153 EFFECTS ON SEX RATIO AND PREGNANCY RATES OF IN VIVO-DERIVED BOVINE EMBRYOS USING LOOP-MEDIATED ISOTHERMAL AMPLIFICATION SEXING METHOD

Abstract

In this study, we examined the effects of developmental stages and quality grades on sex ratio of in vivo-derived bovine embryos. Furthermore, pregnancy rates of fresh frozen-thawed sexed embryos or intact (non-sexed) fresh and frozen-thawed embryos were compared in order to efficiently carry out the sexing of embryos in the field. Embryos were collected from donors at 7 days after estrus following a routine superovulation protocol, and classified into four stages (late morula, early blastocyst, blastocyst, and expanded blastocyst) and two quality grades (Grade 1 and Grade 2–3) by the IETS manual. Embryos were frozen by direct transfer method from 1 to 3 h post-collection in 0.25-mL straws as described previously (Aoyagi et al. 1996 Theriogenology 45, 165 abst). Frozen embryos were thawed in 30°C water for 20 s following 7 s in air. They were then squeezed out into PBS + 5% FCS (PBS), washed twice, and incubated in CR1aa + 5% CS (CR1aa) or PBS. Recently, a commercial embryo sexing program was performed at our laboratory using loop-mediated isothermal amplification (LAMP). The procedure takes 5 min to perform each embryo biopsy and only 40 min for the LAMP process. A few cells of fresh (F; n = 105) and frozen-thawed (Z; n = 143) embryos of Grade 1 (H), and fresh (F; n = 77) embryos of Grade 2–3 (L) were biopsied with a microsurgical blade, and sex was determined by the LAMP method. Embryos were transferred non-surgically into heifers on Day 7 of the estrus cycle. Pregnancies were determined by ultrasonography on Day 30. Data were analyzed by the chi-square test. The sexing of all 325 embryos yielded 148 female (46%), and only 2 embryos were indeterminant (1%). There was no evidence of any effect of developmental stage on sex ratio (female embryos: late morula 69/157 (44%), early blastocyst 42/94 (45%), blastocyst 29/53 (55%), and expanded blastocyst 10/21 (48%)). However, when the sex ratio was examined for embryos of different quality grades, significantly more females were found in the embryos appearing more degenerated (female embryos: FH + ZH vs. FL; 42% vs. 57%, P < 0.05). Pregnancy rates on Day 30 with FH embryos (38/45, 84%) were similar to rates obtained with non-sexed fresh (60/81, 78%) and frozen-thawed embryos (44/54, 82%). The pregnancy rates on Day 30 with ZH embryos incubated in CR1aa (18/40, 45%) were lower than those of FH, non-sexed fresh, and frozen-thawed embryos. However, pregnancy rates of ZH embryos incubated in PBS (13/16, 81.3%) were significantly higher than for those frozen embryos that were thawed and incubated in CR1aa (P < 0.05). After the transfer of embryos sexed by the LAMP method to recipient animals, all 55 calves born were of the predicted sex. In conclusion, the present results showed that with the LAMP method for sexing of the embryos, there were only a few samples for which sex could not be determined. Examination of in vivo-derived Day 7 embryos indicated that female embryos graded lower than male embryos. Furthermore, the removal of a few cells from a fresh or frozen-thawed embryo did not impact its subsequent viability.