The present study aimed to use an in vitro follicle culture (IVFC) biotechnique as a tool to evaluate the influence of whole flaxseed as a feed supplementation in the diet on the in vitro development of caprine early antral follicles (EAFs) and further embryo production. In total, 18 adult goats were homogeneously allocated into two diet groups: Control and Flaxseed. EAFs from both experimental groups (300–400 µm) were isolated and cultured in vitro for 18 days. After IVFC, recovered cumulus–oocyte complexes were submitted to in vitro maturation, and subsequently to IVF and in vitro embryo culture. The endpoints evaluated were follicular growth and morphology, oocyte recovery rate and diameter, sperm penetration, pronuclei formation, embryo development, and estradiol production. The addition of the whole flaxseed in the diet did not affect (P > 0.05) follicular growth and diameter. A higher (P < 0.05) percentage of oocytes ≥ 110 µm was recovered from the flaxseed treatment. However, the sperm penetration rate was higher (P < 0.05) in the control treatment when compared with the flaxseed treatment, but no differences were found regarding the rate of fertilization nor cleaved embryos. In conclusion, dietary flaxseed increased the recovery rate of fully grown oocytes, but it did negatively affect the sperm penetration rate, even though there was no further effect on the cleavage rate.

The aim of this study was to evaluate the influence of two-dimensional (2D) and three-dimensional (3D) alginate culture systems on in vitro development of pre-antral caprine follicles. In addition, the influence of the reproductive age of the ovary donor on the in vitro culture success was investigated. Pre-antral follicles from pre-pubertal or adult goats were isolated and cultured directly on a plastic surface (2D) or encapsulated in an alginate-based matrix (3D). After 18 days, the oocytes underwent in vitro maturation (IVM) and in vitro fertilization (IVF) to produce embryos. The 3D system showed higher rates of follicle survival, lower rates of oocyte extrusion, and a greater number of recovered oocytes for IVM and IVF (P < 0.05). Only pre-antral follicles from adult animals produced MII oocytes and embryos. The estradiol concentrations increased from day 2 to day 12 of culture in all groups tested (P < 0.05). Conversely, progesterone concentrations were lower in 3D-cultured follicles than in 2D-cultured follicles, with differences on days 2 and 6 of culture (P < 0.05). We provide compelling evidence that a 2D or 3D alginate in vitro culture system offers a promising approach to achieving full in vitro development of caprine pre-antral follicles to produce mature oocytes that are capable of fertilization and viable embryos.

To evaluate gene expression of Connexin37 (Cx37) in oocytes from in vitro follicles at different stages, mouse preantral follicles were isolated and cultured for 12 days in vitro. Compared with in vitro follicles, follicles grown in vivo were collected at day 14 (d14), d16, d18, d20, d22 and d24 with the same stages for gene expression of Cx37 in oocytes. Our results showed that Cx37 mRNA increased along with follicular development, reached the highest level at the onset of antrum cavity formation and decreased after antrum formation in both in vivo and in vitro mouse oocytes. However, Cx37 mRNA was significant higher (p < 0.01) in in vitro cultured oocytes than in vivo oocytes. Moreover, significantly higher levels of Cx37 mRNA were found in oocytes from in vitro disrupted follicles (p < 0.01) and non-grown follicles (p < 0.05) than those from normal follicles with a similar size. These data determine temporal gene expression of Cx37 in oocytes from follicules at different stages and indicate that the gene expression level of Cx37 in oocytes could be evaluated as a criterion to the regulatory mechanism of Cx37 in an in vitro model.

Calcium signalling is involved in important events in oocytes, such as meiotic competence acquisition. We have previously demonstrated the positive influence of animal age and gonadotropin stimulation in vivo regarding the ability of oocytes recovered from preantral follicles to exhibit calcium spikes. In the present work we determined whether preantral follicle development in vitro also allows oocytes to acquire calcium signalling activity. We also aimed to verify the influence of animal age, FSH + LH and/or insulin on oocyte calcium spike acquisition during preantral follicle culture. Early preantral follicles were isolated from 12-day-old and 1- to 3-month-old F1 hybrid mice and cultured individually for either 2 or 6 days. At the end of the culture period the oocytes were processed for calcium imaging by confocal microscopy. We show that oocytes recovered from cultured preantral follicles exhibit variable calcium spike activity rates, depending on animal age, culture duration and hormonal supplementation. Oocytes recovered from adult animals continue to exhibit calcium spikes, and those recovered from juveniles acquire that activity after culture. Insulin and gonadotropins in combination account for an early and maintained inhibitory effect on calcium signalling acquisition by oocytes. Insulin alone also leads to an early inhibitory effect, which, however, disappears with longer culture periods. Contrary to the complex in vivo situation, the acquisition of calcium signalling by oocytes in a controlled in vitro environment does not seem to be dependent on gonadotropins alone.