Effects of ascorbic acid on in vitro culture of bovine preantral follicles.

The objective of this study was to evaluate the effects of adding ascorbic acid to the media for in vitro culture of cattle ovarian fragments and to determine their effects on growth activation and viability of early-stage follicles. The ovarian cortex was divided into small fragments; one fragment was immediately fixed (control) and the other fragments were cultured in minimum essential medium (MEM) supplemented or not with various doses of ascorbic acid. Ovarian tissue was processed for histology, transmission electron microscopy (TEM) and immunohistochemical demonstration of proliferating cell nuclear antigen (PCNA). Compared with control fragments, the percentage of primordial follicles was reduced (p < 0.05) and the percentage of growing follicles had increased (p < 0.05) in cultured cortical fragments, independent of the tested medium or incubation time. Furthermore, compared with control tissue, culture of ovarian cortex for 8 days reduced the percentages of healthy, viable follicles (p < 0.05), but not when cultures were supplemented with 25, 50 or 100 µg/ml of ascorbic acid. Ultrastructural and immunohistochemical analysis of 8 day cultured ovarian cortical fragments, however, showed the integrity and viability of follicles only when fragments were cultured in presence of 50 µg/ml of ascorbic acid. In conclusion, this study demonstrated that addition of ascorbic acid to MEM at a concentration of 50 µg/ml not only stimulates the activation of 8 day in vitro cultured cattle primordial follicles and subsequent growth of activated follicles, but also safeguards the viability of these early-stage follicles.

Effects of IAA in combination with FSH on in vitro culture of ovine preantral follicles.

Ovarian cortical fragments from five adult ewes were in vitro cultured for 1, 3 or 5 days in the presence of minimum essential medium either supplemented or not by follicle-stimulating hormone (FSH) (100 ng/ml) or indole-3-acetic acid (IAA) (10, 20, 40 or 100 ng/ml), alone or in combination. After in vitro culture, ovarian fragments were submitted to follicular isolation and viability test was performed using trypan blue. Addition of IAA (10 ng/ml) to a free-FSH medium resulted in the highest percentages of viable follicles, but was progressively deleterious in higher concentrations (20, 40 and 100 ng/ml) if in absence of FSH. Follicular development was observed only when FSH was added to an IAA-free medium. In conclusion, IAA at a concentration of 10 ng/ml increases follicular survival in vitro. However, at high concentrations (20, 40 or 100 ng/ml), this auxin may be deleterious to preantral follicles, the addition of FSH to the medium being necessary.

Trimethylation of histone 3 at lysine 4 in cryopreserved bovine embryos produced in vivo with sexed semen.

The production rates of viable embryos using sexed semen through the conventional methodologies of multiple ovulation and embryo transfer are generally not satisfactory. However, the cryopreservation of these embryos is considered efficient. Knowledge of epigenetics can provide new tools or allow for adapting new protocols that could enhance the efficiency of reproductive biotechnologies. The aim of this study was to characterize the pattern of trimethylation of histone 3 at lysine 4 (H3K4me3) in bovine embryos produced in vivo with sexed semen that were submitted to cryopreservation. Bos taurus × Bos indicus cows (n = 5) were superovulated and inseminated with sexed (two sessions) or conventional (two sessions) semen. A portion of the embryos collected on Day 7 was immediately stored in paraformaldehyde (3%) and another portion was stored in paraformaldehyde after cryopreservation/thawing. All embryos from the four groups (fresh, conventional semen; fresh, sexed semen; cryopreserved, conventional semen; and cryopreserved, sexed semen; 15 embryos per group) were evaluated by immunofluorescence under confocal microscopy to identify and quantify the H3K4me3 status. In total, 190 embryos were recovered, 100 of which were produced with conventional semen and 90 with sexed semen. The use of conventional semen after superovulation yielded 72% (72 of 100) viable embryos, which were mostly (81%; 59 of 72) in advanced stages of development (blastocysts and expanded blastocysts). Embryos produced with sexed semen had a lower viability rate (36.7%; 33 of 90), and most of them were collected at earlier stages of development (morulae and early blastocysts; P < 0.05). The H3K4me3 signal was similar among groups; however, there was a difference between morulae and blastocysts. A high intensity of H3K4me3 was observed in bovine embryos produced in vivo, and this pattern did not vary using sexed semen and the slow cryopreservation process. The lower viability of bovine embryos produced with sexed semen could be not explained by differences in H3K4me. Cryopreservation did not alter the pattern of H3K4me3; in this sense, we suggest that it is a process that exerts minimal damage to the embryos.

Survival and growth of goat primordial follicles after in vitro culture of ovarian cortical slices in media containing coconut water.

The development of culture systems to support the initiation of growth of primordial follicles is important to the study of the factors that control the earliest stages of folliculogenesis. We investigated the effectiveness of five culture media, two supplements and three culture periods on the survival and growth of goat primordial follicles after culturing ovarian cortex. The media were based on minimal essential minimum (MEM) and coconut water solution (CWS) added in the proportion of 0, 25, 50, 75 or 100%. The two supplements were none versus supplemented with insulin-transferrin-selenium, pyruvate, glutamine, hypoxanthine, and BSA. Pieces of goat ovarian cortex were cultured in the media for 1, 3 or 5 days and representative samples were evaluated at day 0 as non-cultured controls. The replicates were the two ovaries of five mixed breed goats. The number of primordial, intermediate, primary and secondary follicles at each period of culture and the number of degenerated follicles were evaluated. Mitotic activity of granulosa cells was studied by immunolocalization of proliferating cell nuclear antigen (PCNA). The number of follicles in each stage and degenerated follicles were statistically analyzed by ANOVA using a factorial design and the significance of differences assessed using Tukey test. Chi-square test was used to compare the percentage of follicles with PCNA positive granulosa cells. As the culture period progressed, the number of primordial follicles fell and there was a significant increase in the number of primary follicles. The fall in the number of primordial follicles was particularly marked after 1 day culture. No effect of media on the number of primordial and primary follicles was observed after culture, but MEM as well as supplements increased the number of intermediate follicles. Follicular degeneration was kept at the same level after culture in the media tested, except for pure CWS that increased the number of degenerated follicles. In contrast, addition of supplements to culture media reduced follicular degeneration. In non-cultured tissue, PCNA was expressed in granulosa cells of 31.6% of the growing follicles. This percentage had not significantly changed after 5 days culture in the various media, indicating the maintenance of proliferation activity of granulosa cells during culture. In conclusion, it is shown that goat primordial follicles may be successfully activated after in vitro culture in all media tested. However, when pure CWS is used the follicular degeneration is enhanced, but the addition of supplements to culture media decrease follicular degeneration.