Spermatozoa co-culture does not improve oocyte nuclear maturation rates in dogs

In canids, in order to overcome the limited efficiency of oocyte in vitro maturation1, several attempts mostly investigating different supplementations of the culture media, culture periods, and variation of the conditions over the course of the culture period (so-called bi-phasic media)2 have been made. In vivo, as canine oocytes are ovulated immature and remain an extended period within the oviducts, spermatozoa are present before full maturation occurs. Taken this into account, it was interesting to verify whether the presence of spermatozoa in in vitro culture medium could trigger meiosis resumption and improve maturation rates. For this purpose, ovaries were collected from bitches at different reproductive status (various breeds, age 1-7 years) by routine ovariohysterectomy and sliced in PBS-PVA to release the cumulus-oocyte complexes (COCs). Grade I COCs (n. 405) were obtained and randomly allocated in four systems with the base medium (BM) consisting of TCM-199 with antibiotics, 10% FBS, 2.2 mg/ml sodium bicarbonate and 20 l/ml pyruvic acid. Hormones were supplemented at the dose of 10 i.u/ml hCG (Sigma Chemical Co., MO, USA), 1μg/ml P4 (Sigma), 1μg/ml E2 (Sigma) as follows: in system A (n.110) oocytes were matured for 48 h in BM with hCG and for additional 24 h in BM with P4; in system B oocytes (n.118) were matured for 48 h in BM with hCG, P4 and E2 and for 24 h in BM with P4; in system A-S (n.92) and B-S (n.85) oocytes were cultured in system A and B, respectively, but with the addition of spermatozoa (5 x 106 sperm/ml) at 48 h of culture. COCs were incubated at 38C, 5% CO2 in air. At the end of maturation period (72 h), oocytes were denuded within 0.2% hyaluronidase solution (Sigma) by repeated pipetting and then, were stained with Hoechst 33342 (Sigma) for evaluation of meiotic configuration. Data were analyzed by ANOVA at p<0.05. The results indicated that the last 24 h of co-culture with spermatozoa did not benefit meiosis resumption (GVBD-AI: 65%, 68%, 44.6%, and 49% for systems A, B, A-S, and B-S, respectively) or full maturation rates (MII: 18.2%, 15.3%, 15.2%, and 13% for systems A, B, A-S, and B-S, respectively), p>0.05. The percentage of degenerated oocytes significantly increased with the presence of spermatozoa (9%, 7.6%, 32.6%, and 28.3%, for systems A, B, A-S, and B-S, respectively; p<0.001). The overall sperm penetration occurred both in immature and mature oocytes, however a higher proportion of matured oocytes (60.5%, 23/38) were penetrated compared to the immature ones (22%, 4/18; p=0.0006). We can speculate that the potential influence of sperm penetration could be on cytoplasmic maturation, rather than nuclear maturation. This aspect should be further investigated and the fact that different periods of co-incubation might influence the results. [1] Luvoni GC, Chigioni S, Allievi E, Macis D. Factors involved in vivo and in vitro maturation of canine oocytes. Theriogenology 2005; 63: 41–59. [2] Apparicio M, Mostachio GQ, Motheo TF, et al. Distribution of cortical granules and meiotic maturation of canine oocytes in bi-phasic systems. Reproduction, Fertility and Development 2015; 27:1082–1087.