262 REPORTS OF IN VITRO PRODUCTION AND PREGNANCY RATES OF BOVINE EMBRYOS PRODUCED BY POST-THAWING SEXED SEMEN

Abstract

Births of predetermined-sex calves illustrates the importance of sperm sexing technology for in vitro production of bovine embryos (Johnson, 2000). Flow cytometry techniques for sperm sexing are usually performed on fresh semen. However, many bulls presenting high genetic value died or became unproductive before development of sperm sexing technologies. The technique of fresh semen sexing and semen reverse sorting method is based on separation of sperm according to differences in DNA content (X or Y). The objective of this study is to examine the blastocysts and pregnancy rates of bovine embryos produced by fertilization with sperm sexed with the post-thawing procedure, called reverse sorting. Oocytes were obtained by ovum pick-up from Dairy Gir, Nelore, and Guzera cows. The COC were transported to the laboratory and matured at 38.5°C and 5% of CO₂ in air, TCM-199 medium with FCS (10% vol/vol), FSH (1.0 μg mL^-1), hCG (50 μg mL^-1), estradiol (1.0 μg mL^-1), sodium pyruvate (0.20 mM), and amicacin (83.4 μg mL^-1). IVF was performed 24 to 26 h after the onset of maturation. Doses of commercialized semen (3 to 5) from the specified breeds were sent to Goyaike Brazil Agropecuária Ltda. The semen sexing process was performed by flow cytometry and semen was returned to the IVF laboratory at 18°C. In the Tecgene laboratory, semen was centrifugated using Percoll gradient and evaluated. After 18 h, the presumptive zygotes were transferred to IVC in SOF medium at 38.5°C and 5% CO₂ in air. The cleavage rate was evaluated 48 h after IVF and embryos were transferred at Day 7. A total of 5213 viable oocytes were obtained from 266 donors, fertilized with 7 different reverse sorted semen samples (1 Dairy Gir, 2 Nelore, and 4 Guzera) resulting in 1333 embryos. From these, 1084 transferred embryos resulted in 260 pregnancies to this time, with 226 females and 34 males. Each bull was evaluated separately according to cleavage rate, production of blastocysts, pregnancy rate, and percentage of females, respectively. Bull 1 presented 100%, 38%, 25%, and 90%; bull 2: 98%, 38%, 21%, and 90%; bull 3: 84%, 23%, 21%, and 84%; bull 4: 87%, 15%, 29%, and 86%; bull 5: 92%, 53%, 70%, and 71%; bull 6: 78%, 15%, 34%, and 71%, and bull 7: 55%, 27%, 50%, and 78%. The average of in vitro blastocysts (26%), the pregnancy rate (24%), and the percentage of females (87%) are similar to rates obtained by commercial fresh semen sexed by Goyaike Brazil Agropecuária Ltda. In conclusion, semen reverse sorting is an alternative method for selecting sex of in vitro production of bovine embryos using thawed semen collected from extinct or unproductive bulls.