Viability and growth of feline preantral follicles in vitro cultured with insulin growth factor and epidermal growth factor supplemented medium

In vitro culture of ovarian preantral follicles has emerged as a reproductive technology aimed at obtaining large amount of oocytes for in vitro embryo production. The addition of growth factors (GF) in the in vitro culture of preantral follicles of different species has provided superior results of follicular development, antrum formation and proliferation of granulosa cells. However, there are only few reports regarding the use of these factors on feline preantral follicle in vitro culture. Thus, the aim of this study was to investigate the effect of a combination of IGF-1 and EGF on in vitro viability and growth of preantral follicles and enclosed oocytes collected from domestic cats. A total of 64 follicles characterized by multilayer granulosa cells were isolated and individually cultured for 6\ua0days (T6) in minimum essential medium supplemented with IGF-1+\ua0EGF (100\ua0ng/ml each) or without (control). A higher percentage of follicles were viable after culture with GF than without, and an increase in size when IGF-1+\ua0EGF were added to the medium (170\ua0\ub1\ua032.4\ua0\u3bcm (T0) vs. 201\ua0\ub1\ua022.3\ua0\u3bcm (T6); p\ua0\ua0.05). These data suggest that the addition of IGF-1 and EGF to the culture medium promotes the in vitro development of preantral follicles of cats

Viability of feline preantral follicles in vitro cultured with Insulin Growth Factor and Epidermal Growth Factor supplemented medium

In vitro culture of ovarian preantral follicles (PF) has emerged as a potential reproductive technology for the production of mature oocytes capable of fertilization [1]. Advances concerning the role of several growth factors (GF) on in vitro activation of primordial follicles have been described. The addition of EGF (Epidermal Growth Factor) and IGF (Insulin-like Growth Factor) in the in vitro culture of PF of different species has provided superior results of follicular development, antrum formation and proliferation of granulosa cells (GC) [2]. However, there are only few reports regarding the use of these factors on feline PF in vitro culture. We previously demonstrated that IGF-1 exerts a positive influence on follicular development [3] and has been recently shown that EGF sustains in vitro primordial follicle viability in the prepubertal cat ovary [4]. Thus, the aim of this study was to investigate the effect of a combination of IGF and EGF on in vitro growth and GC viability of PF collected from domestic cats. A total of 64 PF characterized by multi-layer granulosa cells and theca cells with 150\u2013200 \u3bcm of diameter were isolated and individually cultured for 6 days (T6) in minimum essential medium (MEM; Sigma-Aldrich Co., St. Louis, LO, USA) supplemented with IGF+EGF (100 ng/ml each; Sigma) or without GF (control). Data from follicular and oocyte diameters were submitted to Kolmogorov\u2013Smirnov. Mean values of the increase in follicular size were analysed by Student\u2013Newman\u2013Keuls test, and GC viability was analysed by chi-square test (P 0.05; Sigma). The GC of PF cultured with GF maintained a greater viability (fluorescein diacetate staining; Sigma) than those of PF cultured without (84.3% and 68.7% respectively; P<0.05). These data suggest that the addiction of IGF and EGF to the culture medium promotes better conditions to the in vitro development of preantral follicles of cats. It remains to investigate the precise role of the single growth factor to establish optimal culture conditions. [1] Demeestere I, Centner J, Gervy Y, et al. Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents. Reproduction 2005; 130:147-56. [2] Silva J R V, Van Den Hurk R, Figueiredo J R. Ovarian follicle development in vitro and oocyte competence: advances and challenges for farm animals. Domest Anim Endocrinol 2016, 55:123-5. [3] Alves EA, Padilha L, Savi PA, et al. In vitro survival of follicles recovered from queens at different stages of estrous cycle and cultured with IGF-1. Reprod Domest Anim 2012; 47 (Suppl. 6):109-12. [4] Fujihara M, Comizzoli P, Keefer CL, et al. Epidermal growth factor (EGF) sustains in vitro primordial follicle viability by enhancing stromal cell proliferation via MAPK and PI3K pathways in the prepubertal, but not adult, cat ovary. Biol Reprod 2014; 90:86

Distribution of cortical granules and meiotic maturation of canine oocytes in bi-phasic systems

The aim of this study was to evaluate the influence of different bi-phasic systems with gonadotrophins and steroids on in vitro maturation rates of oocytes obtained from bitches at different reproductive stages (follicular, luteal, anoestrous). In System A (control) oocytes were matured for 72h in base medium (BM) with 10IUmL-1 human chorionic gonadotrophin (hCG), 1?gmL-1 progesterone (P4) and 1?gmL-1 oestradiol (E2); in bi-phasic System B oocytes were matured for 48h in BM with hCG and for 24h in BM with P4; in bi-phasic System C oocytes were matured for 48h in BM with hCG, P4 and E2, and for 24h in BM with P4; in System D, oocytes were cultured in BM without hormonal supplementation. Data were analysed by ANOVA. There was a positive effect of the bi-phasic systems on germinal vesicle breakdown, metaphase I and metaphase II rates, irrespective of reproductive status (PP<0.001). The stage of the oestrous cycle did not influence maturation rates