Dynamics of extracellular vesicle-coupled microRNAs in equine follicular fluid associated with follicle selection and ovulation.

Innumerable similarities in reproductive cyclicity and hormonal alterations highlight the considerable utility of the mare to study aspects of follicular dynamics and reproductive function in view of the largely constricted, human research subjects. The bi-directional communication between the growing oocyte and the surrounding somatic cells embodies the hallmark of mammalian follicular development, partially mediated by extracellular vesicles (EVs) encapsulated with microRNAs (miRNAs) and present in the follicular fluid (FF). Here, we aimed to decipher the dynamics of the miRNAs in EVs from equine FF aspirated in vivo during different stages of follicular development, namely, predeviation (PreDev; 18-20 mm), deviation (Dev; 22-25 mm), postdeviation (PostDev; 26-29 mm), preovulatory (PreOV; 30-35 mm), and impending ovulation (IMP; ∼40 mm). Approximately 176 known miRNAs were found in all groups with 144 mutually detected among all groups. Cluster analysis exhibited 15 different expression patterns during follicular development. Among these patterns, a group of 22 miRNAs (including miR-146b-5p, miR-140, and miR-143) exhibited a sharp reduction in expression from the PreDev until the PreOV stage. Another cluster of 23 miRNAs (including miR-106b, miR-199a-5p, and miR-125a-5p) exhibited a stable expression pattern at the PreDev stage until the PostDev stage, with a significant increase at the PreOV stage followed by a significant decrease at the IMP stage. In conclusion, this study provides greater insights into the stage-specific expression dynamics of FF EV-miRNAs during equine follicular development, which may propose novel approaches to improve ART and provide new biomarkers to facilitate the assessment of ovarian pathophysiological conditions.

Trimethylation profile of histones H3 lysine 4 and 9 in late preantral and early antral caprine follicles grown in vivo versus in vitro in the presence of anethole.

This study assessed the histones methylation profile (H3K4me3 and H3K9me3) in late preantral (PA) and early antral (EA) caprine follicles grown in vivo and in vitro, and the anethole effect during in vitro culture of PA follicles. Uncultured in vivo-grown follicles (PA, n = 64; EA, n = 73) were used as controls to assess the methylation profile and genes' expression related to apoptosis cascade (BAX, proapoptotic; BCL2, antiapoptotic), steroidogenesis (CYP17, CYP19A1), and demethylation (KDM1AX1, KDM1AX2, KDM3A). The isolated PA follicles (n = 174) were cultured in vitro for 6 days in α-MEM+ in either absence (control) or presence of anethole. After culture, EA follicles were evaluated for methylation, mRNA abundance, and morphometry. Follicle diameter increased after culture, regardless of treatment. The methylation profile and the mRNA abundance were similar between in vivo-grown PA and EA follicles. Anethole treatment led to higher H3K4me3 fluorescence intensity in EA follicles. The mRNA abundances of BAX, CYP17, and CYP19A1 were higher, and BCL2 and KDM3A were lower in in vitro-grown EA follicles than in vivo-grown follicles. In conclusion, in vitro follicle culture affected H3K4me3 fluorescence intensity, mRNA abundance of apoptotic genes, and steroidogenic and demethylase enzymes compared with in vivo-grown follicles.

Efficiency of round bale feeders: comparison of Tombstone versus Hay Saver.

Hay wastage when feeding round bales due to contamination, deterioration, and animal refusal can accrue large financial losses for farmers. The present study investigated the efficiency of the conventional Tombstone-style feeder system compared to the Hay Saver feeder system to reduce hay wastage in feeding round hay bales. Mares were distributed equally into two groups, Tombstone and Hay Saver, and fed six bales per group over 48 days. Hay wastage was collected daily, dried, and weighed, while the mares were weighed weekly. Overall, the Hay Saver feeder showed less hay wastage, higher mean mare weight, and higher consumption rate per horse. The results of this study indicated that the Hay Saver feeder system had higher efficiency compared to the Tombstone feeder system.

Dominant follicle and gonadotropin dynamics before ovulation in postpartum lactating mares.

The aim of this study was to compare the dominant follicle (DF) and gonadotropin dynamics for 9 days before ovulation in postpartum lactating (PP Lactating) versus non-postpartum cycling (N-PP Cycling) mares. Every PP Lactating mare on the day of parturition was paired with a N-PP Cycling mare, and the data analyses considered the partum-ovulation interval (POI) and the postpartum interovulatory interval (PPIOI) in PP Lactating mares and two interovulatory intervals in N-PP Cycling mares. The results of the present study revealed several novel and unique aspects of DF development and FSH and LH dynamics before ovulation in PP Lactating mares when compared with N-PP Cycling mares. The most remarkable differences between both groups of mares were the following: (1) a shorter interval to ovulation in PP Lactating mares during the foal heat (POI ≤22 days) compared with all other intervals; (2) a larger DF in PP Lactating mares, and an earlier day of DF at maximum diameter during the foal heat; and (3) lower gonadotropin levels in PP Lactating mares. Regarding the particularities, PP Lactating mares had lower LH levels during the POI than the PPIOI, demonstrating a strong partum effect; spring-foaled mares had longer POIs, larger DF diameters, and lower LH levels; and lower body condition scores and higher body-weight loss led to longer POIs and smaller DF diameters in PP Lactating mares. This study contributes to a broad understanding of ovarian function in the postpartum mare.

Hemodynamic, endocrine, and gene expression mechanisms regulating equine ovarian follicular and cellular development.

Ovulatory follicle development and associated oocyte maturation involve complex coordinated molecular and cellular mechanisms not yet fully understood. This study addresses the relationships among follicle diameter, follicle wall blood flow, follicular-fluid factors, and gene expression for follicle growth, steroidogenesis, angiogenesis, and apoptosis in granulosa/cumulus cells and oocytes during different stages from the beginning of largest/ovulatory follicle to impending ovulation in mares. The most remarkable findings were (i) a positive association between follicle development, follicle blood flow, intrafollicular follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, progesterone, and messenger RNA (mRNA) expression for FSHR and LHCGR in granulosa cells of the largest/ovulatory follicle; (ii) a plateau or decrease in follicle diameter and blood flow and granulosa cell mRNA for FSHR, LHCGR, IGF1R, VEGFR2, CYP19A1, and CASP3 at the preovulatory stage; (iii) higher StAR and BCL2 and lower CASP3 mRNA in granulosa cells at the time of impending ovulation; (iv) greater IGF1R mRNA for granulosa cells at the predeviation stage; and (v) lower FSHR, LHCGR, IGF1R, and VEGFR2 mRNA in cumulus cells and greater LHCGR and IGF1R mRNA in oocytes at the ovulatory stage. This study is a critical advance in the understanding of molecular mechanisms of follicle development and oocyte maturation and is expected to be vital for future studies targeting potential markers.

Follicular-fluid proteomics during equine follicle development.

The complex composition of the follicular fluid (FF), the intimate proximity to the oocyte, and the continual changes in their composition have a major effect on folliculogenesis and oogenesis. To date, the profiling of FF proteomes during follicle selection, development, and ovulation has not been comprehensively investigated. Therefore, a shotgun proteomics approach and bioinformatics analyses were used to profile the proteomes of equine FF harvested in vivo from follicles at the following development stages: predeviation (18-20 mm), deviation (22-25 mm), postdeviation (26-29 mm), preovulatory (30-35 mm), and impending ovulation. A total of 294 proteins were detected in FF (FDR <1%), corresponding to 65 common proteins and 124, 142, 167, 132, and 142 proteins in the predeviation, deviation, postdeviation, preovulatory, and impending ovulation groups, respectively. The higher expression of properdin and several other proteins belonging to the complement system during the deviation time and ovulation suggested their contribution in the selection of the future dominant follicle and ovulation. Apolipoprotein A-1 and antithrombin-III appeared to be important throughout folliculogenesis. The "complement and coagulation cascades" was the major KEGG pathway across all stages of follicle development. The significant expression of several proteins belonging to the serine-type endopeptidase indicated their likely contribution to follicle and oocyte development. Our data provide an extensive description and functional analyses of the equine FF proteome during follicle selection, development, and ovulation. This information will help improve understanding of the ovarian function and ovulatory dysfunctions and might serve as a reference for future biomarker discovery for oocyte quality assessment.

First pregnancy after in vitro culture of early antral follicles in goats: Positive effects of anethole on follicle development and steroidogenesis.

This study aimed to evaluate the role of anethole during the in vitro culture of caprine early antral follicles. Early antral follicles were isolated from caprine ovaries and cultured for 18 days without (control) or with anethole (300 µg/ml). After culture, the cumulus-oocyte complexes were subjected to in vitro maturation, followed by parthenogenetic activation or in vitro fertilization (IVF) and embryo culture. Follicular walls were used for the quantification of messenger RNA (mRNA) of CYP19A1, CYP17, MMP-9, TIMP-2, Bax, and Bcl-2 genes, and culture medium was used for evaluation of ferric reducing antioxidant power (FRAP) and estradiol levels. After in vitro follicle culture (IVFC), anethole induced higher total antioxidant capacity, that is, it produced higher FRAP levels, reduced the Bax/Bcl-2 ratio, and increased the levels of mRNA for CYP19A1 and CYP17, which was associated with a greater estradiol production (p < .05). Also, anethole improved the ability of oocytes to resume meiosis and reach metaphase II stage, as well as yielded higher (p < .05) embryo production (e.g., morulas and blastocysts) in both parthenogenetic activation and IVF techniques. One pregnancy (Day 30) was obtained from IVFC with anethole. In conclusion, anethole promoted in vitro growth and maturation of goat early antral follicles and oocytes and enabled embryo production. Furthermore, this study reports, for the first time in goats, a pregnancy after IVF using oocytes originated from early antral follicles grown in vitro.

Transition to the ovulatory season in mares: An investigation of antral follicle receptor gene expression in vivo.

The inability to obtain in vivo samples of antral follicle wall layers without removing the ovaries or sacrificing the animals has limited more in-depth studies on folliculogenesis. In this study, a novel ultrasound-guided follicle wall biopsy (FWB) technique was used to obtain in vivo follicle wall layers and follicular fluid samples of growing antral follicles. The expression of proliferative, hormonal, angiogenic, and pro-/antiapoptotic receptors and proteins in the follicular wall among three follicle classes were compared during the spring transitional anovulatory (SAN) and spring ovulatory (SOV) seasons in mares. The main findings observed in the granulosa, theca interna, and/or all follicle layers during the SOV season compared with the SAN season were (a) small-sized follicles (10-14 mm) had greater epidermal growth factor receptor (EGFR) and Bcl-2 expression; (b) medium-sized follicles during the expected deviation/selection diameter (20-24 mm) had greater expression of EGFR, Ki-67, luteinizing hormone receptor (LHR), and Bcl-2; and (c) dominant follicles (30-34 mm) had greater EGFR, Ki-67, vascular endothelial growth factor, LHR, and Bcl-2 expression. Estradiol related receptor alpha expression and intrafollicular estradiol concentration increased, along with an increase in follicle diameter in both seasons. In this study, the application of the FWB technique allowed a direct comparison of different receptors' expression among follicles in different stages of development and between two seasons using the same individuals, without jeopardizing their ovarian function. The successful utilization of the FWB technique and the mare as an experimental animal offer a great combination for future folliculogenesis studies on mechanisms of follicle selection, development, and ovulation in different species, including women.

Fat harvesting site is an important determinant of proliferation and pluripotency of adipose-derived stem cells.

To define the optimal fat harvest site and detect any potential differences in adipose-derived stem cells (ASCs) proliferation properties in camels, aspirates from the abdomen and hump sites were compared. Obtained results revealed that ASCs from both abdomen and hump exhibited spindle-shaped and fibroblast-like morphology with hump-derived ASCs being smaller in size and narrower in overall appearance than abdominal ASCs. Abdominal ASCs required a greater time for proliferation than the hump-derived cells. These results were further confirmed with a tetrazolium-based colorimetric assay (MTT) which showed a greater cell proliferation rate for hump ASCs than for the abdomen. Under inductive conditions, ASCs from both abdominal and hump fat deposits maintained their lineage differentiation potential into adipogenic, chondrogenic, and osteogenic lineages during subsequent passages without any qualitative difference. However, expression of alkaline phosphatase was higher in osteogenic differentiated cells from the hump compared with those of the abdomen. Moreover, the increase in calcium content in hump-derived stem cells was higher than that in abdominal-derived stem cells. In conclusion, our findings revealed that ASCs can be obtained from different anatomical locations, although ASCs from the hump fat region may be the ideal stem cell sources for use in cell-based therapies.

Linolenic acid improves oocyte developmental competence and decreases apoptosis of in vitro-produced blastocysts in goat.

The effects of α-linolenic acid (ALA) on developmental competence of oocytes in goats were evaluated in this study. Initially, the level of ALA in small and large antral follicles was determined to be in a range of 0.018-0.028 mg/ml (64.6-100.6 µM, respectively). In vitro maturation was performed in the presence of various concentrations (10, 50, 100, or 200 µM) of ALA. Cumulus expansion, meiotic maturation, levels of intracellular glutathione (GSH), embryonic cleavage, blastocyst formation following parthenogenetic activation (PA) and in vitro fertilization (IVF), number of total and apoptotic cells in blastocyst, and expression of Bax, Bcl-2, and p53 genes in blastocyst cells were determined. Compared with the control, no improvement was observed in cumulus expansion in ALA-treated groups. At 50 µM concentration, ALA increased meiotic maturation rate but had no effect on GSH level. When oocytes treated with 50 µM ALA were subsequently used for PA or IVF, a higher rate of blastocyst formation was observed, and these embryos had a higher total cell number and a lower apoptotic cell number. Expression analyses of genes in blastocysts revealed lesser transcript abundances for Bax gene, and higher transcript abundances for Bcl-2 gene in 50 µM ALA group. Expression of p53 gene was also less observed in ALA-treated blastocysts. Our results show that ALA treatment at 50 µM during in vitro maturation (IVM) had a beneficial effect on maturation of goat oocytes and this, in turn, stimulated embryonic development and regulated apoptotic gene expression.

In vitro culture of bovine preantral follicles: a review.

Preantral follicles are the majority of the ovarian follicle population and their use as a source of homogeneous oocytes for bovine reproductive biotechnologies could result in a substantial advance in this field. However, while in other species embryos and offspring have been produced, in bovine species the results have been limited to the follicular activation of small (primordial) preantral follicles and formation of early antral follicles from large (secondary) preantral follicles after in vitro culture. Therefore, this review will highlight the basic aspects of bovine folliculogenesis by focusing on preantral follicles, the methods of harvesting preantral follicles, the main results from in vitro follicular culture during the last 20 years, and the potential candidate substances (basic supplements, growth factors, and hormones) for improving the efficiency of in vitro follicle growth.

Association of glucose-6-phosphate dehydrogenase activity with oocyte cytoplasmic lipid content, developmental competence, and expression of candidate genes in a sheep model.

PURPOSE: To evaluate associations of glucose-6-phosphate dehydrogenase (G6PDH) activity in sheep oocytes with cytoplasmic lipid content, maturational competence, developmental competence to the blastocyst stage, and gene expression of certain molecular markers. METHODS: Before brilliant cresyl blue (BCB) staining test, oocytes were classified as high, middle, and low cytoplasmic lipid content (HCLC, MCLC, and LCLC) and after the test as having low or high G6PDH-activity (BCB(+) and BCB(-), respectively). After maturation in vitro, a group of oocytes were subjected to IVF followed by in vitro embryo culture and another group was used for evaluation of expression of candidate genes. RESULTS: The cleavage and blastosyst rates were lowest (P < 0.05) in LCLC group, intermediate (P < 0.05) in MCLC group and highest (P < 0.05) in HCLC group. More (P < 0.05) oocytes in HCLC group were BCB(+), and higher (P < 0.05) maturation, cleavage, and blastocyst rates were seen for BCB(+) oocytes than the BCB(-) oocytes. Our gene expression data indicated that mRNA transcript abundance of ITGB2, pZP3, BMP15, and GDF9 genes was similar between BCB oocytes groups. However, the expression of ATP1A1 was higher (P < 0.05) for BCB(+) oocytes compared to BCB(-) oocytes. In addition, BAX transcript abundance was similar (P > 0.05) among BCB(+), BCB(-), and control groups, before and after maturation in vitro. CONCLUSION: Activity of G6PDH in sheep oocytes is highly associated with lipid content, and compared with the morphological parameters might be a more precise and objective predictor for subsequent developmental competence in vitro.

Gene Expression During Early Folliculogenesis in Goats Using Microarray Analysis.

Understanding of gene expression and metabolic, biological and physiological pathways in ovarian follicular development can have a significant impact on the dynamics of follicular atresia or survival. In fact, some oocyte loss occurs during the transition from secondary to early tertiary follicles. This study aimed to understand, by microarray analysis, the temporal changes in transcriptional profiles of secondary and early antral (tertiary) follicles in caprine ovaries. Ovarian follicles were microdissected and pooled to extract total RNA. The RNA was cross hybridized with the bovine array. Among 23,987 bovine genes, a total of 14,323 genes were hybridized with goat mRNAs while 9,664 genes were not. Of all the hybridized genes, 2,466 were stage-specific, up- and down-regulated in the transition from secondary to early tertiary follicles. Gene expression profiles showed that three major metabolic pathways (lipid metabolism, cell death, and hematological system) were significantly differentiated between the two follicle stages. In conclusion, this study has identified important genes and pathways which may potentially be involved in the transition from secondary to early tertiary follicles in goats.

Dominant follicle growth patterns and associated endocrine dynamics in anovulatory and ovulatory waves in women.

Growth patterns and associated endocrine profiles were compared between dominant anovulatory (ADF) and ovulatory follicles (OvF) developing from different waves within and between menstrual cycles in women. Follicular mapping profiles of 49 healthy women of reproductive age and blood samples were obtained every 1-3 days. Sixty-three dominant follicles were classified into wave 1 (W1ADF; n = 8) and wave 2 (W2ADF; n = 6) anovulatory follicles and wave 2 (W2OvF; n = 33) and wave 3 (W3OvF; n = 16) ovulatory follicles. Comparisons were made between W1ADF and W2ADF, W2ADF and W2OvF, and W2OvF and W3OvF. The waves were numbered 1, 2, or 3 based on when the waves emerged relative to the preceding ovulation. W1ADF emerged closer to the preceding ovulation, and W2ADF emerged in the late luteal or early follicular phase. The interval from emergence to maximum diameter was shorter for W2ADF than W1ADF and for W3OvF than W2OvF. Selection of W3OvF occurred at a smaller diameter compared to W2OvF. W1ADF regressed at a faster rate than W2ADF. Also, W1ADF were associated with lower mean FSH and higher mean estradiol than W2ADF. In contrast, W3OvF were associated with higher FSH and LH compared to W2OvF. However, W2OvF were associated with higher progesterone than W3OvF. This study contributes to the understanding of the physiologic mechanisms underlying selection of the dominant follicle, ovulation, and pathophysiology of anovulation in women, as well as optimization of ovarian stimulation protocols for assisted reproduction.

Ethanol, Carnoy, and paraformaldehyde as fixative solutions for histological evaluation of preantral follicles in equine ovarian tissue.

The most adequate fixative solution for equine ovarian tissue is still to be determined as a tool to evaluate the improvement of methodological studies in assisted reproductive techniques and fertility preservation. This study aimed to evaluate a short-time ethanol 70% (ST-EtOH, 45 min) exposure as an alternative fixative compared with two classically fixatives [Carnoy's (CAR) solution and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h). The end points evaluated were morphology and classes of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters in equine ovarian tissue. Ovaries (n = 6) from ovariectomized young mares were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1444 histological slides. The ST-EtOH similarly preserved the preantral follicle morphometry and stromal cell density compared to the PFA fixative, regardless of the exposure time. Nonetheless, the CAR fixative solution had the greatest percentage of normal preantral follicles and the highest stromal cell density among all treatments. In conclusion, Carnoy's solution must be preferred compared with ST-EtOH and PFA fixatives for studies concerning the cellular morphology of equine ovarian tissue. Moreover, ST-EtOH fixative is a good alternative for equine ovarian tissue when a quick histological evaluation is required instead of more time-consuming and expensive techniques. Additional studies concerning the impact of different fixatives on the ultrastructure of cellular populations and their compatibility with IHC and molecular techniques in equine ovarian tissue are warranted.

Emergence and selection of the dominant follicle and gonadotropin dynamics in postpartum lactating versus non-postpartum cycling mares.

Among female livestock, the mare has the shortest interval from parturition to first ovulation. Due to the scarcity of research on postpartum mares, little progress has been made on the characterization of the resumption of ovarian cyclicity after parturition. This study compared follicular and gonadotropin dynamics during follicle emergence and deviation in postpartum lactating (PP Lactating) versus non-postpartum cycling (N-PP Cycling) mares. On the day of parturition, every PP Lactating mare was paired with a N-PP Cycling mare. Comparisons were made by considering the partum-ovulation interval and the postpartum interovulatory interval for the PP Lactating mares, and two interovulatory intervals for the N-PP Cycling mares. The results presented herein demonstrate that during the postpartum period, lactating mares have some similarities in follicular and hormonal profiles around emergence and deviation when compared with non-postpartum cycling mares. However, some peculiar and important characteristics were noticed during the postpartum period in lactating mares: (1) The emergence of the DF occurs around the day of parturition; (2) follicle deviation in the ovulatory wave occurs earlier during the foal heat than in other intervals; (3) lower FSH and LH systemic concentrations were not detrimental enough to prevent the rapid resumption of ovarian activity just after parturition; and (4) the association between parturition and season can have an additional and confounding effect during postpartum ovarian activity in mares. The novel findings of this study provide better knowledge of the resumption of ovarian activity after parturition and may help provide insight into the reproductive management of this species.

Preantral follicle population and distribution in the horse ovary.

Characterization of the ovarian preantral follicle population is a necessary step to improve understanding of folliculogenesis and ovarian physiology. Therefore, in the present study, the preantral follicle population in the equine ovary in young and old mares was investigated according to follicular morphology, follicular class, distance from the geometric center using ovarian maps, and follicular density within ovarian portions (lateral vs intermediary) and regions (dorsal vs ventral). Ovaries were collected from an abattoir and histologically processed for evaluation, and the follicle population was calculated. Overall, in the current detailed study, a higher preantral follicle population per mare ovary (mean: 82,206 ± 50,022; range: 1477 to 773,091) than originally reported was identified. Additionally, a mare age effect was observed in the follicle population (young: 152,664 vs old: 11,750) and the spatial distribution of morphologically normal and abnormal follicles and the density and population of follicular classes. These results demonstrate that, in addition to the preantral follicle population in the mare ovary being comparable to that of other species, the location and spatial distribution of these follicles is dynamic and varies depending on mare age and follicle status (i.e. morphology and developmental stage). The characterization of the distribution and population of preantral follicles in the mare ovary provided by this study can potentially aid in improving reproductive studies and assisted reproductive techniques and may expand the understanding of mechanisms involving ovarian plasticity and follicular migration. Lay summary: Knowledge of the distribution and population of immature eggs within follicles (preantral follicles) in the ovaries of mares can improve approaches to assisted reproductive techniques and fertility preservation. As the existing research on horse preantral follicle population was focused solely on large follicles, the present study provides an updated investigation of small and large preantral follicles in the mare, showing that the population is similar to those in other species. This study also shows that the way these follicles are distributed in the ovary varies depending on age and follicle characteristics. Results from this study may help to highlight which areas of the mare ovary should be looked at to find samples of good-quality follicles.

Tributyltin and the Female Hypothalamic-Pituitary-Gonadal Disruption.

The hypothalamic-pituitary-gonadal (HPG) axis is the principal modulator of reproductive function. Proper control of this system relies on several hormonal pathways, which make the female reproductive components susceptible to disruption by endocrine-disrupting chemicals such as tributyltin (TBT). Here, we review the relevant research on the associations between TBT exposure and dysfunction of the female HPG axis components. Specifically, TBT reduced hypothalamic gonadotropin-releasing hormone (GnRH) expression and gonadotropin release, and impaired ovarian folliculogenesis, steroidogenesis, and ovulation, at least in part, by causing abnormal sensitivity to steroid feedback mechanisms and deleterious ovarian effects. This review covers studies using environmentally relevant doses of TBT in vitro (1 ng-20 ng/ml) and in vivo (10 ng-20 mg/kg) in mammals. The review also includes discussion of important gaps in the literature and suggests new avenue of research to evaluate the possible mechanisms underlying TBT-induced toxicity in the HPG axis. Overall, the evidence indicates that TBT exposure is associated with toxicity to the components of the female reproductive axis. Further studies are needed to better elucidate the mechanisms through which TBT impairs the ability of the HPG axis to control reproduction.

Ovarian and Uterine Dynamics During the Estrous Cycle in Criollo Breed Mares.

After having been brought to America 400 years ago, the Criollo horse reproduced freely for centuries in the southern part of the American continent. Roughness, resistance, and endurance are typical characteristics of this breed of horses. Although the natural selection that occurred over the centuries may have positively influenced the fertility and longevity of this breed, information regarding ovarian function and other reproductive aspects of Criollo breed mares under natural or controlled management conditions is scarce. The objectives of the present study were to (1) characterize the follicular dynamics of Criollo breed mares, (2) characterize growth and regression of the corpus luteum, and (3) study the cervical and uterine tone and ultrasonographic echotexture changes during two consecutive estrous cycles. In this study, 26 interovulatory intervals (IOI) were evaluated in 13 mares. Spontaneous estrous cycles were characterized by the following: (1) the IOI length was 21 days; (2) dominant and subordinate follicles emerged together at day 5 (ovulation = day 0), and their growth rates were slower until the day of deviation; (3) the deviation in growth rates between the two largest follicles of the ovulatory wave occurred when the dominant and subordinate follicles reached 22 and 21 mm in diameter, respectively; (4) the mean diameter of the preovulatory follicle at the day before ovulation was 43 mm; (5) different combinations of follicular waves were observed in each interovulatory interval; (6) the corpus luteum reached its maximum diameter about 2 days after ovulation and decreased gradually afterward; (7) after ovulation, the cervical and uterine tones were positively correlated and remained elevated until the onset of the expected luteolysis (days 12-14); and (8) after the expected luteolysis, the endometrial echotexture started to increase and reached maximum values 5-3 days before ovulation, when it started to decrease. Moderate-to-strong positive correlations between IOIs revealed repeatability within animals for the diameter of the preovulatory follicle at maximum and at the day before ovulation, cervical and uterine tones, endometrial echotexture, and corpus luteum diameter. The findings herein presented are of fundamental importance for a better understanding of the reproductive physiological patterns of the estrous cycle in the Criollo breed mare.

Characterization of preantral follicle clustering and neighborhood patterns in the equine ovary.

Understanding the transition from quiescent primordial follicles to activated primary follicles is vital for characterizing ovarian folliculogenesis and improving assisted reproductive techniques. To date, no study has investigated preantral follicle crowding in the ovaries of livestock or characterized these crowds according to follicular morphology and ovarian location (portions and regions) in any species. Therefore, the present study aimed to assess the crowding (clustering and neighborhood) patterns of preantral follicles in the equine ovary according to mare age, follicular morphology and developmental stage, and spatial location in the ovary. Ovaries from mares (n = 8) were collected at an abattoir and processed histologically for evaluation of follicular clustering using the Morisita Index and follicular neighborhoods in ovarian sections. Young mares were found to have a large number of preantral follicles with neighbors (n = 2,626), while old mares had a small number (n = 305). Moreover, young mares had a higher number of neighbors per follicle (2.6 ± 0.0) than old mares (1.2 ± 0.1). Follicle clustering was shown to be present in all areas of the ovary, with young mares having more clustering overall than old mares and a tendency for higher clustering in the ventral region when ages were combined. Furthermore, follicles with neighbors were more likely to be morphologically normal (76.5 ± 6.5%) than abnormal (23.5 ± 6.5%). Additionally, morphologically normal activated follicles had increased odds of having neighbors than normal resting follicles, and these normal activated follicles had more neighbors (2.6 ± 0.1) than normal resting follicles (2.3 ± 0.1 neighbors). In the present study, it was demonstrated that preantral follicles do crowd in the mare ovary and that clustering/neighborhood patterns are dynamic and differ depending on mare age, follicular morphology, and follicular developmental stage.