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.

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.

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.

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.

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.

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.

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.

Reproductive patterns and follicular waves in postpartum lactating versus non-postpartum cycling mares.

This comparative study between postpartum lactating (PP Lactating) and non-postpartum cycling (N-PP Cycling) mares aimed to characterize reproductive patterns, types and frequencies of follicular waves, corpus luteum and endometrial echotexture dynamics, and the influence of season and body condition. Mares from each group were paired considering the day of parturition of a PP Lactating mare. The partum-ovulation interval (POI) and the postpartum interovulatory interval (PPIOI) were evaluated for PP Lactating mares, and 2 IOIs were evaluated for N-PP Cycling mares. The following observations were made: (i) PP Lactating mares have several different reproductive patterns, such as continuous reproductive activity (i.e., short or long POIs followed by a PPIOI), ovarian inactivity after the first postpartum ovulation, or continuous ovarian inactivity (postpartum anestrous phase); (ii) a greater total number of minor waves was seen in PP Lactating mares; (iii) major primary follicular waves (i.e., ovulatory) emerge around the day of parturition in mares with short POIs; (iv) the season of parturition (spring season), decrease in body condition score, and body-weight loss can have an associated detrimental effect in PP Lactating mares by increasing the total number of minor follicular waves and, consequently, the POI length; (v) endometrial echotexture scores are higher during the POI and can be influenced by the season of parturition; and (vi) corpus luteum development and demise are similar between PP Lactating and N-PP Cycling mares. This study provides, for the first time, detailed information about reproductive physiological aspects during the postpartum period and may facilitate the interpretation of gynecological practices during the foal heat and subsequent IOI in mares.

Heterotopic autotransplantation of ovarian tissue in a large animal model: Effects of cooling and VEGF.

Heterotopic and orthotopic ovarian tissue autotransplantation techniques, currently used in humans, will become promising alternative methods for fertility preservation in domestic and wild animals. Thus, this study describes for the first time the efficiency of a heterotopic ovarian tissue autotransplantation technique in a large livestock species (i.e., horses) after ovarian fragments were exposed or not to a cooling process (4°C/24 h) and/or VEGF before grafting. Ovarian fragments were collected in vivo via an ultrasound-guided biopsy pick-up method and surgically autografted in a subcutaneous site in both sides of the neck in each mare. The blood flow perfusion at the transplantation site was monitored at days 2, 4, 6, and 7 post-grafting using color-Doppler ultrasonography. Ovarian grafts were recovered 7 days post-transplantation and subjected to histological analyses. The exposure of the ovarian fragments to VEGF before grafting was not beneficial to the quality of the tissue; however, the cooling process of the fragments reduced the acute hyperemia post-grafting. Cooled grafts compared with non-cooled grafts contained similar values for normal and developing preantral follicles, vessel density, and stromal cell apoptosis; lower collagen type III fibers and follicular density; and higher stromal cell density, AgNOR, and collagen type I fibers. In conclusion, VEGF exposure before autotransplantation did not improve the quality of grafted tissues. However, cooling ovarian tissue for at least 24 h before grafting can be beneficial because satisfactory rates of follicle survival and development, stromal cell survival and proliferation, as well as vessel density, were obtained.

Spatial distribution of preantral follicles in the equine ovary.

Comprehensive studies on spatial distribution of preantral follicles in the ovary are scarce. Considering that preantral follicles represent the main ovarian reserve, harvesting of these follicles is crucial for the development/use of assisted reproductive techniques. Therefore, knowledge on follicle spatial distribution can be helpful for targeting areas with richer number of preantral follicles through biopsy procedures. The aim of this study was to assess the distribution and localization of equine preantral follicles according to: (i) age, (ii) ovarian portion (lateral and intermediary) and region (dorsal and ventral), (iii) distance from the geometric center, and (iv) follicular class. Ovaries from young and old mares (n = 8) were harvested in a slaughterhouse and submitted to histological processing for further evaluation. For data analyses, a novel methodology was developed according to the geometric center of each histological section for a precise determination of preantral follicle distribution. Results indicated that (i) equine preantral follicles are clustered and located near to the ovarian geometric center, and that aging induced their dispersion through the ovarian cortex; (ii) the distance from the geometric center was shorter for developing follicles than primordial; and (iii) secondary follicles were more distant from the geometric center but closer to the ovulation fossa. In conclusion, the spatial distribution of preantral follicles was successfully determined in the equine ovary and was affected by age, region, and portion.

Central Nervous System and Vertebrae Development in Horses: a Chronological Study with Differential Temporal Expression of Nestin and GFAP.

The neural system is one of the earliest systems to develop and the last to be fully developed after birth. This study presents a detailed description of organogenesis of the central nervous system (CNS) at equine embryonic/fetal development between 19 and 115 days of pregnancy. The expression of two important biomarkers in the main structure of the nervous system responsible for neurogenesis in the adult individual, and in the choroid plexus, was demonstrated by Nestin and glial fibrillary acid protein (GFAP) co-labeling. In the 29th day of pregnancy in the undifferentiated lateral ventricle wall, the presence of many cells expressing Nestin and few expressing GFAP was observed. After the differentiation of the lateral ventricle wall zones at 60 days of pregnancy, the subventricular zone, which initially had greater number of Nestin+ cells, began to show higher numbers of GFAP+ cells at 90 days of pregnancy. A similar pattern was observed for Nestin+ and GFAP+ cells during development of the choroid plexus. This study demonstrates, for the first time, detailed chronological aspects of the equine central nervous system organogenesis associated with downregulation of Nestin and upregulation of GFAP expression.

The Mare Model to Study the Effects of Ovarian Dynamics on Preantral Follicle Features.

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.