Effect of maturation medium onin vitrocleavage of canine oocytes fertilized with fresh and cooled homologous semen

Abstract

This study evaluated the effect of three maturation media on the development of in vitro-matured and in vitro-fertilized dog oocytes. In Experiment 1 (non-comparative experiment) canine cumulus-oocyte complexes (COCs) were matured in vitro in TCM199 supplemented with estrous cow serum (10%) + gonadotropins + steroid (treatment A), TCM199 + estrous cow serum (10%) (treatment B), or TCM199 + polyvinylpyrrolidone (PVP) (4%) (treatment C). All maturation media contained a final concentration of 1 microg/ml of human somatotropin (hST). Oocytes were fertilized with fresh ejaculated sperm and development was assessed by cleavage. The objective of Experiment 2 (comparative experiment) was to compare the rates of cleavage and developmental capacity of COCs matured in vitro in same medium as in Experiment 1, and fertilized either with fresh ejaculated or with cooled extended homologous spermatozoa. In Experiments 1 and 2, oocytes fertilized with fresh semen were in vitro-matured for 48 h, while in Experiment 2 COCs fertilized with cooled semen were matured in vitro for 72 h. The results of Experiments 1 and 2 demonstrated that cleavage was not influenced by the oocyte's maturation environment. The results of Experiment 1 showed that pronucleus formation + cleavage (day 7 after IVF) was similar among treatments A, B and C (p = 0.277). Also, in Experiment 2, pronucleus formation + cleavage (day 7 after IVF) was not different for oocytes fertilized in vitro either with fresh or cooled semen and maturated in media A (p = 0.190), B (p = 0.393) or C (p = 0.687). In both experiments, the numbers of embryos that developed to the 6-8-cell stage were higher for oocytes matured in medium A and fertilized with fresh semen, when compared with numbers of oocytes matured in media B and C. Embryo development to the 6-8-cell stage of oocytes fertilized either with fresh or cooled sperm was observed in treatments A and C in Experiment 2. Cumulus cell expansion was similar among treatments in Experiment 1. In Experiment 2, cumulus cell expansion among treatments A, B and C was similar after 48 h or 72 h of IVM. In both experiments, the greatest expansion category seen was for category 2 (outer cumulus cells slightly expanded). No correlation between cumulus expansion and cleavage were observed. Polyspermy rates in oocytes matured in medium A, and fertilized with fresh sperm were not significantly different from polyspermy rates observed using media B and C, in both experiments. Our findings indicate that treatments A, B and C are similarly effective for the cleavage of dog oocytes. Furthermore, it was demonstrated that canine oocytes matured in vitro could be fertilized by homologous cooled spermatozoa and progress to cleavage.