Small Ovarian Follicles Research Articles

Ovum Pick Up and in vitro Embryo Production using Sex Sorted Semen in Cows

Background: Ovum pick-up, a technique for extracting immature oocytes from antral follicles, with in vitro embryo production, is a way to produce large number of embryos from live donors. Methods: By transvaginal ultrasound-guided ovum pick-up, oocytes were collected from large (9-19 mm), medium (4-8 mm) and small (2-3 mm) ovarian follicles of donor cows (n=8). The recovered oocytes were classified as A, B, C, D and E grades. Oocytes were subjected toin vitro maturation, fertilization (with sex sorted semen) and culture. Cleavage and blastocyst rates were recorded on day 4 and day 7 of culture respectively. Result: Total of 278 follicles were aspirated. The follicles aspirated per cow per OPU session was 12.67±1.86. The oocyte recovery rate was 45.31±7.35. Mean percentage of grade A, B, C, D and E oocytes was 15.69±3.91, 24.97±3.02, 37.53±3.45, 19.44±3.76 and 2.38±2.38 respectively. In vitro maturation rate (IVM%) was 91.71±2.38% and fertilization rate was 90.18±1.52%. Cleavage and blastocyst rates observed was 55.1±5.90 and 24.81±2.95% respectively. The mean percentages of different grades of embryos observed was, code 1: 21.13±2.78, code 2: 14.41±3.25, code 3: 19.56±4.11 and code 4: 44.9±5.90%. Transvaginal ovum pick up can be performed repeatedly without hormonal stimulation. Sex sorted semen can be used for in vitro production of female embryos.

Meiotic and developmental competence of growing pig oocytes derived from small antral follicles is enhanced in culture medium containing FGF2, LIF, and IGF1 (FLI medium)

BackgroundOocytes of large animal species isolated from small ovarian follicles (< 2 mm) are less competent to support early embryonic development after in vitro maturation and fertilization than their counterparts isolated from medium-sized and preovulatory follicles. This study aimed to assess the effect of a new maturation medium containing FGF2, LIF, and IGF1 (FLI medium) on the meiotic and developmental competence of pig cumulus-oocytes complexes (COCs) derived from the small and medium-sized follicles.MethodsThe growing oocytes were isolated from 1 to 2 (small follicle; SF) and the fully-grown ones from 3 to 6 (large follicle; LF) mm follicles and matured in a control M199 medium with gonadotropins and EGF and the FLI medium enriched by the triplet of growth factors. The matured oocytes were parthenogenetically activated and cultured to the blastocyst stage. Chromatin configuration before and during the culture and MAP kinase activity were assessed in the oocytes. Finally, the expression of cumulus cell genes previously identified as markers of oocyte quality was assessed.ResultsThe maturation and blastocyst rates of oocytes gained from LF were significantly higher than that from SF in the control medium. In contrast, similar proportions of oocytes from LF and SF completed meiosis and developed to blastocysts when cultured in FLI. Most of the oocytes freshly isolated from SF possessed germinal vesicles with fine filaments of chromatin (GV0) or chromatin surrounding the nucleolus (GVI; 30%); the oocytes from LF were mainly in GVI (or GVII) exhibiting a few small lumps of chromatin beneath the nuclear membrane. When cultured in the FLI medium for 16 h, an acceleration of the course of maturation in oocytes both from SF and LF compared to the control medium was observed and a remarkable synchrony in the course of chromatin remodeling was noticed in oocytes from SF and LF.ConclusionsThis work demonstrates that the enrichment of culture medium by FGF2, LIF, and IGF1 can enhance the meiotic and developmental competence of not only fully-grown, but also growing pig oocytes and significantly thus expanding the number of oocytes available for various assisted reproductive technology applications.

Transcriptome profiling reveals SLC5A5 regulates chicken ovarian follicle granulosa cell proliferation, apoptosis, and steroid hormone synthesis

The egg-laying performance of hens holds significant economic importance within the poultry industry. Broody inheritance of the parent stock of chickens can result in poor options for the improvement of egg production, and is a phenomenon influenced by multiple genetic factors. However, few studies have been conducted to delineate the molecular mechanism of ovarian regression in brooding chickens. Here, we explored the pivotal genes responsible for the regulation of ovarian follicles in laying hens, using RNA-sequencing analysis on the small ovarian follicles from broody and laying chickens. Sequencing data analysis revealed the differential expression of 200 genes, with a predominant enrichment in biological processes related to cell activation and metabolism. Among these genes, we focused on solute carrier family 5 member 5 (SLC5A5), which exhibited markedly higher RNA expression levels in follicles from laying compared with broody chickens. Subsequent cellular function studies with knockdown of SLC5A5 in chicken ovarian follicle granulosa cells (GCs) led to the down-regulation of genes associated with cell proliferation and steroid hormone synthesis, and concurrent promotion of gene expression linked to apoptosis. These findings indicated that SLC5A5 deficiency led to the inhibition of proliferation, steroid hormone synthesis and secretion, and promotion of apoptosis in chicken GCs. Our study demonstrated a pivotal role for SLC5A5 in the development and function of chicken GCs, shedding light on its potential significance in the broader context of chicken ovarian follicle development, and providing a prospective target to improve the egg-laying performance of chickens via molecular marker-assisted breeding technology.

Assessment of folliculogenesis in ovarian tissue from young patients with Turner syndrome using a murine xenograft model

To study the impact of aneuploid granulosa and stromal cells on folliculogenesis of small ovarian follicles from mosaic Turner syndrome patients using a murine xenograft model. Laboratory study SUBJECTS: Ovarian cortical tissue was obtained by laparoscopic surgery from 18 mosaic TS patients (aged 5-19 years) and 13 controls (aged 5-18 years). Part of each tissue fragment was used to karyotype ovarian cells in non-grafted tissue by fluorescence in situ hybridization. The remaining tissue was xenografted to severe combined immunodeficient mice for 5 months. Grafted tissue was analyzed for aneuploidy, and follicle density and morphology were determined. Expression of proliferating cell nuclear antigen and anti-Müllerian hormone were investigated by immunohistochemistry. The impact of aneuploid granulosa and stromal cells on folliculogenesis. Fluorescence in situ hybridization of ovarian tissue before grafting was performed to determine the level of aneuploidy in stromal cells, and oocytes and granulosa of small follicles. After xenografting the level of aneuploidy of the newly formed layers of granulosa cells was again determined in secondary and antral follicles. Follicle density in ovarian tissue from Turner syndrome patients was significantly lower than in controls before grafting. Fluorescence in situ hybridization analysis confirmed that 101/104 oocytes from non-grafted tissue of Turner syndrome patients showed normal X chromosome content, while granulosa and stromal cells were mainly 45,X. Fragments from 12 Turner syndrome patients contained follicles at all stages after xenografting, including secondary and antral follicles. Follicle density in Turner syndrome patients and controls decreased significantly after grafting. Moreover, a shift from high to low proportions of 45,X granulosa cells was observed during folliculogenesis. Expression of PCNA in follicles from TS patients increased significantly during grafting. Secretion of AMH was impaired before grafting in peri-/postpubertal TS girls, but recovered after grafting. Our study showed that small follicles from mosaic Turner syndrome patients undergo folliculogenesis, despite the presence of aneuploid granulosa and stromal cells. Ovarian tissue cryopreservation could therefore be a valid option to preserve fertility in young mosaic Turner syndrome patients if sufficient numbers of follicles are present, thus preferably before the age of 12.

Effects of asprosin on estradiol and progesterone secretion and proliferation of bovine granulosa cells

Asprosin is an adipokine synthesized by the white adipose tissue that regulates glucose homeostasis and that has been reported to affect bovine theca cell function and follicular growth, but its role on granulosa cell functions remains to be unveiled. Hence, the objective of this study was to investigate asprosin impacts on granulosa cell steroidogenesis. Bovine granulosa cells from small ovarian follicles were cultured in vitro to investigate the effects of asprosin on cell proliferation, production of steroids, mRNA abundance of genes that encode steroidogenic enzymes and cell cycle regulators, and protein relative abundance of steroidogenic signaling pathways. Asprosin was shown to affect granulosa cell functions in a dose-dependent manner. In the presence of FSH, asprosin enhanced estradiol production and stimulated an increase in mRNA expression of FSHR and CYP19A1 in a dose-dependent manner. In the presence of IGF1, asprosin decreased estradiol production, increased progesterone production, altered PKA relative protein expression, and tended to alter the ratio of p-ERK1/2/total ERK1/2 protein expression in a dose-dependent manner. Furthermore, asprosin increased p-53 gene expression in basal culture conditions and with or without FSH and IGF1. Taken together, findings of this study show that asprosin is a regulator of granulosa cell functions and the effects of asprosin depend on dose and cell culture conditions.

Identification of curcumin as a novel potential drug for promoting the development of small ovarian follicles for infertility treatment.

In-vitro fertilization is an effective treatment for various causes of infertility. However, management of women with poor ovarian response or premature ovarian insufficiency remains challenging because these women have underdeveloped small ovarian follicles that do not respond to hormone treatment. In-vitro activation of small follicles has been developed but its efficiency has much room for improvement. In the current study, we provide several lines of evidence showing that curcumin, an FDA-approved traditional medicine, can specifically promote the development of mouse ovarian follicles from the primary to secondary stage, which greatly potentiates these small follicles for subsequent in-vivo development into antral follicles that can be ovulated. Mechanistically, we show that curcumin promotes the proliferation and differentiation of granulosa cells and the growth of oocytes by activating the phosphatidylinositol 3 kinase (PI3K) signaling pathway. Most importantly, we show that in-vitro treatment of human ovarian tissues with curcumin can promote the in-vivo survival and development of small human ovarian follicles, showing that curcumin can be used as a potential drug to increase the success rate of in-vitro activation of small human follicles. We thus identify curcumin as a novel potential drug for promoting the development of small human ovarian follicles for infertility treatment.

Effect of repeated low doses of GnRH on follicular development and ovulation in anovulatory dairy cows with follicle growth to emergence size.

The aim of the study was to evaluate the effect of repeated low doses of GnRH agonist buserelin once a day for 5 days on follicle development and ovulation in anovulatory dairy cows with follicles growth only to emergence. The study was conducted on 71 anovulatory Polish Holstein Friesian cows. Anovulation with growth of follicles to emergence was defined as small ovaries with follicles of ≤ 5 mm in diameter and without corpus luteum on two examinations in a 7-10 day interval between 50-60 days after parturition. Cows were allocated to one of two group. Cows from group 1 (n = 58) received 0.4 μg of buserelin (Receptal, MSD, Poland) i.m. once a day for 5 days. Control cows from group 2 (n = 13) received saline. Ovarian structures were monitored weekly after the end of treatment by ultrasound for 4 weeks. The diameter of ovarian follicles on the ovaries was measured and recorded. Occurrence of ovulation was determined by the presence of corpus luteum. Overall, ovulation occurred in 46.6% (27/58) of cows treated with repeated doses of GnRH, while no corpus luteum was observed in the control group during the study period. There were significantly (p⟨0.05) more follicles 6-9 mm in diameter and 10-20 mm in diameter in cows treated with GnRH than in control cows. In conclusion, repeated low doses of GnRH analogue buserelin once a day for 5 days stimulate the development of ovarian follicles in anovulatory dairy cows with small ovarian follicles and led to ovulation in 46.6% of cows during 4 weeks after the end of the treatment.

Extracellular vesicles from follicular fluid may improve the nuclear maturation rate of in vitro matured mare oocytes

The in vitro maturation (IVM) of equine oocytes is still not efficient and does not yield consistent results. The specific requirements of equine oocytes during this process are still largely unknown, which hinders the development of assisted reproductive techniques (ART) in this species. Because the ovarian follicle microenvironment supports oocytes in their acquisition of developmental competence, follicular fluid seems to be a substantial source of bioactive factors that could support the IVM process. Extracellular vesicles (EVs) are cell-secreted molecules in body fluids that are able to deliver molecular signals and transfer genetic information (mRNA, miRNA) between donor and recipient cells. Hence, our hypothesis is that follicular fluid EVs (ffEVs) from small (<20 mm) ovarian follicles can improve the in vitro maturation rate of mare oocytes. To test our hypothesis, equine ovarian follicular fluid was aspirated and ffEVs were isolated by ultracentrifugation, then characterized using nanoparticle tracking analysis and flow cytometry. Additionally, ffEVs were labeled using the ExoGlow-protein EV labeling kit (System Biosciences, Palo Alto, CA). Cumulus-oocyte complexes (COCs) were matured using a one-step method (Method I, continuous culture for 24–38 h) or a two-step method (Method II, initial denudation after 24 h), in the presence (200 μg protein/ml) or absence of ffEVs. The results show the internalization of ffEVs by equine cumulus cells and, for the first time, also by oocytes. The ffEV treatment during two-step culture had a positive effect on the maturation rate of compacted COCs compared to the control group (45.7% and 20.5%, respectively; p < 0.05). No effect of supplementation was observed on the maturation rate during one-step culture. Our results indicate that the supplementation of culture media with EVs isolated from the follicular fluid of small follicles can improve the IVM rate of mare oocytes, suggesting that ffEVs play an important role during this process and may enhance the development of equine ART.

EFFECTS OF FAC CHEMOTHERAPY ON ANTI MULLERIAN HORMONE IN PREMENOPAUSAL BREAST CARCINOMA

Abstrak: Breast cancer is the most common carcinoma in women in the world. Anti-Mullerian Hormone (AMH) is a hormone secreted by primary, secondary, and small antral ovarian follicles of &lt;4mm. The chemotherapy regimen toxic to ovarian follicles is 5-fluorouracil-doxorubicin-cyclophosphamide (FAC). In Indonesia, there had been no study on the effect of FAC chemotherapy on AMH in premenopausal breast carcinoma patients. This study aims to determine the effect of FAC chemotherapy on AMH in premenopausal breast carcinoma patients. This study employed a prospective cohort study design. Inclusion criteria were breast carcinoma, premenopausal, and history of chemotherapy (-). Exclusion criteria were history of ovarian surgery, pelvic radiation, GnRH-agonist therapy, smoking for ≥10 years, obesity, and chemotherapy dropout. Subjects were examined for AMH before the 1st chemotherapy and after the 4th chemotherapy. Statistical test was done with Wilcoxon test, significant if p≤0.05. The average AMH level before FAC chemotherapy was 1.51±1.957; the average AMH level after the 4th FAC chemotherapy was 0.24±0.587. The result of the Wilcoxon test comparing AMH levels before and after 4th FAC chemotherapy was p&lt;0.0001. FAC chemotherapy reduces AMH patients with premenopausal breast carcinoma.Keywords: Breast carcinoma, chemotherapy, AMH.

Long-Term Changes in Ovarian Follicles of Gilts Exposed Neonatally to Methoxychlor: Effects on Oocyte-Derived Factors, Anti-Müllerian Hormone, Follicle-Stimulating Hormone, and Cognate Receptors.

In this paper, we investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities on ovarian follicles of adult pigs. Piglets were injected with MXC (20 μg/kg body weight) or corn oil (controls) from postnatal Day 1 to Day 10 (n = 5 per group). Then, mRNA expression, protein abundance and immunolocalization of growth and differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), anti-Müllerian hormone (AMH) and cognate receptors (ACVR1, BMPR1A, BMPR1B, TGFBR1, BMPR2, and AMHR2), as well as FSH receptor (FSHR) were examined in preantral and small antral ovarian follicles of sexually mature gilts. The plasma AMH and FSH levels were also assessed. In preantral follicles, neonatal exposure to MXC increased GDF9, BMPR1B, TGFBR1, and BMPR2 mRNAs, while the levels of AMH and BMP15 mRNAs decreased. In addition, MXC also decreased BMP15 and BMPR1B protein abundance. Regarding small antral follicles, neonatal exposure to MXC upregulated mRNAs for BMPR1B, BMPR2, and AMHR2 and downregulated mRNAs for AMH, BMPR1A, and FSHR. MXC decreased the protein abundance of AMH, and all examined receptors in small antral follicles. GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles of control and treated ovaries. All analyzed receptors were detected in the oocytes and granulosa cells of preantral follicles, and in the granulosa and theca cells of small antral follicles. The exception, however, was FSHR, which was detected only in the granulosa cells of small antral follicles. In addition, MXC decreased the plasma AMH and FSH concentrations. In conclusion, the present study may indicate long-term effects of neonatal MXC exposure on GDF9, BMP15, AMH, and FSH signaling in ovaries of adult pigs. However, the MXC effects varied at different stages of follicular development. It seems that neonatal MXC exposure may result in accelerated initial recruitment of ovarian follicles and impaired cyclic recruitment of antral follicles.

Modification of O-ring intrauterine devices (IUDs) in mares: Contraception without estrus suppression

A significant welfare concern for those within the horse industry is that of the overpopulation in free-roaming horses in the western US. The goal of the present study was to identify a humane and inexpensive means of providing contraception and reducing pregnancy rates in a way that could be applied to free-roaming horses on the western U.S. open range lands. A series of studies was conducted utilizing the previously-described silastic O-ring intrauterine devices (IUDs). Throughout these studies pregnancy was successfully prevented in mares with an O-ring IUD, but collective retention rates failed to exceed 50% regardless of size or durometer (material hardness/malleability). When the O-ring IUDs were compared to the recently-described Y-design IUDs (Holyoak et al., 2021) the Y-design IUDs had a markedly greater rate of retention; therefore, the Y-design IUDs would be most appropriate for potential use in free-roaming horses. Assessment of mare records revealed that the vast majority of mares continued to have ovulations and mating continued to occur while the IUDs were in place. A few mares had multiple small ovarian follicles when the IUD was in utero. Findings indicate that utilizing an IUD with the expectation of estrus suppression in mares would fail to be fruitful.

Deslorelin Slow-Release Implants Delay Ovulation and Increase Plasma AMH Concentration and Small Antral Follicles in Haflinger Mares.

Simple SummaryIn horses, oocyte collection followed by intra-cytoplasmatic sperm injection is increasingly used. The yield of oocytes is a limiting factor and depends on the number of follicles present on the ovary during oocyte collection. Therefore, the aim of this study was to analyze the effect of slow-release implants containing the GnRH analogue deslorelin on the number of follicles and on hormones regulating follicular development. Six mares received a deslorelin implant and six mares served as controls. The interval to the first spontaneous ovulation was prolonged in treated mares. The treatment changed the release pattern of the gonadotrophins LH and FSH. Changes in the number of follicles 10 to 15 mm in diameter were detected in deslorelin-treated mares. These changes were also reflected by increasing plasma anti-Muellerian hormone concentrations, a hormone produced by growing follicles. In conclusion, deslorelin implants induce changes in ovarian follicle subpopulations and could be a promising tool for the preparation of mares for assisted reproductive procedures.There is an increasing interest in the manipulation of ovarian follicular populations in large domestic animals because this could prove beneficial for assisted reproductive techniques such as ovum pick-up (OPU). The aim of the present study was to evaluate the effects of deslorelin slow-release implants (SRI) on the interovulatory interval, antral follicle count (AFC), number of follicles of different size ranges and plasma anti-Muellerian hormone (AMH) concentration in mares. To synchronize their estrous cycles, Haflinger mares (n = 12) were treated twice with a PGF2α analogue. One day after the second injection (day 0), mares received a 9.4 mg deslorelin SRI (group DES, n = 6) or 1.25 mg deslorelin in a short-acting formulation (CON; n = 6), respectively. Regular transrectal ultrasonography of the genital tract was performed and blood samples were collected for the analysis of progesterone, AMH and gonadotrophins. The interval from implant insertion to the first spontaneous ovulation was 23.8 ± 10.5 days in group DES compared to 17.0 ± 3.9 days in group CON (p < 0.05). For the concentrations of LH, FSH and AMH, interactions between time and treatment were detected (p < 0.05). The AFC and the mean number of follicles with 5 to 10, 10 to 15 and 15 to 20 mm in diameter changed over time (p < 0.05). A time x treatment interaction was demonstrated for follicles of 10 to 15 mm in diameter (p < 0.05). The changes in this follicular subpopulation were reflected by increased plasma AMH concentration in group DES. In conclusion, 9.4 mg deslorelin implants show minor effects with regard to estrus suppression in mares, whereas the changes in the subpopulation of small ovarian follicles could be a promising tool for preparation of mares for OPU.

Development of a Chemical Reproductive Aging Model in Female Rats.

Women are born with an abundant but finite pool of ovarian follicles, which naturally and progressively decreased during their reproductive years until menstrual periods stop permanently (menopause). Perimenopause represents the transition from reproductive to non-reproductive life. It is usually characterized by neuroendocrine, metabolic and behavioral changes, which result from a follicular depletion and reduced number of ovarian follicles. During this period, around 45-50 years old, women are more likely to express mood disorders, anxiety, irritability and vasomotor symptoms. The current animal models of reproductive aging do not successfully replicate human perimenopause and the gradual changes that occur in this phase. While the traditional rat model of menopause involves ovariectomy or surgical menopause consisting of the rapid and definitive removal of the ovaries resulting in a complete loss of all ovarian hormones, natural or transitional menopause is achieved by the selective loss of ovarian follicles (perimenopause period). However, the natural aging rodent (around 18-24 months) model fails to reach very low estrogen concentrations and overlaps the processes of somatic and reproductive aging. The chronic exposure of young rodents to 4-vinylcyclohexene diepoxide (VCD) is a well-established experimental model for perimenopause and menopause studies. VCD induces loss of ovarian small follicles (primary and primordial) in mice and rats by accelerating the natural process of atresia (apoptosis). The VCD, ovary-intact or accelerated ovarian failure (AOF) model is the experimental model that most closely matches natural human progression to menopause mimicking both hormonal and behavioral changes typically manifested by women in perimenopause. Graphical abstract: The female reproductive system is regulated by a series of neuroendocrine events controlled by central and peripheral components. (A). The mechanisms involved in this control are extremely complex and have not yet been fully clarified. In female mammals whose ovulation (the most important event in a reproductive cycle) occurs spontaneously, reproductive success is achieved through the precise functional and temporal integration of the hypothalamus-pituitary-ovary (HPO) axis. (B). In women, loss of fertility appears to be primarily associated with exhaustion of ovarian follicles, and this process occurs progressively until complete follicular exhaustion marked by the final menstrual period (FMP). (C). While in female rodents, reproductive aging seems to begin as a neuroendocrine process, in which changes in hypothalamic/pituitary function appear independently of follicular atresia. The traditional rat model of menopause, ovariectomy or surgical menopause consists of the rapid and definitive removal of the ovaries resulting in a complete loss of all ovarian hormones. (D). The chronic exposure (15-30 days) to the chemical compound 4-vinylcyclehexene diepoxide (VCD) in young rodents accelerates gradual failure of ovarian function by progressive depletion of primordial and primary follicles, but retains residual ovarian tissue before brain alterations that occurs in women in perimenopause. Low doses of VCD cause the selective destruction of the small preantral follicles of the ovary without affecting other peripheral tissues.

Weaned Sows with Small Ovarian Follicles Respond Poorly to the GnRH Agonist Buserelin.

Simple SummaryThis study evaluated the influence of mean ovarian follicle size and the season of weaning on the effectiveness of administering the GnRH agonist buserelin to synchronize ovulation in weaned sows. The results from 352 sows demonstrated that sows with small follicles (<0.5 cm in diameter) at treatment are poor responders, a condition more frequent among sows weaned in summer–autumn than in those weaned in winter–spring.The GnRH agonist buserelin (GnRH), used to synchronize ovulation in weaned sows, attains only 70–80% effectivity, owing to several reasons of ovarian origin. This study evaluated in particular whether mean ovarian follicle size at treatment and the season of weaning are among those influencing GnRH responsiveness. The experiment was carried out in a temperate-region farm with 352 sows of 1–6 parities weaned either in winter–spring (WS, 174 sows) or in summer–autumn (SA, 178 sows). The sows were randomized into two groups: GnRH (10 µg of buserelin acetate at 86 h after weaning, 172 sows) and control (180 sows). The ovaries were transrectally scanned from weaning to ovulation and the sows clustered according to their mean follicular size at treatment time: small (<0.5 cm in diameter), medium (0.5 to 0.64 cm) and large (0.65 to 1.09 cm). In total, 88.33% of the GnRH-treated sows ovulated, with 82% of them within the expected time window (120–132 h after weaning). In contrast, 95.45% of the unresponsive sows had small follicles at the time of treatment and were mostly weaned in SA (20.45%) than in WS (4.76%). In conclusion, the conspicuous presence of sows having small ovarian follicles at treatment time compromises the efficiency of the GnRH agonist buserelin to synchronize ovulation in weaned sows, which occurs more frequently in summer–autumn weaning.

Factors Affecting Ovarian Activity in Donkeys: The Effect of Season, Age and Body Condition Score (BCS)

During the period of year (representing spring, summer, autumn, and winter) Jennies at Giza Zoo abattoir were checked for body condition score (from 1 to 5), age (1 to 7 years; 8 to 14 years; and &gt;15 years old). After culling, ovaries of 377 Jennies were collected, two ovaries were weighed, and the number and size of ovarian follicles were recorded. Cumulus-Oocyte Complexes (COCs) were collected after the ovarian follicles have been sliced and scraped, and COCs number and quality were determined. Data indicated that right and left ovaries and overall ovarian weight were significantly increased (P&lt;0.05) in summer than the other seasons. The number of small size ovarian follicles was decreased (P&lt;0.05) in spring than other seasons, while oocytes number and quality were not significantly affected by season. Jennies of 1 to 7 years old showed a significant (P&lt;0.05) smaller left ovary and total ovarian weight than the two other groups. Jennies of 8-14 years old) have a significant high (P&lt;0.05) small size and a whole number of ovarian follicles than the other groups. Jennies with BCS-3 possess a significantly (P&lt;0.05) higher total ovarian weight, total number of ovarian follicles and the total number of COCs recovered than BCS-1 or BCS-4. While the BCS-4 group showed the lowest (P&lt;0.05) number of ovarian follicles and produced less (P&lt;0.05) number of COCs than the other groups. Conclusion: In Egyptian Jennies, ovarian activity was affected by season, BCS and age of the animal, while, COCs yield and quality were more affected by BCS.

Global transcriptome analysis of porcine oocytes in correlation with follicle size.

Although our knowledge regarding oocyte quality and development has improved significantly, the molecular mechanisms that regulate and determine oocyte developmental competence are still unclear. Therefore, the objective of this study was to identify and analyze the transcriptome profiles of porcine oocytes derived from large or small follicles using RNA high-throughput sequencing technology. RNA libraries were constructed from oocytes of large (LO; 3-6 mm) or small (SO; 1.5-1.9 mm) ovarian follicles and then sequenced in an Illumina HiSeq4000. Transcriptome analysis showed a total of 14,557 genes were commonly detected in both oocyte groups. Genes related to the cell cycle, oocyte meiosis, and quality were among the top highly expressed genes in both groups. Differential expression analysis revealed 60 up- and 262 downregulated genes in the LO compared with the SO group. BRCA2, GPLD1, ZP3, ND3, and ND4L were among the highly abundant and highly significant differentially expressed genes (DEGs). The ontological classification of DEGs indicated that protein processing in endoplasmic reticulum was the top enriched pathway. In addition, biological processes related to cell growth and signaling, gene expression regulations, cytoskeleton, and extracellular matrix organization were among the highly enriched processes. In conclusion, this study provides new insights into the global transcriptome changes and the abundance of specific transcripts in porcine oocytes in correlation with follicle size.

Effect of long-term food restriction on reproductive performances in female yellowtail, Seriola quinqueradiata

In aquaculture of species with a large body size, such as tuna and yellowtail, a major profitability concern is that broodstock management for egg collection incurs high feeding costs and labor. In order to reduce feeding costs, development of restricted feeding techniques without affecting the reproductive performance may be useful. To gain a better understanding of the potential significance of capital energy in reproduction of large fish with income breeding patterns, we examined the effects of long-term restricted feeding prior to the onset of oocyte development on reproductive performance in female yellowtail (Seriola quinqueradiata). A control group was fed to satiety, while experimental groups were restricted to 50 and 25% of the ration administered to the control group from July to December. The females subjected to restricted feeding had lower ovary weight and smaller ovarian follicles than did the control females in February. Moreover, plasma 17β-estradiol levels in food-restricted females were significantly lower than those in normally fed females in January and February. Furthermore, we found that food-restricted females had lower pituitary gene expression levels of gonadotropins (follicle-stimulating hormone and luteinizing hormone) in January. These results suggest that severe food restriction during the immature phase delays initiation of oocyte development via an alteration in pituitary gonadotropin production in yellowtail females. Once food intake was sufficient after switching to the satiation feeding regime, however, oocyte development rapidly advanced, resulting in the production of fully functional eggs. Overall, these findings demonstrate that the yellowtail displays a mixed strategy of capital-breeding patterns at the onset of sexual maturation, but stronger income-breeding patterns with its progression in reproduction with energy acquired during the vitellogenic phase. Additionally, food deprivation over 6months by 50% of the amount of food required for satiation during the immature phase did not change reproductive performance in female yellowtail, although the females in the 25% feeding group showed a lower percentage of normally hatching eggs. Therefore, a 50% restricted feeding regime before the onset of sexual maturation is acceptable for reducing feeding costs in broodstock management without affecting reproductive performance.

Mitotic index and morphological characteristics of ovarian small follicles from goats submitted to nutritionally unbalanced regimens.

The objective of this study was to assess the influence of nutritional regimens such as adequate feeding, restricted feeding, and underfeeding-refeeding on the follicle growth and development from caprine ovaries. Goats were divided into three different groups (n = 5 per group). For 24 weeks, goats received elephant grass plus concentrate to provide 1.5 (n = 5) and 0.72 (n = 10) times the energy requirements for maintenance of live weight. Underfed goats were subsequently refed for 6 weeks with the diet of the nourished group (1.5 times the energetic requirements of maintenance). Follicular morphology and morphometry, as well as granulosa cells mitotic index were assessed. Ovarian follicles were classified as small or large preantral follicles, or as small or large antral follicles. Ovarian volume was smaller in animals from both underfed and refed groups than in those animals from fed group. Although no difference in the total number of normal follicles was observed among the nutritional groups, underfed animals presented higher percentages of atretic preantral and small antral follicles when compared with fed animals. Large antral follicles from underfed and refed goats presented a lower mitotic index when compared with fed ones. In conclusion, ovaries from goats challenged with prolonged undernutrition will be functionally compromised, which is characterized by atresia of preantral and small antral follicles and decreased mitotic index of large antral follicles. Refeeding those animals will not recover ovarian function to a same level experienced by goats fed a diet with adequate energy requirements.

Three-dimensional culture of buffalo granulosa cells in hanging drop mimics the preovulatory follicle stage.

Granulosa cell (GC) culture models mimicking the intrafollicular environment are limited. Such models have a great potential in reproductive toxicity studies. The buffalo, a monovulatory species like humans, could be a better model than polyovulatory rodents. Therefore, we targeted the development and characterization of three-dimensional (3D) culture systems for buffalo GCs. The GCs from small ovarian follicles (SF) maintained the CYP19 gene expression for 144 hr in a 2D culture system. Hence, GCs from SF were cultured directly in 3D using hanging drop and Poly-([2-hydroxyethyl methacrylate]) (polyHEMA) methods in the DMEM media containing 1 ng/ml FSH and 10 ng/ml IGF-1 for 144 hr. The expression profile of nine GC-specific transcripts; CYP19, TNFAIP6, AMH, PTI, NR4A1, FSHR, RUNX, LHR, and COX2/PTGS2; revealed that 3D-spheroids developed in hanging drop method maintained the GC phenotype of preovulatory follicles. Therefore, hanging drop method is a best method for culturing GCs to mimic the intrafollicular environment.

Glutamate Cysteine Ligase Modifier Subunit (Gclm) Null Mice Have Increased Ovarian Oxidative Stress and Accelerated Age-Related Ovarian Failure.

Glutathione (GSH) is the one of the most abundant intracellular antioxidants. Mice lacking the modifier subunit of glutamate cysteine ligase (Gclm), the rate-limiting enzyme in GSH synthesis, have decreased GSH. Our prior work showed that GSH plays antiapoptotic roles in ovarian follicles. We hypothesized that Gclm(-/-) mice have accelerated ovarian aging due to ovarian oxidative stress. We found significantly decreased ovarian GSH concentrations and oxidized GSH/oxidized glutathione redox potential in Gclm(-/-) vs Gclm(+/+) ovaries. Prepubertal Gclm(-/-) and Gclm(+/+) mice had similar numbers of ovarian follicles, and as expected, the total number of ovarian follicles declined with age in both genotypes. However, the rate of decline in follicles was significantly more rapid in Gclm(-/-) mice, and this was driven by accelerated declines in primordial follicles, which constitute the ovarian reserve. We found significantly increased 4-hydroxynonenal immunostaining (oxidative lipid damage marker) and significantly increased nitrotyrosine immunostaining (oxidative protein damage marker) in prepubertal and adult Gclm(-/-) ovaries compared with controls. The percentage of small ovarian follicles with increased granulosa cell proliferation was significantly higher in prepubertal and 2-month-old Gclm(-/-) vs Gclm(+/+) ovaries, indicating accelerated recruitment of primordial follicles into the growing pool. The percentages of growing follicles with apoptotic granulosa cells were increased in young adult ovaries. Our results demonstrate increased ovarian oxidative stress and oxidative damage in young Gclm(-/-) mice, associated with an accelerated decline in ovarian follicles that appears to be mediated by increased recruitment of follicles into the growing pool, followed by apoptosis at later stages of follicular development.