81 EFFECT OF IN VITRO CULTURE SYSTEM MODIFICATION USING CR1aa MEDIUM ON EMBRYO DEVELOPMENT AND PREGNANCY RATE IN CATTLE

Abstract

The culture of in vitro matured and fertilized oocytes is a critical step of in vitro production of bovine embryos. Generally, oocytes are co-incubated with sperm in TALP medium containing different additions and then zygotes are transferred to a medium with another composition. At the same time the effect of the medium alteration on the development of early embryos is unknown. Continual adjustment of fertilized oocytes to the changing culture environment may result in a reduction of their developmental potential. The aim of the present study was to compare effects of two different culture systems on the embryo development and subsequent pregnancy rate in cattle. Slaughterhouse-derived cumulus–oocyte complexes were matured for 24 h in TCM-199 supplemented with 10% FCS, 0.2 mM sodium pyruvate, 10 μg mL^–1 porcine FSH, and 5 μg mL^–1 ovine LH. Frozen/thawed sperm from different Russian Black Pied bulls were prepared in Sperm-TALP medium by swim-up procedure. In vitro matured oocytes were co-incubated for 18 h with prepared sperm in the modified Fert-TALP medium containing 10 μg mL^–1 heparin, PHE (20 μM penicillamine, 10 μM hypotaurine, 1 μM epinephrine), and 0.1% MEM nonessential amino acids. The embryo culture was carried out using 2 systems. A total of 340 presumptive zygotes were incubated in CR1aa medium (Rosenkrans et al. 1994 J. Anim. Sci. 72, 434–437) up to Day 5 post-insemination (System 1) and a total of 442 presumptive zygotes were incubated for 24 h in a fresh Fert-TALP medium without PHE and heparin and then cleaved embryos were transferred to CR1aa medium and incubated until Day 5 post-insemination (System 2). Thereupon, the embryos were transferred to a fresh CR1aa medium supplemented with 5% FCS and cultured for 3 or 5 days. The embryo development was evaluated at Days 2, 8, and 10 for cleavage and blastocyst formation and hatching rates, respectively. A portion of blastocysts (of Grade 1 according to IETS classification) obtained at Day 8 were immediately transferred to recipients or were frozen in 1.5 M ethylene glycol and stored in liquid nitrogen until transplantation. The embryo development data (from 6–8 replicates) were analysed by ANOVA and the embryo transplantation data were analysed using the chi-squared test. The cleavage rates did not differ among Systems 1 and 2 and were 63.6–65.7%. On the other hand, the significant differences between culture Systems 1 and 2 were detected in rates of blastocysts (21.9 ± 1.4 v. 28.8 ± 2.8; P < 0.05) and hatched blastocysts (7.2 ± 1.2 v. 12.3 ± 1.6; P < 0.05). The pregnancy rate for frozen embryos was also higher (but not significantly) in System 2 than in System 1 [26.3% (9/34) v. 16.7% (2/12)], whereas for fresh embryos the similar values of the pregnancy rate were observed [on average 42.9% (6/14)]. Thus the additional 24-h culture of zygotes in Fert-TALP medium favourably affects bovine embryo development in vitro.