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.

Altered Cell Cycle Gene Expression and Apoptosis in Post-Implantation Dog Parthenotes

“…Although successful cloning of dogs was first reported in 2005 [32], conventional assisted reproductive technologies such as in vitro oocyte maturation, fertilization and embryo development are not available in dogs due to lack of efficient protocols [40], [41], [42]. Because of this, knowledge of preimplantation events in this species is very limited.…”

Kinetics of Nuclear Status and Kinase Activities During In Vitro Maturation of Canine Oocytes

“…Oocyte preparation and IVM were carried out following the procedure described by Rodrigues et al [10] with some modifications. Ovaries within the ovarian bursa were harvested from more than 30 bitches at different stages of the oestrous cycle at local veterinary clinics.…”

“…Many researchers have reported the in vitro maturation (IVM) of canine oocytes, and the discussion in the literature now focuses on how to improve development. For example, the age of the donor bitch [3], oocyte diameter [4,5], protein [6][7][8][9][10] and hormonal supplementation of maturation media [11][12][13][14][15][16] have all been examined. However, the proportion of MII oocytes remains low (0-43.4%) [9,[15][16][17][18][19][20], and a number of essential issues have yet to be resolved.…”

The in vitro culture supplements and selected aspects of canine oocytes maturation

“…In vitro culture (IVC) systems differ between each species of mammals in the type of various media application as well as with regards to different supplements (Hewitt et al 1998, Rodrigues et al 2007, Bukowska et al 2008. Various media supplements lead to different efficiency of in vitro maturation (IVM), which is presented by different species specific number of oocytes that reach the MII (metaphase II) stage.…”