168 CORRELATION OF DEVELOPMENT KINETICS AND SEX OF IN VITRO-PRODUCED BOVINE EMBRYOS

Abstract

Approaches to improve the culture medium for in vitro production (IVP) of bovine embryos have been continuous because of the high commercial demand and a portion of this attempts the production of female cattle (dairy cows and stud cattle). However, in some embryonic in vitro culture systems, the development kinetics is faster in male than in female embryos (Avery 1992 Mol. Reprod. Dev. 32, 265–70; Xu 1992 Mol. Reprod. Dev. 31, 249–50). The aim of this work was to relate the kinetics of blastocyst expansion with the production rates of male and female embryos. Cumulus–oocyte complexes (n = 917; classes I and II) of cows from a slaughterhouse were matured with TCM-199 bicarbonate and 10% FCS (38.5°C, 5% CO₂) for 24 h and fertilized with frozen-thawed semen in TALP-IVF medium for 18 h. Presumptive zygotes were culture in SOF medium supplemented with 10% FSB (5% O₂, 38.5°C). Seven days after IVF, embryos were divided in 2 groups according to their kinetic stage of development: nonexpanded blastocysts (n = 175), or hatched and expanded blastocysts (n = 146). Hence, embryos were individually frozen in LN and stored in cryotubes. After thawing, Proteinase K (16 mg mL^–1) was added to each tube and the tubes were incubated for 60 min at 37°C. Proteinase was denatured at 98°C for 10 min and the contents of each tube were divided into 2 samples (A and B) and subjected to the PCR technique. Two pairs of primers for the specific sequence of the Y chromosome were used to amplify the sequence of 210 and 250 bp for the male bovine and 1 pair of primers was used for the autosomal bovine sequence with a 280-bp fragment. Female embryos with a 280-bp product were observed in sample A and none were observed in sample B. The presence of 2 amplicons (280 and 210 bp) in sample A and 1 amplicon of 250 bp in sample B indicated that the embryo was male. A chi-square test was used to evaluate homogeneity. An analysis of the percentage of males and females between the experimental groups was performed by logistic regression and significance was considered when P < 0.05. There was no difference in the proportions of males and females in the nonexpanded blastocyst group (49.71 and 50.29%; P > 0.05). In the hatched and expanded blastocyst group, the proportion of males (65.75%) was statistically different from the proportion of females (34.25%); that is, the chance of the embryo being male was twice as high (P < 0.0038). These results suggest that there is a difference in the kinetics of embryo development between male and female embryos and that blastocyst expansion can point that out. In vitro culture media with FCS support the development of expanded male blastocysts. Further research in culture medium modifications (FCS, the energy source, amino acids and others) are needed to respond to the trend in the production of sex-defined embryos.