The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

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Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient [Validação comparativa utilizando PCR quantitativo em tempo real (qPCR) ABSTRACTThe objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR) of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05). The percentage of female embryos in the Percoll and OptiPrep groups was 62.0% and 47.1%, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.

The effectiveness of induction of the acrosome reaction (AR) test as a parameter to in vitro estimate embryo production (IVP) in Nelore breed and the AR pattern by the Trypan Blue/Giemsa (TB) stain were evaluated. Frozen semen samples from ten Nelore bulls were submitted to AR induction and were also evaluated for cleavage and blastocyst rates. The treatments utilized for AR induction were: control (TALP medium), TH (TALP medium + 10μg heparin), TL (TALP medium + 100μg lysophosphatidylcholine) and THL (TALP medium + 10μg heparin + 100μg lysophosphatidylcholine). Sperm acrosomal status and viability were evaluated by TB staining at 0 and after 4h incubation at 38°C. The results obtained for AR presented a significant difference (P<0.05) in the percentage of acrosome reacted live sperm after 4h of incubation in the treatments that received heparin. The cleavage and blastocyst rates were 60% and 38% respectively and a significant difference was observed among bulls (P<0.05). It was founded a satisfactory model to estimate the cleavage and blastocyst rates by AR induction test. Therefore, it can be concluded that the induction of the AR test is a valuable tool to predict the IVP in Nelore breed.Keywords: cattle, fertility, semen, acrosome reaction, fertilization Palavras-chave: bovino, fertilidade, sêmen, reação acrossomal, fertilização RESUMO Avaliou-se a eficiência da técnica de indução da reação acrossomal (RA) como parâmetro para estimar a produção in vitro (PIV) de embriões Nelore e analisou-se o padrão de RA pela técnica de coloração Azul de Tripan/Giemsa (TB). Amostras de sêmen congelado de dez touros foram submetidas à indução da RA e avaliadas quanto a taxa de clivagem e blastocisto. Os tratamentos utilizados para indução da RA foram: controle (meio TALP), TH (meio TALP + 10μg heparina), TL (meio TALP + 100μg lisofosfatidilcolina) e THL (meio TALP + 10μg heparina + 100μg lisofosfatidilcolina

The purpose of this work was to associate the modified swim-up method with centrifugation in density gradient for the separation of X-bearing spermatozoa. Sperm viability and integrity were evaluated through the Trypan Blue/Giemsa staining method. Quality control of centrifuged spermatozoa was performed in in vitro produced embryos. The results were validated by the sex ratio of in vitro produced embryos using PCR by Y-specific sequences present in bovine male genomic DNA. After determining genetic sex of in vitro produced embryos, the results showed difference (P<0.05) in deviation of sex ratio when comparing the control group (45.2% females) with the other spermatozoa selection procedures (60.6% females) (P<0.05). The sperm selection methods are capable of selecting X-bearing spermatozoa without compromising the spermatozoa fertility (cleavage and blastocyst rates, 70% and 26%, respectively) and were considered relevant methods to be introduced in bovine in vitro produced embryo programs.Keywords: density gradient centrifugation, embryo sex ratio, Percoll™, swim-up RESUMO

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