271 USE OF SEXED SEMEN FOR BOVINE IVF IN MEXICO: PRELIMINARY RESULTS

Abstract

The use of sexed embryos in dairy cattle is useful for the genetic and economic improvement of production. The aim of this study was to determine differences in in vitro maturation (IVM), in vitro fertilization (IVF), and in vitro development (IVD) with the use of sexed and unsexed sperm. Semen from one Holstein bull was used for the experiment. The semen was sexed and frozen by X-Y, Mexico (subsidiary of X-Y Inc., Fort Collins, CO, USA). Only the X fraction of spermatozoa was sorted and frozen in 0.25-mL plastic straws with 2.0 × 10⁶ spermatozoa/straw. A modified protocol for IVF was used. A total of 334 ovaries were obtained from a local slaughterhouse and transported to the laboratory in physiological saline (25°C). From these, 1019 cumulus-oocyte complexes (COCs) were obtained and used for the procedures of IVM, IVF, and IVD. The average number of follicles aspirated per ovary was 3.6, and an average of 3.05 COCs were recovered per ovary. The oocyte recovery rate was 85%. For IVM, COCs were incubated in TCM-199 supplemented with BSA, pyruvate, FSH, and LH for 24 h. All incubations were performed at 38.5°C in a humidified atmosphere of 5% CO₂ in air. After this period, COCs were placed in fertilization medium (TALP supplemented with BSA, heparin, penicillamine, and hypotaurine). For IVF, oocytes were randomly assigned to two groups: sexed semen (Holstein) or treatment group (TG), and non-sexed semen (Brahman, used as control in our laboratory) or control group (CG). For insemination, frozen-thawed semen from the Holstein and Brahman bulls was washed by centrifugation in two concentration gradients of a silicone solution. In both groups, sperm concentration used for IVF was 1 × 10⁶ spermatozoa/mL. After insemination, oocytes and semen were co-incubated for 18 h. For IVD, presumptive zygotes were incubated for 7 days in a modified IVD medium (Barc-1) supplemented with BSA. The degree of IVD was evaluated according to the number of divisions of the embryos produced, and the number of embryos that developed to the morula and blastocyst stages. Five replicates were made. The rates of IVM, IVF, and IVD were analyzed by logistic regression. The COCs produced 890 fertilized oocytes. Of these, 442 were from the TG and 448 from the CG. A total of 393 embryos from the TG and 372 from the CG developed in vitro; embryos were evaluated for development on Day 7. A total of 108 morulae (21%) were produced in the TG and 100 (19%) in the CG, whereas 99 (19%) blastocysts developed in the TG and 105 (20%) in the CG. There were no statistically significant differences between the two groups studied for embryo IVD (P > 0.05). It is concluded that IVM, IVF, and IVD procedures used for conventional non-sexed semen can be used for similar results with sperm sexed by flow cytometry. This is the first report of sexed semen use for bovine IVF in Mexico and is a precedent for future investigations on in vitro embryo production in Mexico. More experiments are needed to confirm these preliminary findings.

Sexed semen was provided by Rancho El Nacimiento, Establo 196, Tizayuca, State of Hidalgo, México. Funding for J. A. was provided by CONACYT and UNAM.