372 X-CHROMOSOME-BEARING SPERM SEPARATION BY Percoll™ DISCONTINUOUS DENSITY GRADIENT AND SEX RATIO DEVIATION IN IN VITRO PRODUCED BOVINE EMBRYOS

Abstract

The aims of this study were to separate X-chromosome-bearing bovine sperm by discontinuous Percoll ™ (GE Healthcare Bio-Science AB, Uppsala, Sweden) density gradients, validate the sexing of resultant IVF embryos by PCR, replace the bovine fetal serum (BFS) with BSA in the culture medium, to decrease male development advantage, and verify whether the gradient can be used in an IVF laboratory routine. The gradient was prepared by mixing Dubelcco’s Modified Eagle’s Medium (DMEM; Sigma Aldrich, St. Louis, MO, USA) with Percoll™ isotonic solution with 0.3% BSA, for different densities obtained ranging from 1.110 to 1.123 g mL-1, disposed in 3 layers into 15-mL conical tubes. For sexing, 40 million thawed sperm were overlaid on density gradients. The tubes were centrifuged at 500 g, for 15 min, at 22°C. After centrifugation, sperm sediment was used for IVF. For the control group, a Percoll™ 45, 90% gradient was used. The oocytes were selected from ovaries from slaughterhouse and maturated for 24 h in TCM-199 medium. After fertilization, oocytes and sperm were incubated for 20 h in 5% CO2, in humidified air at 38.5°C. Presumptive zygotes were denuded of cumulus cells, and washed in modified SOF medium and then transferred to 500 μL SOF in four well dishes. Embryo culture was carried out under mineral oil in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 38.5°C, and the cleavage assessed at 46 h and development to the blastocyst stage at Day 7. To obtain embryonic cell DNA for sex determination by PCR, 115 embryos of the sexed and 82 of the control group were used. Two pairs of primers of Y-specific sequences were split in two distinct samples. The first pair detected a sequence of 210 bp, and the second one 196 bp of the bovine Y-chromosome. A third one detected an autosomal sequence of 280 bp, indicating the presence of bovine genomic DNA. PCR multiplex was carried out in the same tube with first and third primers and the PCR of the second one was carried out in another tube. The results were analyzed by X2. Of the sexed group, from a total of 373 oocytes, the cleavage rate was 58.2% (n = 217); 35.6% (n = 133) produced embryos; 36.5% (n = 42) were male embryos and the female embryo rate was 63.5% (n = 73). From a total of 268 control oocytes, the cleavage rate was 63.8% (n = 171); produced embryos 37.3% (n = 100); 57.3% (n = 47) were male embryos and the female rate was 42.7% (n = 35). The Percoll™ density gradient for sperm sexing altered the proportion of IVF embryos toward more females. Because of fast and easy preparation, the gradient can be used routinely in an IVF laboratory and also, BSA can replace FBS for the IVF. FAPESP process number 59357-9 and CAPES