Cryopreservation of canine ovarian tissue by slow freezing and vitrification: Evaluation of follicular morphology and apoptosis rate.

In this study, we aimed to evaluate the efficacy of cryopreserving canine ovarian tissue using vitrification and slow freezing methods while investigating potential differences in cryotolerance based on follicular type and cryopreservation technique. Twenty-eight ovaries were collected from 14 anoestrus bitches of various breeds, aged between 2 and 5 years, and undergoing elective ovariohysterectomy. The ovaries were sectioned into small fragments and randomly assigned to three groups: vitrification, slow freezing, and a control group (fresh tissue). Vitrification was performed using cryotubes containing DAP 213 solution (2M DMSO, 1M acetamide, 3M propylene glycol) in two stages, while slow freezing involved cryotubes with 1.5M DMSO solution inserted into a programmable machine. The effects of cryopreservation were evaluated by histology and immunohistochemistry (cleaved caspase-3), to determine the percentage of cells undergoing apoptosis. Histological examination revealed that the slow freezing group exhibited a significantly higher percentage of intact follicles (45.75 %) compared to those subjected to vitrification (38.17 %; P = 0.01). Immunohistochemical evaluation further indicated that 84.21 % of the follicles in the slow freezing group did not express caspase-3, suggesting the absence of apoptosis. Conversely, vitrified samples exhibited significantly more apoptotic cells compared to other groups (P < 0.001). Furthermore, early antral follicles displayed a higher susceptibility to degeneration regardless of the cryopreservation method employed. Nevertheless, when comparing the cryopreserved groups, early antral follicles showed greater degeneration in slow freezing group, while preantral follicles were the most affected in the vitrification group. In conclusion, slow freezing demonstrated superior preservation of viable follicles compared to vitrification and emerged as the preferred technique for cryopreserving canine ovarian tissue. These findings contribute valuable insights into optimizing cryopreservation methods for canine ovarian tissue, potentially benefiting reproductive technologies and fertility preservation in canines.

Actomyosin contraction in the follicular epithelium provides the major mechanical force for follicle rupture during Drosophila ovulation.

Ovulation is critical for sexual reproduction and consists of the process of liberating fertilizable oocytes from their somatic follicle capsules, also known as follicle rupture. The mechanical force for oocyte expulsion is largely unknown in many species. Our previous work demonstrated that Drosophila ovulation, as in mammals, requires the proteolytic degradation of the posterior follicle wall and follicle rupture to release the mature oocyte from a layer of somatic follicle cells. Here, we identified actomyosin contraction in somatic follicle cells as the major mechanical force for follicle rupture. Filamentous actin (F-actin) and nonmuscle myosin II (NMII) are highly enriched in the cortex of follicle cells upon stimulation with octopamine (OA), a monoamine critical for Drosophila ovulation. Pharmacological disruption of F-actin polymerization prevented follicle rupture without interfering with the follicle wall breakdown. In addition, we demonstrated that OA induces Rho1 guanosine triphosphate (GTP)ase activation in the follicle cell cortex, which activates Ras homolog (Rho) kinase to promote actomyosin contraction and follicle rupture. All these results led us to conclude that OA signaling induces actomyosin cortex enrichment and contractility, which generates the mechanical force for follicle rupture during Drosophila ovulation. Due to the conserved nature of actomyosin contraction, this work could shed light on the mechanical force required for follicle rupture in other species including humans.

Spatiotemporal single-cell RNA sequencing reveals the role of steroid hormone pathway during chicken primordial follicle formation.

The size of the initial primordial follicle pool in the ovary depends on primordial follicle formation, which determines the female reproductive lifespan. However, the molecular regulation of primordial follicle formation in chickens remains unclear. In this study, the left ovaries of chickens were collected at 2 d posthatch (dph), 5.5 dph, and 10.5 dph to examine the formation of primordial follicles. Single-cell mRNA sequencing (scRNA-seq) and spatial transcriptomic analysis were performed to explore the ovarian microenvironment and identify regulatory pathways involved in the formation of primordial follicles in chickens. Histomorphological analysis of chicken ovary tissues revealed the presence of germ cell cysts at 1 dph, which began to disintegrate at 2 dph. Primordial follicles appeared at 5.5 dph and continued to develop into larger-diameter follicles. scRNA-seq and spatial transcriptomic analysis revealed 24 cellular clusters involved in chicken primordial follicle formation. The metabolic pathway of steroid hormone synthesis was found in pregranulosa and pretheca cells. Histological analysis showed that chicken ovaries did not form primordial follicles after the inhibition of the steroid hormone synthesis pathway by simvastatin or tamoxifen. In addition, mRNA transcriptomic and bioinformatics analyses revealed that GREB1 was a downstream gene of the steroid hormone synthesis pathway during the formation of chicken primordial follicles. This study provides a valuable foundation for investigating primordial follicle formation in avian species and optimizing their reproductive performance.

USP13 regulates ferroptosis in chicken follicle granulosa cells by deubiquitinating ATG7.

The development and maturation of follicles are intricately linked to egg production and reproductive performance of chickens. Granulosa cells death directly affects the development and maturation of follicles, thereby impacting the reproductive performance of hens. Ferroptosis is a new type of cell death, it is unknown how it affects the growth and development of chicken follicles. In this study, RNA-seq analysis revealed significant differences in the expression of ferroptosis-related genes between normal follicles and atretic follicles, suggesting a potential role for ferroptosis in follicle growth and development. In addition, we found that ubiquitin-specific protease 13 (USP13) was significantly upregulated in atrophic follicles. Overexpression of USP13 results in depletion of glutathione (GSH), peroxidation of lipids, accumulation of iron, and activation of ferroptosis in chicken granulosa cells. In contrast, USP13 knockdown significantly inhibited ferroptosis events. Mechanistically, USP13 prevents the degradation of autophagy related 7 (ATG7) by deubiquitinating it, thereby enhancing the stability of ATG7 protein and ultimately promoting ferroptosis. In conclusion, this study elucidates the crucial role of the USP13-ATG7 axis in regulating ferroptosis in chicken follicle granulosa cells, thereby presenting a novel avenue for molecular breeding research in chickens.

Effect of active immunization with OPN5 on follicular development and egg production in quail under different photoperiods.

OPN5 is one of the main deep brain photoreceptors (DBPs), converting photoperiodic information into neuroendocrine signals to regulate reproduction in birds. This study investigated the mechanism of OPN5-mediated photoperiodic regulation of reproduction by active immunization against OPN5. 96 female quail were divided into OPN5-immunized and control group under the same photoperiod: 16 L:8 D (d 1 to d 35), 8 L:16 D (d 36 to d 70) and 12 L:12 D (d 71 to d 126). OPN5-immunized group was conducted with OPN5 protein vaccination and control group was given a blank vaccine. Samples were collected on d 1, d 30, d 60, and d 126. Results showed switching photoperiod to 8 L:16 D decreased the laying rate, GSI%, numbers of YFs and WFs, serum levels of PRL, P4 and E2, and pituitary PRL and TSHß protein expressions in both groups (P < 0.05). Whereas the OPN5-immunized group exhibited higher laying rates than the control group (P < 0.05). The control group showed reduced GnRHR and TSHß gene expressions in the pituitary and increased GnIH and DIO3 transcript and/or protein abundance in the hypothalamus. (P < 0.05). The OPN5-immunized group had lower DIO3 expression at both mRNA and protein levels. (P < 0.05). Switching photoperiod from 8 L:16 D to 12 L:12 D increased the laying rates, GSI%, numbers of YFs and WFs, serum levels of PRL, and PRL protein expression in both groups (P < 0.05), and the responses were more pronounced in OPN5-immunized group (P < 0.05). In contrast to the control group, quail with OPN5-immunization had higher OPN5 and DIO2 transcript and/or protein levels but lower DIO3 expressions in the hypothalamus along the transition photoperiods (P < 0.05). The results revealed that OPN5 responds to photoperiod transition, and its activation mediates related signaling to up-regulate TSH-DIO2/DIO3 pathway and VIP-PRL secretion to prime quail reproductive functions.

Reproductive morphophysiology of an iteroparous batch spawner fish: from early follicle development to ovulation.

Batch spawner fishes develop successive clutches of oocytes which allows them to participate in many reproductive cycles during their adult life (iteroparous) and spawn in multiple events within each breeding cycle. Here, ovarian follicular development was morpho-functionally analyzed in females of the iteroparous batch spawner fish Gymnocorymbus ternetzi. To obtain better insights into the reproductive morpho-physiology in batch spawners, the objective of this research was to analyze the dynamics of the follicular development, with its hormonal regulation between two active reproduction events. We found that over 16 days, follicles progressed asynchronously to chromatin nucleolus, Primary and Secondary growth stages of oogenesis with progressive secretion of 17ß-estradiol (E2). During the end of secondary growth, the increase in 17α,20ß-dihydroxy-4-pregnen-3-one (17,20ß-p) was measured relative to the maturation process of the ovarian follicles (e.g., nuclear migration and its rupture during the resumption of meiosis). Interestingly, an additional increase in E2 was observed after fish reproduction, probably related to the recruitment of new batch follicles for secondary growth. We also measured the high values of multiple condition factor post-reproduction measurements, reflecting more energy invested during the pre-reproductive process. We also quantified high concentrations of 17,20ß-p, probably related to the recruitment of a new batch of oogonia to meiosis, presumably secreted by post-ovulatory follicles, after fish reproduction. We finally found that fish without exposure to reproductive stimulus developed a regression phase at day 24, characterized by massive follicle atresia, that allow them to recycle energy and constitutive materials of the follicles invested during oogenesis for another reproductive cycle.

Investigations into the role of platelet-activating factor in the peri-conception period of the mare.

In Brief: In many mammals, the lipid platelet-activating factor (PAF) has important functions in female reproduction and fertility. This study shows that PAF is present in the reproductive tissues of mares and is involved in processes related to ovulation and early pregnancy. Abstract: Platelet-activating factor (PAF) has been implicated in a number of reproductive processes ranging from ovulation to embryo motility but has not been widely explored in the mare. To identify the presence and examine the role of PAF in the equine periconception processes, targeted mass spectrometry coupled with chromatographic separation was performed on equine follicular fluid (FF), and PAF was quantitatively detected. Subsequently, untargeted high-resolution mass spectrometry-based lipidomic analysis was carried out to quantify PAF in different-sized pre-ovulatory follicles, whereby different molecular species of PAF, PAF (14:0) and PAF (16:1), were both seen to be increasing with follicle diameter. These findings suggest that PAF within FF is increasing as preovulatory follicles approach ovulation. Additionally, immunofluorescence staining identified the PAF receptor in the luminal pericellular, apical, and basal aspect of equine oviductal epithelial cells. Lastly, an equine oviductal epithelial organoid model was generated and showed that the addition of PAF significantly increased the ciliary beat frequency (CBF) (Hz), an action consistent with a role for PAF in embryo migration. It is proposed that the local action of PAF on the ciliated cells of the oviduct propels both the oocyte and the conceptus towards the uterus. In the mare, it appears that PAF is a contributor during the periconception period, potentially being a mediator in the mechanisms of ovulation and in the dialogue of very early pregnancy.

Effect of the gonadotropin surge on steroid receptor regulation in preovulatory follicles and newly formed corpora lutea in the cow.

The objective of the study was to characterize the mRNA expression patterns of specific steroid hormone receptors namely, estrogen receptors (ESRRA-estrogen related receptor alpha and ESRRB-estrogen related receptor beta) and progesterone receptors (PGR) in superovulation-induced bovine follicles during the periovulation and subsequent corpus luteum (CL) formation. The bovine ovaries (n = 5 cow / group), containing preovulatory follicles or early CL, were collected relative to injection of the gonadotropin-releasing hormone (GnRH) at (I) 0 h, (II) 4 h, (III) 10 h, (IV) 20 h, (V) 25 h (preovulatory follicles) and (VI) 60 h (CL, 2-3 days after induced ovulation). In this experiment, we analyzed the steroid receptor mRNA expression and their localization in the follicle and CL tissue. The high mRNA expression of ESRRA, ESRRB, and PGR analyzed in the follicles before ovulation is significantly reduced in the group of follicles during ovulation (25 h after GnRH), rising again significantly after ovulation in newly formed CL, only for ESRRA and PGR (P < 0.05). Immunohistochemically, the nuclei of antral follicles' granulosa cells showed a positive staining for ESRRA, followed by higher activity in the large luteal cells just after ovulation (early CL). In contrast, the lower PGR immunopresence in preovulatory follicles increased in both small and large luteal cell nuclei after follicle ovulation. Our results of steroid receptor mRNA expression in this experimentally induced gonadotropin surge provide insight into the molecular mechanisms of the effects of steroid hormones on follicular-luteal tissue in the period close to the ovulation and subsequent CL formation in the cow.

Treatment alternatives to induce follicular wave emergence for timed-AI in lactating dairy Cows.

Two experiments evaluated the effect of different hormonal treatments to synchronize follicle wave emergence on follicle dynamics and pregnancies per AI (P/AI) in estradiol (E2)/progesterone (P4) timed-AI (TAI) protocols in lactating dairy cows. In Experiment 1, lactating, primiparous Holstein cows (n = 36) received a P4 releasing device (Day 0) and were allocated at random to one of the following three treatment groups: Group EB received 2 mg E2 benzoate (EB) intramuscularly (i.m.), Group EB + GnRH received 2 mg EB+20 µg buserelin (GnRH) i.m., or Group EB + P4 received 2 mg EB + 100 mg of injectable P4 (iP4) in oil i.m. All cows received 0.150 mg D-Cloprostenol on Days 7 and 8 followed by P4 device removal, 400 IU eCG and 1 mg ECP on Day 8. Daily ultrasound examinations revealed that although the interval from P4 device removal to ovulation was not affected by treatment, cows that received EB + GnRH had an earlier (P < 0.05) emergence of the new follicular wave (Day 2.6 ± 0.2) than the other two treatment groups (Days 3.5 ± 0.3 and 6.1 ± 0.3, for EB and EB + P4, respectively). In Experiment 2, 808 lactating cows were assigned randomly to the three treatments evaluated in Experiment 1, and all the cows were TAI to determine P/AI. Cows in the EB + GnRH group had greater P/AI (57.4 %, P < 0.01) than those in the EB (44.6 %) or EB + P4 (45.7 %) groups. In conclusion, the administration of GnRH, but not iP4, on the day of insertion of a P4 device improves P/AI in lactating dairy cows synchronized for TAI with an estradiol/P4-based protocol.

In vitro production of meiotically competent oocytes from early antral follicles in sheep.

The potential of using long in vitro culture (LIVC) of cumulus-oocyte complexes (COCs) from early antral follicles (EAFs) as an assisted reproductive technology in cattle has shown promising results. This study explored the feasibility of applying this technology to sheep as seasonal breeding animals. Ovaries from sheep were collected during both the breeding and non-breeding seasons. COCs were isolated from EAFs (350-450 µm) and cultured in TCM199 medium supplemented with 0.15 µg/mL Zn sulfate, 10-4IU/mL FSH, 10 ng/mL estradiol, 50 ng/mL testosterone, 50 ng/mL progesterone, and 5 µM Cilostamide. After five days of LIVC, the COCs were submitted to an in vitro maturation procedure. The results indicate successful in vitro development of COCs, evidenced by a significant increase in oocyte diameter (p < 0.000) and the preservation of gap junction communication between oocyte and cumulus cells. The gradual uncoupling was accompanied by a progressive chromatin transition from the non-surrounded nucleolus (NSN) to the surrounded nucleolus (SN) (p < 0.000), coupled with a gradual decrease in global transcriptional activity and an increase in oocyte meiotic competence (p < 0.000). Maintenance of oocyte-cumulus investment architecture, viability, and metaphase II capability was significantly higher in COCs collected during the breeding season (p < 0.000), suggesting higher quality than those obtained during the non-breeding season. In conclusion, our study confirms LIVC feasibility in sheep, emphasizing increased effectiveness during the breeding season in isolating higher-quality COCs from EAFs. These findings can influence improving the LIVC system in mammals with seasonal reproduction.

CircRNAs involved in the red light of effect on follicle selection in pigeons.

Red light (RL) can enhance egg production in poultry. CircRNAs play a crucial role by serving as transcriptional regulators. However, their role in influencing follicle development in White King pigeons remains unexplored. In this study, 54 paired White King pigeons were chosen and divided into RL and white light (WL) groups, each with 3 subgroups. The egg production of paired pigeons in each replicate was recorded for 45 d, and the characteristics of follicle development were monitored during the laying interval (LI). The granulosa cell layer from follicles of the second-largest follicle (F2) was collected, and high-throughput sequencing was performed to elucidate the molecular mechanism of follicle development in pigeons. The study confirmed that RL enhances egg production in pigeons. Additionally, under RL, the F2 follicle was selected, while under WL, small follicles were kept on the third day (LI3). A total of 5,510 circRNAs were identified across all samples, revealing differentially expressed circRNAs (DECs) in various comparisons: 627 in RF1 vs. WF1, 900 in RF2 vs. WF2, 606 in RF1 vs. RF2, and 937 in WF1 vs. WF2. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that host genes of DECs were enriched in pathways like steroid hormone biosynthesis, oocyte meiosis, GnRH signaling pathway, and apoptosis pathway. Moreover, circRNA_5497, circRNA_2016, and circRNA_3328 were common DECs across 4 groups, sharing miRNA binding sites with follicle selection-associated genes. In conclusion, our findings suggest that RL promotes egg production by stimulating follicle selection during LI, offering insights into the regulatory mechanisms of circRNAs in follicle selection under RL. This knowledge can help enhance the reproductive performance of pigeons.

Epigenetic modifications of gonadotropin receptors can regulate follicular development.

The spatiotemporal transcription of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone/human chorionic gonadotropin receptor (LHCGR) are crucial events for follicular development. However, their regulatory mechanisms are unclear. DNA methylation and histone acetylation are the main epigenetic modifications, and play important roles in transcriptional expression, which regulate cell responses including cell proliferation, senescence and apoptosis. This review will discuss the dynamic epigenetic modifications of FSHR and LHCGR that occur during the process of follicular development and their response to gonadotropins. In addition, some alteration patterns that occur during these epigenetic modifications, as well as their retrospect retrotransposons, which regulate the gene expression levels of FSHR and LHCGR will be discussed.

Serum anti-Müllerian hormone is an indirect predictor of ovarian reserve in domestic cats.

Anti-Müllerian hormone (AMH) serves as an indirect marker for predicting primordial follicles that are representative of ovarian reserve. In this study the possibility of using AMH and age to predict the ovarian reserve in domestic cats. Ovaries and blood were collected from 30 cats undergoing routine ovariohysterectomy. The animals were divided into three age groups: prepubertal (<4 mo, n = 10), adult (1-5 y, n = 10), and senior (>5 y, n = 10). Blood was collected at surgery for serum AMH measurements using the AMH Gen II ELISA kit. The intra-assay coefficient of variation (CV) and inter-assay CV were 3.56 % and 7.68 %, respectively. One side of the ovary was processed to determine AMH localization using immunohistochemistry and for a histological count of follicles, which is the gold standard. The expression of AMH protein was quantified from the contralateral ovary by Western blot analysis. Primordial follicles exhibited the most pronounced inverse relationship with age (rho = -0.779, P < 0.05), followed by a positive association with serum AMH concentration (rho = 0.490, P < 0.05), indicating that both age and AMH are potential markers indicative of primordial follicles. Furthermore, secondary (rho = 0.651, P < 0.05) and small antral follicles (rho = 0.648, P < 0.05) were identified as the major sources of circulating AMH, as indicated by the stronger correlation with serum AMH concentrations compared with primary follicles. However, there was no significant correlation between the expression of AMH protein and other factors, including age, primordial follicles, primary follicles, secondary follicles, small antral follicles, and serum AMH concentration. A model for predicting primordial follicle number using serum AMH concentration (AIC = 672.66, P < 0.05) and age (AIC = 668.93, P < 0.05) was established. In conclusion, both serum AMH concentration and age may serve as comparable markers of ovarian reserve in domestic cats. Moreover, AMH is particularly useful in situations where age information is not available.

Nonlinear compartmental modeling to monitor ovarian follicle population dynamics on the whole lifespan.

In this work, we introduce a compartmental model of ovarian follicle development all along lifespan, based on ordinary differential equations. The model predicts the changes in the follicle numbers in different maturation stages with aging. Ovarian follicles may either move forward to the next compartment (unidirectional migration) or degenerate and disappear (death). The migration from the first follicle compartment corresponds to the activation of quiescent follicles, which is responsible for the progressive exhaustion of the follicle reserve (ovarian aging) until cessation of reproductive activity. The model consists of a data-driven layer embedded into a more comprehensive, knowledge-driven layer encompassing the earliest events in follicle development. The data-driven layer is designed according to the most densely sampled experimental dataset available on follicle numbers in the mouse. Its salient feature is the nonlinear formulation of the activation rate, whose formulation includes a feedback term from growing follicles. The knowledge-based, coating layer accounts for cutting-edge studies on the initiation of follicle development around birth. Its salient feature is the co-existence of two follicle subpopulations of different embryonic origins. We then setup a complete estimation strategy, including the study of structural identifiability, the elaboration of a relevant optimization criterion combining different sources of data (the initial dataset on follicle numbers, together with data in conditions of perturbed activation, and data discriminating the subpopulations) with appropriate error models, and a model selection step. We finally illustrate the model potential for experimental design (suggestion of targeted new data acquisition) and in silico experiments.

Chicken ovarian follicular atresia: interaction network at organic, cellular, and molecular levels.

Most of follicles undergo a degenerative process called follicular atresia. This process directly affects the egg production of laying hens and is regulated by external and internal factors. External factors primarily include nutrition and environmental factors. In follicular atresia, internal factors are predominantly regulated at 3 levels; organic, cellular and molecular levels. At the organic level, the hypothalamic-pituitary-ovary (HPO) axis plays an essential role in controlling follicular development. At the cellular level, gonadotropins and cytokines, as well as estrogens, bind to their receptors and activate different signaling pathways, thereby suppressing follicular atresia. By contrast, oxidative stress induces follicular atresia by increasing ROS levels. At the molecular level, granulosa cell (GC) apoptosis is not the only factor triggering follicular atresia. Autophagy is also known to give rise to atresia. Epigenetics also plays a pivotal role in regulating gene expression in processes that seem to be related to follicular atresia, such as apoptosis, autophagy, proliferation, and steroidogenesis. Among these processes, the miRNA regulation mechanism is well-studied. The current review focuses on factors that regulate follicular atresia at organic, cellular and molecular levels and evaluates the interaction network among these levels. Additionally, this review summarizes atretic follicle characteristics, in vitro modeling methods, and factors preventing follicular atresia in laying hens.

Autophagy is involved in granulosa cell death and follicular atresia in ewe ovaries.

In mammalian ovaries, most follicles do not ovulate and are eliminated by atresia, which primarily depends on granulosa cell (GC) apoptosis. Autophagy is an alternative mechanism involved in follicle depletion in mammals through independent or tandem action with apoptosis. However, follicular autophagy has not yet been investigated in sheep; therefore, the present study aimed to investigate the involvement of autophagy in atresia among a pool of growing antral follicles in ewe ovaries. The abundance of the autophagic marker LC3B-II was determined using western blotting in GCs collected from ewe antral follicles. The antral follicles were classified as healthy or atretic based on morphological criteria and steroid measurements in follicular fluid (FF). Immunofluorescence and confocal microscopy analyses were performed on GCs to evaluate the presence of autophagic proteins and their subcellular localisation. Caspase-3 and DNA fragmentation were assessed using western blotting and TUNEL assays, respectively, in the same GC population to investigate the simultaneous apoptosis. The novel results of this study demonstrated enhanced LC3B-II protein expression in GCs of atretic follicles compared to that of healthy ones (1.3-fold increase; P = 0.0001, ANOVA), indicating a correlation between autophagy enhancement in GCs and antral follicular atresia. Autophagy, either functioning independently or in tandem with apoptosis, may be involved in the atresia of growing antral follicles in ewe ovaries because atretic GCs also showed high levels of apoptotic markers. The findings of this study might have important implication on scientific understanding of ovarian follicle dynamics.

Reduced anti-Müllerian hormone action in cumulus-oocyte complexes is beneficial for oocyte maturation without affecting oocyte competence.

Anti-Müllerian hormone (AMH) is a key paracrine/autocrine factor regulating folliculogenesis in the postnatal ovary. As antral follicles mature to the preovulatory stage, AMH production tends to be limited to cumulus cells. Therefore, the present study investigated the role of cumulus cell-derived AMH in supporting maturation and competence of the enclosed oocyte. Cumulus-oocyte complexes (COCs) were isolated from antral follicles of rhesus macaque ovaries for in vitro maturation with or without AMH depletion. Oocyte meiotic status and embryo cleavage after in vitro fertilization were assessed. In vitro maturation with AMH depletion was also performed using COCs from antral follicles of human ovarian tissue. Oocyte maturation and morphology were evaluated. The direct AMH action on mural granulosa cells of the preovulatory follicle was further assessed using human granulosa cells cultured with or without AMH supplementation. More macaque COCs produced metaphase II oocytes with AMH depletion than those of the control culture. However, preimplantation embryonic development after in vitro fertilization was comparable between oocytes derived from COCs cultured with AMH depletion and controls. Oocytes resumed meiosis in human COCs cultured with AMH depletion and exhibited a typical spindle structure. The confluency and cell number decreased in granulosa cells cultured with AMH supplementation relative to the control culture. AMH treatment did not induce cell death in cultured human granulosa cells. Data suggest that reduced AMH action in COCs could be beneficial for oocyte maturation. Cumulus cell-derived AMH is not essential for supporting oocyte competence or mural granulosa cell viability.

Influence of early progesterone removal on follicular development, expression of estrus, and pregnancy rates in presynchronized postpartum beef cows.

The objective of this study was to evaluate the impact of early progesterone removal on pregnancy rates to fixed-time artificial insemination (FTAI) in presynchronized beef cows. Postpartum beef cows (n = 882) were randomly assigned to 1 of 2 treatments: 1) 7&7 Synch: cows received a controlled internal drug release insert (CIDR) and a 25-mg injection of prostaglandin F2α on day 0, 100 µg of GnRH on day 7, a second injection of prostaglandin F2α (PG2) at CIDR removal on day 14, and a second injection of GnRH at FTAI 60-66 h after PG2 (day 17); 2) 7&6 Synch: cows received the same treatment as 7&7 Synch; however, CIDR removal occurred in conjunction with PG2 on day 13, while FTAI remained at 60-66 h after CIDR removal (day 16). Ovarian ultrasonography was performed to determine follicle diameter at PG2 and FTAI in a subset of cows (n = 40). Cows exposed to the 7&7 Synch tended to have larger follicle diameter at PG2 compared with 7&6 Synch cows (P = 0.09); however, there were no differences in follicle diameter at FTAI. No differences were determined between treatments for the expression of estrus (7&7 Synch: 61.6 ± 5.30; 7&6 Synch: 54.1 ± 5.45; P = 0.31) or pregnancy rates to FTAI (7&7 Synch: 60.8 ± 3.83; 7&6 Synch: 57.0 ± 3.84; P = 0.42). In conclusion, early removal of progesterone did not impact pregnancy rates in presynchronized beef cows.

Effect of prostaglandin alone and in combination with trace minerals on the follicular and luteal dynamics, estrus response and pregnancy in sub-estrus buffaloes (Bubalus bubalis).

Sub-estrus is a condition when buffaloes do not display behavioural estrus signs, despite being in estrus and causes a delay in conception and increases the service period. The present study describes the effect of synthetic prostaglandin (PGF2α) alone and in combination with trace minerals on the follicular and corpus luteum (CL) dynamics, serum estradiol (E2) and progesterone (P4) concentration correlating estrus response and pregnancy outcome in sub-estrus buffaloes during the breeding season. A total of 50 sub-estrus buffaloes, identified through ultrasonography (USG) examination, were randomly allocated into three groups, viz. T1 (Synthetic PGF2α, Inj. Cloprostenol 500 µg, i.m, n = 17), T2 (Synthetic PGF2α + Trace mineral supplementation, Inj. Stimvet 1 mL/100 kg body weight, i.m., n = 17) and control (untreated; n = 16). Following treatment, 100% of sub-estrus buffaloes were induced estrus in the T1 and T2 groups, while only 18.75% were induced in the control. The CL diameter and serum P4 concentration were significantly lower at post-treatment, whereas the pre-ovulatory follicle (POF) size and serum E2 concentration were significantly higher in the T1 and T2 groups as compared to the control (p < .05). The buffaloes of the T2 group had a greater proportion of moderate intensities estrus than those of T1. Moreover, the proportion of buffaloes conceived in the T1 and T2 were 41.2% and 52.95%, respectively. The larger POF diameter and higher serum E2 concentration were associated with intense intensity estrus and higher conception rate (66.7%) in sub-estrus buffaloes. Similarly, CL regression rate, POF size and serum E2 concentration were relatively higher in the buffaloes conceived as compared to those not conceived. It is concluded that synthetic PGF2α in combination with trace minerals induces moderate to intense intensities estrus in a greater proportion of sub-estrus buffaloes and increases the conception rate during the breeding season. Moreover, behavioural estrus attributes correlating follicle and luteal morphometry, serum E2 and P4 concentration could be used to optimise the breeding time for augmenting the conception rate in sub-estrus buffaloes.

The Reproductive Lifespan of Ovarian Follicle.

The functional unit within mammalian ovaries is the ovarian follicle. The development of the ovarian follicle is a lengthy process beginning from the time of embryogenesis, passing through multiple different stages of maturation. The purpose of this review is to describe the most basic events in the journey of ovarian follicle development, discussing the importance of ovarian reserve and highlighting the role of several factors that affect oocyte quality and quantity during aging including hormonal, genetic and epigenetic factors. Novel, promising anti-aging strategies are also discussed.