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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 p0.05). Compared to the medium replacement at six days of culture, there was a statistically significant difference for T24 (68.9%, p

Dairy farming plays an important role in the socioeconomic scenario, and several characteristics point to an enormous potential for growth in the productivity of the herd. This is a complex activity that needs to be carried out efficiently in all the links that integrate it. Moreover, due to the physiological and metabolic aspects of the animals with the aptitude for the milk production, several challenges arise, especially in the reproductive scope. In this context, breeding biotechniques appear as a good alternative to the increase in reproductive rates and genetic improvement of dairy cattle. In addition, ovarian antral follicle counts (AFC) have been studied as a way to improve the selection of females to compose reproductive programs, because it has been shown to be a factor of high repeatability over the reproductive life in the same animal. Although studies have shown controversial results regarding the relationship between AFC and fertility, it is still necessary to investigate the possible variables that may interfere with the performance of donors in each biotechnology used. Thus, this review intends to gather and discuss the relationship of AFC with physiological aspects of dairy cows, its application as a parameter of selection of animals with good fertility and, the challenges for the application of reproductive biotechniques in dairy cattle.

The goal of this study was to determine the distribution of pre-antral follicles in the ovarian parenchyma of mares. For Experiment 1, each ovary was cut longitudinally at the greater curvature, performing two hemiovaries. After that, six fragments from each hemiovary were obtained, resulting in 12 fragments, which were divided into the innermost region of the parenchyma, the middle region and the outermost region. All the three obtained sections were cut transversally to obtain two fragments from each one. For Experiment 2, each ovary also submitted to a longitudinal cut on the greater curvature, forming two hemiovaries. Each hemiovary was sectioned into four symmetrical fragments, resulting in eight fragments per ovary. The fragments were related as being near to or far from the ovulatory fossa. The fragments of both experiments were immediately fixed in Carnoy for 12 hr and kept in 70% ethanol for 24 hr. Follicles were classified according to the stages of development and for morphological integrity according to oocyte morphology and granulosa cells. After the histological assessment, a total of 1,130 follicles were visualized from Experiment 1, being 1,054 (93.3%) primordial follicles and 76 (4.7%) follicles in development. The innermost region had the highest percentage of pre-antral follicles compared to the other regions (p < .05). The middle and outermost regions showed higher percentages of intact primordial and developing follicles than the innermost region (p < .05). Considering Experiment 2, 938 follicles were found, being 894 (95.3%) primordial and 44 (4.7%) follicles in development. The region near the ovulatory fossa presented higher (58.7%; 551 of 938) follicular concentration compared to the region far from the ovulatory fossa (41.3%; 387 of 938; p < .05). As a conclusion, distribution of pre-antral follicles in the equine ovary has a specific pattern through the parenchyma. Also, the follicular integrity differed in the studied ovarian areas.

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.

In follicular aspiration, physical aspects are of high significance for the technique to succeed, such as vacuum pressure, caliber of the needle and the way the follicular wall curettage is performed. The aim of this study was to investigate the recovery rate of equine oocytes aspirated by scraping of the follicular wall, testing different calibers of disposable needles, as well as the morphological evaluation of the cumulus oophorus complexes (COCs). Mares ovaries (n=447) obtained at a local slaughterhouse were transported to the laboratory in a thermal container (20 °C) and had the tunica albuginea and connective tissues dissected. The aspirated follicles had 10 to 25 mm in diameter, and 30x8 (21G 1 ¼) or 40x12 (18G 1 ½) needles were used for the aspiration, forming group A (G-A) and group B (G-B), respectively. In G-A and G-B, 480 and 548 follicles were aspirated, respectively. Under the stereomicroscope, the oocytes were evaluated according to the quality of the ooplasm and characteristics of the cumulus cells (grade I, II, III and denuded). The statistical analysis was performed using the Student’s t-test, logistic regression and test of proportions, and differences were considered significant when P

Contents This study investigated the effects of different concentrations of FSH (10, 50, 100 and 200 ng/ml) in supplemented MEM+ on the development of equine pre-antral follicles that were cultured in vitro for 2 or 6 days. The ovaries (n = 5) from mares in seasonal anoestrus were collected from a local abattoir. Ten ovarian tissue fragments of approximately 3 × 3 × 1 mm were obtained from each animal. The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ supplemented with FSH at four different concentrations, establishing the following 11 groups: control (D0); MEM + (D2); MEM + (D6); MEM + 10 ng/ml of FSH (D2); MEM + 10 ng/ml of FSH (D6); MEM + 50 ng/ml of FSH (D2); MEM + 50 ng/ml of FSH (D6); MEM + 100 ng/ml of FSH (D2); MEM + 100 ng/ml of FSH (D6); MEM + 200 ng/ml of FSH (D2); and MEM + 200 ng/ml of FSH (D6). Follicles were observed in only 9.65% (388 of 4,018) of the histological sections. Of the 861 follicles evaluated, 488 were in the primordial stage, and 373 were in various developmental stages; 59.7% were morphologically normal. Regarding the integrity of the pre-antral follicles, the groups with 100 ng/ml FSH of 2-days culture as well as 50, 100 and 200 ng/ml FSH of 6-days culture provided the best results. In conclusion, the in vitro culture of abattoir-derived equine ovarian fragments presented better morphological integrity when supplemented with FSH for 6 days, in comparison with the MEM culture group. However, no clear effects were observed with FSH regarding the promotion of activation from a primordial to a developing follicle.

O objetivo deste estudo foi investigar a eficácia dos fixadores teciduais Bouin, Carnoy ou Formol 10% em fragmentos ovarianos equinos. Ovários (n=4) de éguas, sem raça definida, foram obtidos de abatedouro local e transportados em recipiente térmico a 20 ºC. Imediatamente após a coleta, os ovários foram lavados com solução de PBS modificado (Cultilab®, Campinas-SP, Brasil), e divididos em nove fragmentos com aproximadamente 5x5x1 mm, retirados do parênquima de cada ovário. Em seguida, os fragmentos ovarianos foram imersos em um dos três diferentes fixadores, Bouin (B), Carnoy (C) ou Formol 10% (F), por 6, 12 ou 24 horas. Cada fragmento foi acondicionado individualmente em um frasco contendo aproximadamente 20 vezes o volume da solução fixadora. Após este período, foram mantidos em álcool 70% por 24 horas. Para cada fixador e tempo foram realizadas quatro réplicas. No processamento histológico, os fragmentos foram desidratados em concentrações crescentes de álcool, diafanizados em xilol e incluídos em parafina. Em seguida, foram feitos cortes seriados de 5 ?m em micrótomo rotativo (Leica®, Wetzlar-Alemanha), seguidos da montagem de lâminas e coloração com ácido periódico de Schiff (PAS) e hematoxilina. Foram avaliadas 540 lâminas com 1.620 cortes histológicos, contendo 465 folículos pré-antrais que foram classificados como íntegros ou degenerados. A degeneração foi detectada pela presença de pelo menos um dos seguintes aspectos: retração do citoplasma, núcleo picnótico, vacúolos citoplasmáticos, deslocamento das células da granulosa e/ou rompimento da membrana basal. Um teste de regressão logística foi utilizado para a análise estatística, e as diferenças foram consideradas significativas quando P