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

In vitro Survival of Follicles Collected from Domestic Cats&#8217; Ovaries at Different Stages of Oestrous Cycle and Cultured with IGF-1

Optimal conditions for in vitro culture of feline ovarian follicles have not yet been defined. Follicular development is regulated by intraovarian growth factors, as insulin-like growth factor (IGF-1), and during the different stages of the oestrous cycle, follicles are exposed to specific hormonal environments. The aim of this study was to investigate the effect of IGF-1 on in vitro growth and granulosa cell (GC) viability of preantral follicles collected from domestic cats at follicular and luteal phases of the oestrous cycle. Oestrus and ovulation were induced in 12 cats. A total of 39 and 32 follicles collected at the follicular and luteal phases, respectively, were individually cultured in vitro for 6 days in minimum essential medium media supplemented with or without IGF-1 (100 ng/ ml). Follicles collected during the follicular phase and cultured without IGF-1 displayed a significant increase in size and higher GC viability (46.5 \ub1 22.1 lm, 66.7%, respectively) than that of follicles collected at the luteal phase and cultured without IGF-1 (26.7 \ub1 14.4 lm, 50%, respectively; p < 0.05). In contrast, when IGF-1 was added to the culture medium, no differences were observed in size or GC viability between follicles collected at the two phases of the cycle. Nonetheless, follicles collected at the luteal phase and cultured with IGF-1 had a significant increase in their diameter and GC viability (31.9 \ub1 15.9 lm, 63.6%, respectively) than that cultured without IGF-1 (26.7 \ub1 14.4 lm, 50%, respectively; p < 0.05). These data suggest that in vitro growth and GC survival of feline preantral follicles are affected by the oestrous cycle phase, and the IGF-1 exerts a positive effect on follicles collected at the luteal phase

In vitro survival of preantral follicles recovered from queens at different stages of estrous cycle and cultured with IGF-1

OBJECTIVE AND METHODS. In vitro growth of preantral follicles may supply high numbers of competent oocytes that can be destined to in vitro embryo production. However, optimal culture systems that support follicular and oocyte development in vitro have not yet been defined. Ovarian follicular development is regulated mainly by endocrine, autocrine, and paracrine factors and follicles are exposed to specific hormonal environments during the different stages of the estrous cycle. The intraovarian insulin-like growth factor-I (IGF-1) regulates in vivo follicular development and there are important evidences that it stimulates in vitro growth of preantral follicles in domestic animals (1). Feline preantral follicles have been previously cultured in vitro (for review 2) and it has been suggested that IGF-1 enhances oocyte metabolism (3). However, no information are available on the response of preantral follicles recovered in different phases of the estrous cycle to cultural conditions. Thus, the aim of this study was to investigate the in vitro survival of preantral follicles recovered from ovaries of queens in follicular or luteal phase of the estrous cycle and cultured in presence of IGF-1. Twelve queens were housed with 12 hours of light and submitted to estrous induction with IM injection of 100 UI eCG (Novormon\uae- Intervet) and 100 UI hCG (Vetecor\uae- Hertape Calier) 82 hours later (4). Six queens were spayed 96 h after eCG administration (follicular phase), the others 36 h after the hCG injection (luteal phase). Estrous phases were confirmed by vaginal cytology prior to the surgical procedure and macroscopic evaluation of ovarian structures after excision. Preantral follicles surrounded by complete basal membrane and containing more than one layer of granulosa cells were retrieved from excised ovaries and selected as previously described (5). A total of 72 follicles were cultured for 6 days at 38.5 \u2daC and 5% CO2 in air in Minimum Essential Medium (Sigma Chemical Co., USA) with IGF-1 100 ng/ml (Sigma) or without (control). Before and after culture, follicular diameter was recorded and follicular viability was assessed by fluorescein diacetate (FDA, Sigma) staining. Increase (%) in diameter was analyzed by Tukey\u2019s and Fisher\u2019s test, viability rates by Chi-square test (P<0.05). RESULTS. After 6 days of culture, preantral follicles retrieved during follicular phase showed a significant increase in the size and a higher viability rate than those retrieved in the luteal phase of the estrous cycle (18.8% vs.11.5%; P= 0.0001 and 75% vs. 62.5%; P=0.004). However, when IGF-1 was added to the culture medium, follicles retrieved in follicular or luteal phase showed similar increase in diameter (14.9% vs.13.4%; P>0.05) and viability (73.8% vs. 76%; P>0.05). Regardless of the stage of the estrous cycle, overall results showed that the increase in diameter was not different in follicles cultured with or without IGF-1 (14.6% vs. 15.8%; P>0.05), but follicular survival was enhanced when IGF-1 was added to the culture medium (75% vs. 69.4%; P=0.0001). CONCLUSIONS: Present data suggest that in vitro survival of preantral follicles is affected by the estrous stage of the donor and IGF-1 improves survival of follicles retrieved in luteal phase of the estrous cycle. Thus, the hormonal environment of the follicles within the ovary might impact their potential development when isolated and cultured. Further investigations on growth factors are needed to evaluate their effect on follicles with reduced in vitro developmental capability. REFERENCES (1) Giudice LC. Insulin-like growth factors and ovarian development. Endocr Rev 1992; 13: 641-669. (2) Jewgenow K, Paris MCJ. Preservation of female germ cells from ovaries of cat species. Theriogenology 2006; 66: 93-100. (3) Jewgenow K. Impact of peptide growth factors on the culture of small preantral follicles of domestic cats. Theriogenology 1996; 45: 889-895. (4) Villaverde AI, Melo CM, Martin I, Ferreira TH, Papa FO, Taconeli CA, Lopes MD. Comparison of efficiency between two artificial insemination methods using frozen-thawed semen in domestic cat (Felis catus): artificial insemination in domestic cats. Anim Reprod Sci 2009; 114: 434-442. (5) Lima AK, Bezerra MB, Oliveira LC, Figueiredo JR, Silva LDM. Isolamento e caracteriza\ue7\ue3o de fol\uedculos ovarianos pr\ue9-antrais em gatas dom\ue9sticas (Felis catus). Rev Bras Reprod Anim 2003; 27: 396-397

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