higher than the relative expression of TP1 and 8.9 fold higher than P2. The relative expression of P2 was 41.8 fold lower than TP2 (P < 0.05). For sperm samples, the relative expression of P1 was 4 fold higher than TP1 and 8 fold higher than P2. The relative expression of P2 was 15.3 fold lower than TP2 (P < 0.05). Until now, this is the first work that shows the P2 gene expression in bovine sperm. According to these results, we verified that there was a pattern of expression between P1-TP1 and P1-P2 in the testis and semen samples. However, the relative expression between P2-TP2 did not show a pattern of expression between samples of testis and bovine semen. [Financial support. FAPESP (2010/ 09145-3)]. Keywords: bovine, gene expression, protamine. s342
Acta Scientiae Veterinariae, 2011. 39(Suppl 1): Abstracts - 25th Annual Meeting SBTE-Brazil. August 2011. A013 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY AGE AT PUBERTY IN BOARS SUBMITTED TED TO PORCINE SOMATOTR TROPIN (PST) TREATMENT Camila Piz izoni, Cláudia F. Demar emarco, Ismael Mateus Cavazini, azini, Viviane Rohr ohrig Rabassa abassa, August ugusto Schneider, Ivan Bianchi & Mar arcio N. Cor orrêa rêa UNIVERSIDADE FEDERAL DE PELOTAS, CAPÃO DO LEÃO, RS, BRAZIL. Growth hormone (GH) or somatotropin (ST) is widely used in animal production. Several studies in humans relate the use of exogenous GH to testicular development and age at puberty when there is a deficiency in endogenous GH production. In veterinary applications GH use in healthy animals has also been investigated. The aim of this work was to evaluate the effect of exogenous pST administration on age at puberty in healthy boars. Starting at 22 days of age, fifteen young boars were used (Landrace x Large White): 7 animals in Control Group, that received 0.5 mL of saline solution each 3 days and 8 animals in GH Group, that received 90 µg/Kg IM of pST (Reporcin ® , Zamira Life Sciences Pty Ltd., Australia) also each 3 days. The animals were trained to ejaculate for semen collection three times a week starting at 150 days of age. For the training a dummy sow was used and the boars were allowed to ejaculate and semen was submitted to motility analysis (%). The animals were considered as reaching puberty when motility was higher than 10%. For statistical analysis, means were compared by analysis of variance with Tukey adjustment using the SAS software. Control Group reached puberty at 177.0 (± 3.1) days of age with 47.1% (± 11.2) of motility, while GH Group reached puberty at 182.0 (± 2.0) days with 50.0% (± 8.0) motility. There was no difference for age at puberty (P = 0.19) and motility in the first ejaculate (P = 0.83). In conclusion, growth hormone had no effect on age at puberty when injected in healthy prepuberal boars. Keywords: puberty, boars, growth hormone. A014 MALE REPRODUCTIVE PHYSIOLOGY AND SEMEN TECHNOLOGY EVAL ALUATION OF SPERM MEMBRANE INTEGRITY OF BUFF UFFAL ALOES ( (BUB UBAL ALUS BUB UBALIS ALIS) ) USING DIFFERENT LABOR ABORATORIAL ORIAL TECHNIQUES G. Rocha Silva 1 , Alexandre Rossetto Garcia 2 , Sâmia Rubielle Silva De Castro 3 , Benjamim de Souza Nahúm 2 , Alessandra Ximenes Santos 1 , Arnaldo Algaranhar Gonçalves 1 & Daniel Vale Barros 4 1 UNIVERSIDADE FEDERAL DO PARÁ, BELEM, PA, BRAZIL. 2 EMBRAPA AMAZÔNIA ORIENTAL, BELÉM, PA, BRAZIL. 3 FACULDADES INTEGRADAS DO TAPAJÓS, SANTARÉM, PA, BRAZIL. 4 UNIVERSIDADE FEDERAL RURAL DA AMAZÔNIA, BELÉM, PA, BRAZIL. Simple techniques, reliable and easy to execute are critical to laboratory analysis of semen. The assessment of integrity of sperm membrane is an important element for andrology, because it allows evaluate the cell functionality, even when morphological defects are not previously diagnosed. The eosin used in eosin-nigrosin (EN) seminal staining doesn’t penetrate the sperm presenting intact membrane. However, when membrane are damaged, eosin stains sperm in pink, and nigrosin gives the contrast to visualize the unstained cells (Brito 2007, N Clinical Technique Equine Practice 6, 249-264). The hypoosmotic swelling test (HOST) is able to evaluate the integrity of the cell membrane considering the liquid transportation that occurs when membrane is intact. Consequently, tails become bent when the sperm cell is subjected to hypoosmotic medium (Jeyendran et al., 1992, Archives of Andrology 29, 105-116). These research aimed was to compare the use of EN and HOST in diagnosis of sperm membrane integrity on buffalo raw semen. The experiment was executed at Embrapa Eastern Amazon, in Belém, Pará. Five buffalo bulls had semen collected by artificial vagina, and each bull provided six ejaculates (n = 30). Semen was assessed and each ejaculate was subjected to EN and HOST tests HOST (Jeyendran et al. 1984, Journal of Reproduction and Fertility 70, 219-228). The plasma membrane integrity (%) obtained with EN or HOST was correlated with progressive motility (%). Data were analyzed using t-Test and the level of association between variables was calculated by Pearson correlation (P < 0.01). The percentage of spermatozoa with intact membrane detected by the techniques EN and HOST was 85.4 ± 8.3% and 73.1 ± 10.5%, respectively (P < 0.01). Results were higher than those published by Iqbal et al (2010, Journal of Animal Science 88, 922-925) using EN (69.1 ± 0.6%) and HOST (59.4 ± 0.8%) for analysis seminal of Nili-Ravi buffaloes. The correlation of motility with EN was highly significant and of medium intensity (r=0.66, P < 0.0001), but it was not observed using HOST (r = 0.19, P = 0.31). These findings indicate superior efficiency of EN technique for raw buffalo semen evaluation, when sperm motility is used as a parameter for predicting the potential fertility of an ejaculate. Thus, it was concluded that EN and HOST techniques are reliable, especially the first, which doesn’t require sophisticated equipment to be executed, such as phase-contrast microscope. Therefore, EN is recommended for the analysis of the sperm membrane integrity of buffalo semen, that makes it profitable for laboratory routines. Keywords: eosin-nigrosin, hypoosmotic swelling test, raw semen. s343
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