Culture of preantral ovarian follicles of Bos taurus indicus with alpha-lipoic acid.

The aim of this study was to evaluate follicular development, morphological integrity, and antioxidant potential of preantral ovarian follicles from Bos taurus indicus females grown in vitro with alpha-lipoic acid. Ovaries (n = 24) of Bos taurus indicus (n = 12) females were collected during slaughter and fragmented. A randomly obtained fragment from each pair of ovaries was fixed in Bouin (non-cultivated control; D0). These fragments were intended for classical histology (morphology and evaluation of follicular growth), and a fragment from each pair of ovaries was frozen at -80°C (non-cultivated control; D0), and assigned for analysis of oxidative stress [thiobarbituric acid reactive substances (TBARS), nitroblue tetrazolium (NBT), and ferric reducing antioxidant power (FRAP)]. The remaining fragments were cultured in vitro for 6 (D6) or 12 (D12) days, containing only minimum essential medium (MEM) or MEM supplemented with alpha-lipoic acid (50, 100, or 250 ng/ml), on an extracellular matrix of agarose gel, in an oven at 38.5ºC. Every 2 days, 100% of the culture medium was replaced. Supplementation with 100 ng/ml was effective for maintaining follicular integrity after 6 days of culture (primordial: 51.28%; development: 36.88%; P < 0.0001). There was no difference (P > 0.05) between treatments compared with the non-cultivated control treatment (D0), using the NBT and TBARS assays. Therefore, supplementation of the in vitro culture medium of bovine preantral ovarian follicles with a concentration of 100 ng/ml of alpha-lipoic acid at 6 days of culture was effective for maintaining follicular integrity and, after 6 days, maintaining stable levels of reactive oxygen species.

Follicular development, morphological integrity, and oxidative stress in bovine preantral follicles cultured in vitro with ascorbic acid.

The aim of this study was to evaluate the follicular development, morphological integrity, and oxidative stress of preantral ovarian follicles from Bos taurus indicus females grown in vitro with ascorbic acid. Ovaries (n = 20) from Bos taurus indicus females were collected, fragmented, and were cultured in vitro for 6 or 12 days in minimum essential medium (MEM), or MEM supplemented with 50 or 100 ng/ml ascorbic acid, with an extracellular matrix of agarose gel, in an incubator at 38.5°C; every 2 days, 100% of the culture medium was replaced. The data were analyzed using the chi-squared test and/or Fisher's exact test. In the event of a significant effect, the proportions were compared using a 2 × 2 proportion test. The oxidative stress analysis data were submitted to analysis of variance followed by the Bonferroni test. Values were considered significant when P ≤ 0.05. The addition of 100 ng/ml of ascorbic acid to the in vitro culture medium of preantral ovarian follicles from bovine females promoted follicular development, was efficient in maintaining morphological integrity, as well as the stability of reactive oxygen species, after 6 days of in vitro culture.

Influence of age and ovarian antral follicle count on the reproductive characteristics of embryo donor mares.

BACKGROUND: Age and antral follicle count (AFC) are related to fertility in cattle, but this information remains limited in mares. METHODS: To verify the influence of age and AFC on the reproductive characteristics of mares, 15 Quarter Horse donors, with 5-15 antral follicles, ranging from three to 17 years old, healthy and in good nutritional status, were divided into groups with low AFC (≤9 follicles) or high AFC (≥10 follicles) and mares considered young (≤9 years) or old (≥10 years). Mares were submitted to ultrasonography to determine the dominant follicle diameter, follicular growth rate, degree of uterine oedema and embryonic recovery for a minimum of three oestrous cycles. RESULTS: AFC was higher (P=0.001) in young mares compared with old mares. An interaction (P=0.001) between AFC and age was observed with regard to follicular growth rate, being that mares with low AFC and are old showed the lowest follicular growth compared with those of low AFC and young, high AFC and old, and high AFC and young. Younger mares and those with high AFC exhibited higher degree of uterine oedema (P<0.05) on the third day of oestrus compared with older mares and with low AFC (3 and 2, respectively). However, in both groups, the degree of oedema reduced by the time of ovulation. CONCLUSION: Follicular growth rate, degree of uterine oedema and the number of antral follicles are higher in young mares with high AFC. However, the rate of embryonic recovery is not influenced by donors' age or AFC.

Growth and Differentiation Factor-9 Supplementation Affects Viability and Morphology of Preantral Follicles in Equine Ovarian Fragments During Short-term in vitro Culture

Abstract This study aimed to evaluate different concentrations of growth and differentiation factor-9 (GDF-9) on the development and maintenance of equine preantral follicle morphology during short-term in vitro culture. Ovaries (n=5) from five mares were collected from a local slaughterhouse and transported to the laboratory, where nine fragments (5x5x1mm) were procured from each ovary. One fragment from each was immediately fixed and submitted for histological analysis (control group; D0). The other eight fragments were cultured in situ for two (D2) or six (D6) days in MEM+ or MEM+ supplemented with GDF-9 at different concentrations (i.e., 50, 100 and 200 ng/mL the GDF-9). After culturing with different concentrations of GDF-9 for 2 or 6 days, the fragments were processed for histological analysis. After two days of cultivation, we observed an increase in the percentage of developing follicles for 0 (MEM+), 50, 100 and 200 ng/mL GDF-9 compared to control (D0; P<0.05). When we evaluated all treatments that preserved follicular integrity, the GDF-9 concentration of 100 ng/mL presented results superior to those of the other cultures (P<0.05). While, at six days of culture, the concentration of 200 ng/mL of GDF-9 appeared to be more efficient in providing development compared to MEM+ (P<0.05). The percentage of morphologically intact follicles in the 6 days culture samples treated with 50 ng/mL of GDF-9 indicated that this concentration was effective in maintaining the integrity of the follicle (P<0.05). We conclude, therefore, that graduated GDF-9 addition to the medium ensure follicular development and is sufficient maintain the architecture.

Interval in the replacement of in vitro culture medium affects the integrity and development of equine preantral follicles / Intervalo na substituição do meio de cultivo in vitro afeta a integridade e o desenvolvimento de folículos pré-antrais equinos

The efficiency of a culture system is related to the elaboration and replacement of a medium with conditions suitable for follicular development. Recent investigations suggested that in vitro culture medium should be replaced after specific time periods in various species. However, the suitable interval for the exchange of in vitro culture medium has not yet been established in equine species. The objective of this investigation was to evaluate the effect of medium exchange intervals of 24 hours (T24) or 48 hours (T48) for in vitro culture of preantral follicles at 2 or 6 days. At the end of the culture period, the fragments were processed using classical histology. Equine preantral follicles were classified according to morphological integrity and developmental stage. Data analysis was performed using Fisher's test with a significance level of p<0.05. Out of a total of 399 follicles evaluated, 174 (43.6%) were primordial follicles, 225 (56.4%) were in development, and 63.76% were morphologically intact. In the in vitro culture performed over two days, there was no significant difference in relation to follicular integrity after medium replacement (p>0.05). Compared to the medium replacement at six days of culture, there was a statistically significant difference for T24 (68.9%, p<0.05). Therefore, we suggest changing the medium for equine species at 48 hours after the start of culture followed by subsequent daily replacements.(AU)

A eficiência de um sistema de cultivo está relacionada à elaboração e substituição do meio de cultivo com condições adequadas ao desenvolvimento folicular. Pesquisas recentes sugerem que o meio de cultivo in vitro deve ser substituído após períodos de tempo específicos para várias espécies. No entanto, o intervalo adequado para a troca de meio de cultivo in vitro ainda não foi estabelecido na espécie equina. O objetivo desta investigação foi avaliar o efeito de intervalos de troca média de 24 horas (T24) ou 48 horas (T48) para cultivo de folículos pré-antrais aos 2 ou 6 dias. No final do período de cultivo, os fragmentos foram processados ​​usando histologia clássica. Os folículos pré-antrais equinos foram classificados de acordo com a integridade morfológica e o estágio de desenvolvimento. A análise dos dados foi realizada utilizando o teste de Fisher com um nível de significância de p<0,05. De um total de 399 folículos avaliados, 174 (43,6%) foram folículos primordiais, 225 (56,4%) estavam em desenvolvimento e 63,76% estavam morfologicamente intactos. No cultivo in vitro realizado ao longo de dois dias, não houve diferença significativa em relação à integridade folicular após a substituição do meio (p>0,05). Comparado com a substituição média aos seis dias de cultivo, houve diferença estatisticamente significativa para T24 (68,9%, p<0,05). Portanto, sugerimos alterar o meio para as espécies equinas às 48 horas após o início da cultura, seguindo as subsequentes substituições diárias.(AU)

In vitro culture supplementation of EGF for improving the survival of equine preantral follicles.

Folliculogenesis is a process of development and maturation of the ovarian follicles, being essential for the maintenance of fertility. In in vivo conditions, 99.9% of the follicles of an ovary do not ovulate and undergo atresia. In order to minimize this loss and to clarify the existing mechanisms, a technique was developed that allows for the in vitro follicular development. The objective of this study was to evaluate the effects of different epidermal growth factor (EGF) concentrations on the in vitro culturing of equine preantral follicles. Ovaries (n = 10) were collected from a local slaughterhouse of mares in seasonal anestrus, washed with 70% alcohol and PBS, and transported. The inner portion of the ovary was divided into 11 fragments of approximately 3 × 3 × 1 mm. A fragment of each ovary was immediately fixed in Bouin (control group). The remaining 10 fragments were individually cultured for 2 and 6 d. The medium was supplemented with different concentrations of EGF (0, 10, 50, 100, and 200 ng/mL). After cultivation, the fragments were processed and classified according to the developmental stage and morphology. In total, 1065 slides containing 6105 tissue sections were evaluated. Within 2 d of culture, there was a higher proportion of intact follicles at the EGF concentrations of 0 and 100 ng/mL (p > 0.05). After 6 d of culture, only the EGF concentration of 100 ng/mL demonstrated a difference when compared to the other treatments (0, 10, 50 and 200 ng/mL of EGF, p > 0.05). There was follicular development after 2 d at all EGF concentrations. Thus, we suggest that EGF promotes follicular survival in equines at a concentration of 100 ng/mL in in vitro cultures of ovarian fragments for 2 d. In addition, we suggest that EGF promotes follicular survival in equines at a concentration of 100 ng/mL in situ cultivation.

Intensified use of TAI and sexed semen on commercial farms.

The livestock sector has achieved many technological advances, which have resulted in continued improvements in animal production systems and in the reproductive efficiency of herds. The associated use of reproductive biotechnology and genetic improvements combined with adequate sanitary and nutritional management are essential conditions for sustainable intensified animal production and financial autonomy within farms. Timed artificial insemination (TAI) represents one of the strategies with the greatest impact of expansion in providing genetic improvements and increased reproductive efficiency at a decreased cost. Despite the high proportion of cows receiving TAI, this market still exhibits considerable potential for expansion. After a TAI procedure, approximately 40 to 60% of females become pregnant. This result can vary depending on such factors as the hormonal protocol employed, female category, body condition score, ovarian status, farm management and aspects related to bulls and semen. The fertility and genetic quality of the bull plays an important role in the herd because a single bull can influence the entire production system. Another important strategy is the use of sex-sorted semen associated with TAI, primarily when associated with management practices to improve the pregnancy rate. This paper presents a review of the intensification of TAI, supplying practical information regarding the implementation of TAI commercial programs.