Dynamic Medium Produces Caprine Embryo From Preantral Follicles Grown In Vitro

Abstract

The aim of this study was to develop a dynamic culture medium containing FSH, LH and EGF to promote the in vitro development of oocytes obtained from goat preantral follicles to complete maturation and to improve the capacity of these oocytes for in vitro fertilization (IVF) and embryo production. For experiment I, preantral follicles were cultured for 18 days in medium supplemented with increasing concentrations of FSH (T1 - control) or in control medium added LH alone or in association with EGF: T2 (LH 50 ng/ml), T3 (LH 50 ng/ml + EGF 50 ng/ml), T4 (LH 50 ng/ml + EGF 100 ng/ml), T5 (LH 100 ng/ml), T6 (LH 100 ng/ml + EGF 50 ng/ml) and T7 (LH 100 ng/ml + EGF 100 ng/ml). For experiment II, preantral follicles were cultured only in the culture medium used in T7, and after 18 days, their oocytes underwent in vitro maturation (IVM) followed by IVF. At the end of the culture period, T3, T4 and T7 had a positive influence on the daily follicular growth rate. Oocytes grown in T4 and T7 had a meiosis resumption percentage significantly superior to the other treatments. Two embryos were obtained, in which preantral follicles in medium supplemented with 100 ng/ml LH and 100 ng/ml EGF (T7). In conclusion, our sequential culture system was able to promote the in vitro growth of preantral follicles, promoting their oocyte maturation and caprine embryo production from preantral follicles.

In vitro culture of goat preantral follicles from fetal ovaries

“…However, among the individual concentrations of the growth factors and hormones, the extrusion of the oocytes from cultured PFs was observed only in GH or EGF supplemented medium (Table 1). There were reports that EGF, FSH, GH and IGF-I induced the growth of small and large PFs of adult sheep (Cecconi et al, 1999;Hemamalini et al, 2003;Tamilmani et al, 2005) and goats (Rajarajan et al, 2006;Matos et al, 2007;Martins et al, 2010;Saraiva et al, 2010Saraiva et al, , 2012Frota et al, 2011;Magalhaes et al, 2012). Similarly, in our earlier studies, T 4 , FSH, IGF-I and GH, individually or in combinations (T 4 + FSH; IGF-I + GH; T 4 + FSH; IGF-I + GH) supported better in vitro growth, antrum formation, extrusion of oocytes and subsequent maturation of oocytes from cultured adult sheep PFs (Arunakumari et al, 2007(Arunakumari et al, , 2010.…”

“…Multiple ovulation and embryo transfer (MOET) and in vitro production (IVP) of embryos using oocytes from antral follicles of slaughter house ovaries has enhanced the utilization of female genetic resources only to a limited extent. In addition to MOET and IVP of embryos from antral follicles, IVP of oocytes/embryos obtained from cultured primordial or preantral follicles (PFs) have the potential to maximize the utilization of female gametes (Eppig and O'Brien, 1996;Wu et al, 2001a, b;O'Brien et al, 2003;Gupta et al, 2008;Arunakumari et al, 2010;Saraiva et al, 2010;Magalhaes et al, 2011). Lack of a repeatable culture system for efficient production of competent oocytes from cultured PFs hampers the maximum utilization of oocytes.…”

Long-term in vitro culture of bovine preantral follicles: Effect of base medium and medium replacement methods

“…However, to our knowledge, in vitro experiments with preantral follicles of cattle are few and with limited success, compared to other species such as goats (Saraiva et al, 2010;Magalhães et al, 2011), sheep (Arunakumari et al, 2010;Luz et al, 2012), and mice (Eppig and O'Brien, 1996;O'Brien et al, 2003). The in vitro follicular culture performance may be affected by different factors such as follicle size (Katska and Rynska, 1998), type of base culture medium (Rossetto et al, 2012), addition of supplements (Figueiredo et al, 1994), gas concentration (Gigli et al, 2006), method of medium replacement (i.e., interval, removal, and addition of culture medium; Araújo et al, 2011), and presence or absence of mineral oil (Fukui et al, 1996).…”

“…The difference between supplementation and conventional medium change methods for estradiol production with use of the ␣-MEM + base medium may suggest there were beneficial impacts of a certain degree of dilution, as well as the removal of content by discarding of the culture media when the conventional media was used, which could be considered harmful to follicle development throughout the 32 days of culture. The renewal of medium throughout the in vitro culture of preantral follicles has been used with success, leading to embryo production in goats (Saraiva et al, 2010;Magalhães et al, 2011), sheep (Arunakumari et al, 2010;Luz et al, 2012), buffalo (Gupta et al, 2008), and monkeys (Xu et al, 2011). However, the periodic renewal of culture medium is laborious and may be detrimental to embryonic development by removing embryo-derived growth factors, changing pH and gas atmosphere, and lowering the temperature of culture medium (Fukui et al, 1996).…”

Goat ovarian follicles express different levels of mRNA for inhibin-ßA subunit and activin-A stimulates secondary follicle growth in vitro

“…To avoid this huge loss of oocytes, some studies (ROMERO & SMITZ, 2009; secondary follicles (~200µm) can grow up to antral stages in a long term culture system. In these studies, the effects of various hormones and epidermal growth factor have been evaluated and recently oocyte maturation and in-vitro embryo production have been described for goats (SARAIVA et al, 2010;. Despite the success of these studies, the in-vitro maturation of isolated preantral follicles and fertilization of in-vitro matured oocytes still have a very low efficiency, resulting in a small production rate of embryos.…”