273 BOVINE EMBRYO SEXING IN FIELD CONDITIONS: EFFICACY OF THE POLYMERASE CHAIN REACTION METHOD AND PREGNANCY RATES IN DAIRY HERDS LOCATED IN THE SOUTH AND SOUTHEAST REGIONS OF BRAZIL

Abstract

Currently, artificial insemination with sexed semen is the simplest and most economical strategy for choosing the sex of the offspring. However, sexed semen is not available for certain valuable bulls. Therefore, the use of unsexed semen and subsequent sexing of embryos before transfer into recipients can be an interesting approach to take advantage of these bulls. This study aimed to evaluate the efficiency of embryo sexing under field conditions, as well as the technical performance of dairy farms in the south and southeast regions of Brazil. Day 7 embryos (n = 2447) collected from superovulated (porcine FSH-prostaglandin F_2α protocol) cows kept in 8 herds in the states of Paraná and São Paulo were morphologically classified (grades 1, 2, and 3) and subjected to micromanipulation to remove a biopsy of trophoblast cells. Biopsied embryos were kept on standby in holding medium (TQC, Nutricell, Campinas, São Paulo, Brazil) until sexing results were known. Each biopsy was introduced into individual plastic microtubes containing 8 mL of lysis solution and proteinase K (Invitrogen, Darmstadt, Germany) maintained at 55°C for 5 min, followed by 5 min at 95°C. After this step was added 20 µL of a solution composed of primers specific to the Y chromosome (5′-CCTCCCCTTGTTCAAACGCCCGGAATCATT-3′; 5′-TGCTTGACTGCAGGGACCGAGAGGTTTGGG-3′) and for an autosomal gene (5′-AAGACCTCGAGAGACCCTCTTCAACACGT-3′; 5′-AGGTCGCGAGATTGGTCGCTAGGTCATGCA-3′), deoxynucleotide 5′-triphosphates, reaction buffer, and a Taq DNA polymerase kit (Embrapa, Brasília, Brazil). The samples were transferred to a thermal cycler and subjected to 40 cycles of temperature variation for DNA amplification. After PCR amplification, 20 µL of each sample was applied in 2% agarose gel and subjected to electrophoresis (100 V, 40 mA) for approximately 35 min. After identifying the sex, the biopsied embryos as well as additional intact embryos (n = 3945) were transferred into recipients programmed to be on Day 7 of oestrous. The diagnosis of pregnancy and confirmation of the sex of fetuses were performed by ultrasonography between 60 and 70 days after embryo transfer. Pregnancy rate differences after the transfer of intact and biopsied embryos were analysed by a chi-squared test. The reading of the electrophoresis band for sex identification was clear for 2202 (90%) embryos and doubtful for 245 embryos. The overall pregnancy rates were 55.9 and 58.3% for biopsied and intact embryos, respectively (P > 0.05). Pregnancy rate from sexed Grade 3 embryos (32.5%, 67/206) was lower (P < 0.05) than those from Grade 1 (60.1%, 834/1388) and Grade 2 (53.3%, 463/853) embryos. The accuracy of sex identification (sex predicted by PCR and sex observed by ultrasonography) was 93.2%. These results indicate that embryo sexing by PCR can be used successfully in on-farm conditions.