Evaluation of two methods of in vitro production of ovine embryos using fresh or cryopreserved semen

Abstract

Successful production of high quality blastocysts depends on the use of a culture system that ensures the acquisition of developmental competence by the maturing oocyte followed by an efficient in vitro fertilization. In the present work the effect of FSH and pyruvate in an EGF containing medium for ovine oocyte maturation prior to insemination with fresh (F) or frozen–thawed (FT) semen on embryo developmental competence and cryosurvival was determined. Sheep oocytes were matured in two culture media (M1 and M2, respectively; M1=CM+EGF, n=836 and M2=CM+EGF+pyruvate+FSH, n=850) for 22h and then fertilized using FT or F spermatozoa (M1×FT=371, M2×FT=359, M1×F=353 and M2×F=372, 9 replicates) from Merino rams (n=3). After embryo culture and evaluation, good quality blastocysts (grade 1) were vitrified in OPS. Post-thawed embryo integrity, re-expansion and number of total and viable cells were assessed. Oocyte maturation rates presented no differences (P>0.05) between treatments (M1=87.0±4.1 and M2=86.7±3.9%) as well as embryo developmental rates either for maturation media or semen status. However, fresh semen improved blastocyst quality (grade 1 embryos F=52.5±4.8% and FT=39.0±4.4%, P=0.01). Grade 1 blastocysts presented similar post-thawed integrity and re-expansion rates. After 3h of culture, expansion rates were higher (P=0.05) for M2×F warmed embryos (80.0±8.3%) than for M1×F (54.3±10.4%). Results seem to confirm the existence of a synergistic effect between FSH, EGF and pyruvate upon cytoplasmic maturation of ovine oocytes. Moreover, in vitro fertilization by fresh semen clearly improves ovine embryo developmental competence by enhancing morphological blastocyst quality. The beneficial effect of M2 on cryosurvival was only observed in embryos derived from fresh semen. Therefore these combined strategies enhance embryo cryosurvival.