Failure of sexing by developmental arrest of bovine embryos in vitro produced with H-Y antisera

Embriões bovinos produzidos in vitro, em estádio de mórula, foram cultivados em meio contendo anticorpos anti H-Y de alto título proveniente de ratos por 24h e, após este tempo, classificados em dois grupos: 1) embriões inibidos em estádio de mórula (classificados como machos) e 2) embriões que se desenvolveram e formaram a blastocele (classificados como fêmeas). O sexo de 311 embriões, distribuídos em três grupos de concentração dos anticorpos, 3%, 5% ou 7%, foi identificado pela reação em cadeia da polimerase. Não houve desvio da proporção entre machos e fêmeas (P>0,05) nos grupos em que se utilizaram os anticorpos anti H-Y, quando comparadas ao grupo-controle, sem adição de anticorpos anti H-Y. Diferentemente dos resultados obtidos utilizando-se embriões bovinos produzidos in vivo, a sexagem com anticorpos anti H-Y de alto título em embriões produzidos in vitro não propiciou sucesso.In vitro produced bovine embryos at morula stage were cultured in medium containing high titer of rat H-Y antisera for 24h. The embryos were classified in two groups: 1) embryos arrested at morula stage (classified as males); and 2) embryos that developed and formed a blastocoele (classified as female). The sex of 311 embryos, divided in three groups of concentration of H-Y antisera, 3%, 5% or 7%, was identified by polimerase chain reaction. The results showed no difference (P>0.05) on sexual deviation in groups in which the H-Y antisera was added, in relation to control group, in which no H-Y antisera was added. In contrast with results obtained with in vivo produced bovine embryos, the sexing of in vitro produced bovine embryos with high H-Y antisera titer did not succed.CNP

Comparative validation using quantitative real-time PCR (qPCR) and conventional PCR of bovine semen centrifuged in continuous density gradient

The 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

Assessment of swim-up and discontinuous density gradient in sperm sex preselection for bovine embryo production

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

Sexagem de espermatozoides bovinos por centrifugação em gradiente descontínuo de densidade de Percoll Sex selection in bovine spermatozoa by using Percoll discontinuos density gradient centrifugation

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.<br>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 12 fractions 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