87 Population estimate and morphology of ovarian preantral follicles in fetal and adult alpacas (Vicugna pacos)

In mammals, folliculogenesis begins at the fetal stage and is a complex, dynamic process that involves follicular quiescence, activation, growth, follicular migration, and cell interactions. At birth, the preantral ovarian follicular population, drastically reduced, constitutes >90% of all ovarian follicles, representing the ovarian reserve that will be used throughout the reproductive life. In alpacas, changes in follicular wave growth patterns and ovulation are different from other species; thus, it is important to know the ovarian reserve at fetal stage, as a starting point for future studies. Therefore, the aim of the present study was to establish the morphological characterisation and estimate the population of alpaca preantral follicles in the fetal stage. Ovaries from alpacas (n=5) in the fetal stage (fetus during the last third of gestation) were collected at a local slaughterhouse. Whole ovaries were individually fixed in 4% paraformaldehyde phosphate-buffered saline overnight at room temperature for routine histology. Ovaries were dehydrated in alcohol, cleared with xylene, and embedded in paraffin, and all tissue was serially sectioned at 7μm with a rotating microtome (Leica). The histological sections were mounted and stained with periodic acid-Schiff and hematoxylin. Preantral follicles were classified according to their developmental stage: primordial (one layer of flattened granulosa cells surrounding the oocyte), primary follicle transition (flattened cuboidal granulosa cells surrounding the oocyte), primary (single layer of cuboidal granulosa cells around the oocyte), or secondary (oocyte surrounded by more than one complete layer of cuboidal granulosa cells). We estimated the number of preantral follicles by counting all follicles in each histological section. Only follicles in which the oocyte nucleus was visible were counted. In addition, for each follicle category (n=40 per group), oocyte and follicle diameters were measured using an ocular micrometer. The variable means were compared using unpaired Student's t-test analysis, with significance set at P ≤ 0.05. Estimation of preantral follicular population (mean±standard deviation) was 80 516±14 575 in the ovaries of alpaca fetuses. Most of the follicles found belong to the primordial (49.2%) or primary follicle transition (39.2%) categories, followed by primary (10.8%) and secondary (0.8%) stages. Follicle and oocyte diameters of primordial (33.3±7.2; 21.5±4.6μm) and primary follicle transition stages (36.7±3.0; 23.4±2.6μm) were significantly (P<0.05) smaller than those of primary-stage follicles (77.9±15.8; 50.02±11.1μm). Finally, preantral follicles classified with normal morphological integrity appearance for each developmental stage were 98.2% (primordial), 96.7% (primary follicle transition), 91.5% (primary), and 88.1% (secondary), respectively. In conclusion, this study shows for the first time an estimation of the population of preantral follicles in alpaca fetal ovaries and establishes follicle and oocyte diameters and their normal morphological integrity.

Number and density of equine preantral follicles in different ovarian histological section thicknesses

The correlation between the number of antral follicles and ovarian reserves (preantral follicles) in purebred Bos indicus and Bos taurus cows

The sustainability and production of collared peccary (Pecari tajacu) has been studied in the last few years; however, further information on its reproduction is necessary for breeding systems success. Understanding folliculogenesis aspects will contribute to effective reproductive biotechniques, which are useful in the preservation and production of wildlife. The aim of this study was-to evaluate the ovarian folliculogenesis in collared peccary. Ovaries from six adult females of collared peccary were obtained through ovariectomy and analyzed. These were fixed in aqueous Bouin's solution and sectioned into 7 microm slices, stained with hematoxilin-eosin and analyzed by light microscopy. The number of pre-antral and antral follicles per ovary was estimated using the Fractionator Method. The follicles, oocytes and oocyte nuclei were measured using an ocular micrometer. Results showed that the length, width, thickness, weight, and the gross anatomy of the right and left ovaries were not significantly different. However, the mean number of corpora lutea was different between the phases of the estrous cycle (p<0.05), with the highest mean in the luteal phase. Primordial follicles were found in the cortex; the oocytes were enveloped by a single layer of flattened follicular cells. In the primary follicles, proliferation of the follicular cells gave rise to cuboidal cells (granulosa cells). The secondary follicle was characterized by two or more concentric layers of cuboidal cells (granulosa), beginning of antrum formation, and the presence of pellucid zone and theca cells. Antral follicles were characterized by a central cavity (antrum), the presence of cumulus oophorus and theca layers (interna and externa). In the right ovary, the values of the primordial and primary follicles were similar, but significantly different from the secondary ones (p<0.05). In the left ovary, significant differences were observed between all follicles in the follicular phase (p<0.05); the mean number of primordial and primary follicles was similar in the luteal phase. The mean number of pre-antral follicles and antral follicles in the follicular phase was higher in the left ovary (p<0.05). The mean number of antral follicles in the luteal phase was similar in both ovaries. We also found significant differences in mean diameter of preantral follicles, oocyte, granulosa layer and oocyte nucleus during the estrous cycle. In the antral follicles a significant difference was observed only in follicular diameter (p<0.05). The predominance of active primordial and primary follicles was found in both phases; otherwise the secondary follicles and antral follicles showed a high degree of degeneration. The results obtained in the present work will strengthen the development of biotechnology programs to improve the productive potential and conservation of the collared peccary.

Population estimate of the preantral follicles and frequency of multioocyte follicles in prepubertal and adult bitches

The mare has been strongly advocated by several research groups as an important comparative animal model to study antral follicular dynamics in women due to some similarities in reproductive events. More recently, the mare has also been suggested as a potential model for studies related to preantral follicles. The search for an appropriate animal model for comparative studies of preantral follicle population, density, and distribution has been a major focus of recent ovarian translational studies. The aim of the present study was to investigate the influence of reproductive phase (anestrous v. diestrous) and ovarian structures (antral follicles and corpus luteum) on the quality, class distribution, number, and density of preantral follicles, and stromal cell density. Ovarian fragments were harvested in vivo from young, healthy mares (n = 10) during both reproductive phases using a biopsy pickup method and submitted to histological analysis. All evaluations and measurements were performed by a single operator and only follicles with a visible nucleus was considered for follicle counting. Data were analysed by Kruskal-Wallis test, Wilcoxon-Mann-Whitney test, and Spearman’s correlation. A total of 142 ovarian biopsy fragments were collected (mean, 3.7 fragments per mare in each reproductive phase) and 13 462 histological sections were evaluated. Overall, 1493 preantral follicles were recorded with a mean of 10.5 ± 1.7 follicles per ovarian fragment [range, 0 to 165; mean coefficient of variation (CV) = 195%]. The mean follicle density was 2.7 follicles per cm2 (range, 0 to 39; CV = 214%) and differed (P &lt; 0.05) among mares. The mean preantral follicle and ovarian stromal cell densities were greater (P &lt; 0.05) in the diestrous phase and a positive correlation of stromal cell density with the number and density of preantral follicles was observed. The mean area (mm2) of ovarian structures increased (P &lt; 0.05) in the diestrous phase and had positive correlations with number of preantral follicles, follicle density, and stromal cell density. Biopsy fragments collected from ovaries during the diestrous phase had a higher (P &lt; 0.05) follicle density, stromal cell density, and proportion of normal preantral follicles. In conclusion, our results showed (1) the diestrous phase influenced positively the preantral follicle quality, class distribution, and follicle and stromal cell densities; (2) the area of ovarian structures was positively correlated with the follicle and stromal cell densities; and (3) the presence of an active corpus luteum had a positive effect on the quality of preantral follicles and follicle and stromal densities. Therefore, herein we demonstrated that reproductive phases and ovarian structures induce changes in preantral follicle features and stromal cell density favouring the harvesting of ovarian fragments containing an appropriate number of healthy preantral follicles. These findings reinforce the concept of the use of the mare as an appropriate model to provide comparative insights about preantral follicle density and ovarian plasticity.

The aims of the study were to investigate the influence of FSH and the ovary cutting treatments on the development of primordial follicles into preantral and antral follicles. Ovaries were grouped as whole ovary (without cutting); partially cut ovary and completely cut ovary (hemi ovary). All ovary groups were cultured in Dubellco’s Modified Eagle Medium (DMEM) containing 5ug/ml insulin, 10ug/ml transferrin, 5ug/ml selenium (ITS), 5% FBS, 50ug/ml gentamycin with and without 100uIU/ml Follicle Stimulating Hormone (FSH). Cultures were done in 5% CO2 incubator at 37oC. Results showed that after 8 days in vitro cultured in DMEM containing FSH, the average number of preantral follicles isolated from each whole, partially-cut and completely-cut ovaries were 16.1 + 3.3, 26.8 ± 7.7 and 12.3 + 1.9, respectively. On the other hand, those cultured in DMEM without FSH they were 17.3 + 3.8, 23.3 ± 5.2 and 17.8 + 2.8, respectively. After additional cultured for 8 days, the percentage of preantral follicles developing into the antral follicles in DMEM with and without FSH were 26.7 + 5.7 and 11.7 + 2.9, respectively. In conclusion, the supplementation of FSH in the culture medium did not increase the number of preantral follicles, but significantly increased the number of antral follicles. The ovary cutting treatment significantly increased the average number of collected preantral follicles.

The present study aimed to obtain quanti-qualitative data about the follicular ovarian population in Cebus apella females. Seven ovaries were obtained from 4 C. apella adult females. The ovaries were subjected to light microscopy. The number of preantral and antral follicles for each ovary was estimated using the Fractionator method. The preantral follicles were classified into primordial, transitional, primary and secondary follicles. Antral follicles were those that presented an antral cavity. All counted follicles were classified as normal or degenerated. The diameter of the follicles, oocytes and their nuclei were determined to accompany the follicular development. All results were represented as mean ± SE. The number of preantral follicles was 56,938 ± 21,888 and 49,133 ± 26,896 for the right and left ovaries, respectively. The percentage of normal follicles was 80 ± 4.95%. The follicular diameter ranged from 22 ± 0.5 µm to 61.2 ± 4.0 µm. Regarding the antral follicles, the number of normal and degenerate follicles per ovary were 60.0 ± 19.0 and 3 ± 1.8 follicles, respectively. The antral follicular diameter was 514.4 + 56.6 µm. In conclusion, the information obtained in this study can be used as a parameter for subsequent in vivo or in vitro studies about folliculogenesis in non-human neotropical primates of the C. apella species.

Zebu ( Bos indicus) ovarian preantral follicles: morphological characterization and development of an efficient isolation method

The present study aimed to determine the relationship among changes in the number of preantral follicles and concentrations of follicle-stimulating hormone (FSH), luteinizing hormone (LH), progesterone (P4), androstenedione (A) and estradiol-17beta (E2) in the fetal heart, umbilical cord and maternal blood. Primordial follicles had already appeared in a 20 cm fetus and primary follicles were observed in a 50 cm fetus. In a 70 cm fetus, the number of primordial and primary follicles increased rapidly and secondary follicles were present. The concentrations of LH and FSH did not change between 20 cm and 160 cm in fetal length. When the fetal length became > 70 cm, serum levels in the fetus, umbilical cord and mothers, and E2 levels in umbilical cord increased synchronously (p < 0.05). These results showed increases in the number of preantral follicles in the Antarctic minke whale fetal ovary along with fetal growth during the early gestation period. These findings suggest that the change in preantral follicles was associated with changes in the concentration of steroids in early gestation periods. The changes in steroid concentrations in the fetal and umbilical cord blood and the increased number of preantral follicles were coincident at around 70 cm in fetal length, whereas the growth and differentiation of primordial and primary follicles appeared to be independent of FSH and LH.

Ontogeny of the ovary in polycystic ovary syndrome

Ovarian tissue collected by biopsy procedures allows the performance of many studies with clinical applications in the field of female fertility preservation. The aim of the present study was to investigate the influence of reproductive phase (anestrous vs. diestrous) and ovarian structures (antral follicles and corpus luteum) on the quality, class distribution, number, and density of preantral follicles, and stromal cell density. Ovarian fragments were harvested by biopsy pick-up procedures from mares and submitted to histological analysis. The mean preantral follicle and ovarian stromal cell densities were greater in the diestrous phase and a positive correlation of stromal cell density with the number and density of preantral follicles was observed. The mean area (mm2) of ovarian structures increased in the diestrous phase and had positive correlations with number of preantral follicles, follicle density, and stromal cell density. Biopsy fragments collected from ovaries containing an active corpus luteum had a higher follicle density, stromal cell density, and proportion of normal preantral follicles. In conclusion, our results showed: (1) the diestrous phase influenced positively the preantral follicle quality, class distribution, and follicle and stromal cell densities; (2) the area of ovarian structures was positively correlated with the follicle and stromal cell densities; and (3) the presence of an active corpus luteum had a positive effect on the quality of preantral follicles, and follicle and stromal densities. Therefore, herein we demonstrate that the presence of key ovarian structures favors the harvest of ovarian fragments containing an appropriate number of healthy preantral follicles.

Cryopreservation and characterization of canine preantral follicles

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

BackgroundThere is substantial evidence both in humans and in animals that a prolonged reduction in plasma thyroid hormone concentration leads to reproductive problems, including disturbed folliculogenesis, impaired ovulation and fertilization rates, miscarriage and pregnancy complications. The objective of the present study is to examine the consequences of chronic hypothyroidism, induced in adulthood, for the size of the ovarian follicle pool. In order to investigate this, adult female rats were provided either a control or an iodide deficient diet in combination with perchlorate supplementation to inhibit iodide uptake by the thyroid. Sixteen weeks later animals were sacrificed. Blood was collected for hormone analyses and ovaries were evaluated histologically.ResultsAt the time of sacrifice, plasma thyroid-stimulating hormone concentrations were 20- to 40-fold increased, thyroxine concentrations were negligible while tri-iothyronin concentrations were decreased by 40% in the hypothyroid group, confirming that the animals were hypothyroid. Primordial, primary and preantral follicle numbers were significantly lower in the hypothyroid ovaries compared to the euthyroid controls, while a downward trend in antral follicle and corpora lutea numbers was observed. Surprisingly the percentage of atretic follicles was not significantly different between the two groups, suggesting that the reduced preantral and antral follicle numbers were presumably not the consequence of increased degeneration of these follicle types in the hypothyroid group. Plasma anti-Müllerian hormone (AMH) levels showed a significant correlation with the growing follicle population represented by the total ovarian number of primary, preantral and antral follicles, suggesting that also under hypothyroid conditions AMH can serve as a surrogate marker to assess the growing ovarian follicle population.ConclusionsThe induction of a chronic hypothyroid condition in adult female rats negatively affects the ovarian follicular reserve and the size of the growing follicle population, which may impact fertility.