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.

RESUMO -O objetivo neste trabalho foi desenvolver um método de seleção do sexo de espermatozoides bovinos por centrifugação em gradiente de densidade de Percoll. Utilizou-se sêmen congelado de touros mantidos em regime de colheita de sêmen. A fração de espermatozoides X ou Y foi separada por centrifugação em treze diferentes gradientes de densidade de Percoll formados por 1 a 12 camadas com densidades que variaram de 1,004 g/mL a 1,123 g/mL. As soluções com diferentes densidades foram preparadas misturando-se, em proporções diferentes, meio de cultura Hank's e uma solução estoque composta de NaCl 1,5 M e Percoll (1:9, v/v). Sobre cada gradiente foi colocado um total de 50 × 10 6 espermatozoides descongelados em 0,7 mL de meio Hank's e centrifugados a 250 X g por 30 minutos, em rotor horizontal, a 25°C. Os espermatozoides das frações superior e inferior foram tratados com Quinacrina Mustarda e analisados (200 deles) quanto à presença do corpúsculo-F. Dos espermatozoides encontrados no sedimento de dois gradientes, compostos de 8 e 12 frações com densidades variando entre 1,050 a 1,120 g/mL e 1,044 a 1,123 g/mL, respectivamente, visualizaram-se 25% com corpúsculo-F e os 75% restantes prováveis portadores do cromossomo X. O aumento na porcentagem de espermatozoides X após a centrifugação em gradiente de densidade permitirá que esse método de sexagem seja usado em larga escala na produção comercial de carne e leite bem como no teste de progênie. Palavras-chave: Bos taurus, seleção do sexo, sêmenSex selection in bovine spermatozoa by using Percoll discontinuos density gradient centrifugation ABSTRACT -The objective of this work was to develop a bovine spermatozoid sex selection method by using Percoll density gradient centrifugation. It was used frozen semen of bulls kept in semen collection regime. Fraction X or Y was separated by centrifugation in three different Percoll density gradient formed by 1 to 12 layers with densities varying from 1.004 g/mL to 1.123 g/mL. Solutions with different densities were prepared by mixing, at different proportions, Hank's culture medium and a stock solution composed of NaCl 1.5 M and Percoll (1:9, v/v). On each gradient, it was put 50 × 10 6 spermatozoids thawed in Hank's medium and centrifuged at 250 X g for 30 min in a horizontal rotor, at 25°C. The spermatozoids in the superior and inferior fractions were treated with Mustard Quinacrine and analyzed (200 spermatozoids) for the presence of F-body. Of the spermatozoids found in the sediment of the two gradients, composed of 8 and 12fractions with densities varying from 1.050 to 1.120 g/mL and 1.044 to 1.123 g/mL, respectively, it was identified 25%with the F-body and the other 75% were probably X chromosome-bearing spermatozoids. Increase in percentage of the X-spermatozoid after density gradient centrifugation will allow this system of spermatozoid sexing to be used in large scale in commercial production of meat and milk as well as for progeny tests in bovines. IntroduçãoA sexagem de espermatozoides tem valor econômico signifi...

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

The low cost of sperm sexing methods combined with in vitro embryo production in genetic improvement programs can increase the profitability of cattle production, in particular when it does not decrease reproductive efficiency. The aim of this work was to evaluate the sex ratio deviation of thawed bovine semen processed by density gradient centrifugation and swim-up. Semen doses were collected from ten bulls of different breeds, and each experimental group was replicated ten times.A Percoll™ gradient was prepared by mixing Dulbecco’s Modified Eagle Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) isotonic solutions with Percoll™ (GE Healthcare Bio-Science AB, Uppsala, Sweden) stock solution with 0.3% BSA, resulting in densities ranging from 1.110 to 1.123 g mL-1. The layers of discontinuous density gradient were disposed from the larger density (bottom of the tube) to the smaller into 15-mL conical centrifuge tubes. About 40 million sperm were overlaid on Percoll™ gradient and were centrifuged at 500 g for 15 min, at 22°C.The thawed semen samples were deposited in 15-mL conical centrifuge tubes containing 5 mLof DMEM and centrifuged twice at 300 g for 5 min for extender removal. After the second centrifugation, the supernatant was discarded and the sediment diluted in 1.0 mL of DMEM supplemented with 0.3% BSA. The tube was maintained in an incubator at 38.5°C and 5% CO2 for 1 h. One milliliter of supernatant was recovered and evaluated for the sperm motility and vigor. Eighty million sperm were submitted to the swim-up method. For the association of density gradient and swim-up, the swim-up supernatant was recovered and overlaid on Percoll™ gradient. The gradients were centrifuged and the sperm pellet recovered. The recovered sperm were submitted to DNA extraction with phenol-chloroform. Quantitative Real Time PCR (Parati et al. 2006 Theriogenology 66, 2202-2209) was used for the determination of the proportion of X-chromosome-bearing sperm, after centrifugation through density gradient and after swim-up. The results of amplification were analyzed in 7500 Sequence Detection System Software (Applied Biosystems, Foster City, CA, USA), using Relative Quantification (Ct) Study assay. The results of X-sorted sperm samples analyzed by multiple pairwise comparisons (Tukey) were not different (P > 0.05). The percentage of X-chromosome-bearing sperm after the density gradient comprised 50.3% of the sample, after the swim-up was 49.9% and after swim-up combined with Percoll™ gradient centrifugation was 56.6%. Therefore, sperm sex selection using Percoll™ gradient centrifugation and swim-up was not effective.

ResumoObjetivou-se determinar o efeito e o melhor nível de inclusão de ácido ascórbico em meio diluidor TRIS-Gema para criopreservação de sêmen caprino. Foram utilizados cinco ejaculados de três bodes da raça Anglo Nubiana. Cada ejaculado foi dividido em quatro alíquotas de 200 µL, compondo quatro tratamentos: um sem adição de ácido ascórbico (controle) e os demais com 0,0528; 0,1056 e 0,1584mg/mL de ácido ascórbico no diluidor TRIS-Gema. Avaliaram-se motilidade espermática progressiva e vigor espermático pós-diluição, pós-resfriamento e pós-descongelamento; integridade de membrana (HOST); integridade acrossomal e teste de termorresistência lento (TTR). Os dados foram submetidos à Análise de Regressão a 5% de probabilidade. Não houve diferença para motilidade progressiva e para vigor espermático, repectivamente, na pós-diluição; pós-resfriamento e pós-descongelamento, assim como para os testes complementares HOST e após 120 minutos do TTR, para motilidade e vigor, no pós-descongelamento (P>0,05). Houve comportamento quadrático com a inclusão do ácido ascórbico para integridade acrossomal (61,58%), com nível ótimo de 0,1006mg/mL de ácido ascórbico (P<0,05). A inclusão de 0,1006mg/mL de ácido ascórbico no diluidor TRIS-Gema melhorou a integridade acrossomal durante o processo de criopreservação, podendo ser uma alternativa na composição dos diluidores seminais caprinos. Palavras-chave: antioxidantes; espermatozoide; estresse oxidativo; TRIS-gema. AbstractThe objective of this study was to determine the optimal inclusion level of ascorbic acid in TRISyolk extenders for cryopreservation of goat semen. We used five ejaculates of three Anglo Nubian goats. Semen from each animal was divided into four 200μL aliquots, composing four treatments: no

Resumo:Este trabalho teve como objetivo avaliar a eficiência de gradientes de densidade contínuos de Percoll e OptiPrep para a separação de espermatozoides portadores do cromossomo X, e avaliar a influência sobre a viabilidade espermática, taxa de prenhez e proporção do sexo. Para isso, 100 milhões de espermatozoides foram depositados em cada tubo contendo o gradiente de densidade, previamente preparados, em tubos de poliestireno, 24 horas antes da centrifugação e mantidos a 4 °C. Centrifugou-se a 500 x g por 15 minutos a 22 °C e, posteriormente, os sobrenadantes foram aspirados e os espermatozoides recuperados no fundo dos tubos. Uma amostra foi avaliada antes a após a centrifugação para verificação da motilidade e vigor espermático. Os espermatozoides foram utilizados para a inseminação artificial em tempo fixo. Os resultados indicaram que a centrifugação não afetou a viabilidade espermática e a taxa de prenhez, entretanto não houve desvio da proporção do sexo para fêmeas. Mais estudos são necessários para determinar a necessidade de adaptação da técnica de centrifugação para a espécie ovina e assim obter o desvio da proporção do sexo como na espécie bovina. Palavras-chave: carneiro; sêmen; sexagem; viabilidade espermática. Abstract:The objective of this study was to evaluate the efficiency of continuous density gradients of Percoll and OptiPrep for separation of X-bearing sperm, and assess the influence on sperm viability, pregnancy rate and sex ratio. With this purpose 100 million spermatozoa were deposited into each tube containing density gradient, which were previously prepared in polystyrene tubes, 24 hours