Search Results (52)

Ghrelin, the endogenous ligand of the growth hormone secretagogue receptor, is released pre-prandially and during periods of negative energy balance, exhibiting anti-fertility properties. In this study, twenty ewes were divided into two groups: a ghrelin-treated group receiving 1.25 μg/kg body weight (BW) of ghrelin per day via mini-pumps for 28 days and an untreated control group. Estrus was synchronized, superovulation was induced with FSH, and embryos and follicular fluid were collected six days post-estrus. Blood samples were taken to measure LH, progesterone, and anti-Müllerian hormone (AMH) concentrations. Results indicated that in treated animals, preovulatory LH surge was weaker, and progesterone levels were lower than in controls. Differences were observed in the superovulatory response and the number of collected embryos, both being higher in controls. While AMH levels did not differ between groups at the beginning of the experiment, they were lower in treated animals at the time of FSH administration. Treated ewes exhibited a reduced number of small follicles, and their follicular fluid contained lower AMH concentrations than the controls. These findings suggest that ghrelin plays a direct role in regulating LH secretion from the pituitary and in controlling ovarian follicle development, highlighting the strong interaction between nutrition and fertility. Full article

Semen quality plays a crucial role in bovine in vivo embryo production. This study aimed to compare the effects of 0 °C-refrigerated semen and liquid nitrogen-frozen semen on embryo quality in Simmental cattle. Semen collected from five bulls was equally divided into two groups: one diluted with a 0 °C refrigeration solution and stored at 0 °C, and the other diluted with a cryopreservation solution and stored in liquid nitrogen for 24 h. We evaluated sperm motility, progressive motility (assessed via a computer-assisted sperm analyzer), acrosome integrity, and plasma membrane integrity in both groups. Fifty superovulated Simmental cows were artificially inseminated with semen from both groups. Embryos were non-surgically flushed on day seven, followed by BrdU proliferation staining and TUNEL apoptosis staining. Proliferation and apoptosis levels were quantified using marker genes. Results showed that 0 °C-refrigerated semen exhibited significantly higher sperm motility, progressive motility, acrosome integrity, and plasma membrane integrity compared to liquid nitrogen-frozen semen (p < 0.05). While total embryo numbers showed no significant difference between groups (p ≥ 0.05), embryos from 0 °C-refrigerated semen contained significantly more proliferative cells (p < 0.05) and fewer apoptotic cells (p < 0.05) than those from frozen semen. These findings demonstrate that 0 °C-refrigerated semen outperforms liquid nitrogen-frozen semen in both sperm quality parameters and resultant embryo quality. Full article

Selective serotonin reuptake inhibitors (SSRIs), like fluoxetine, are increasingly used by women of a reproductive age, raising concerns about their impact on oocyte quality and early embryonic development. This study investigated the effects of chronic fluoxetine exposure on oocyte maturation, ovulation, and embryonic development in a mouse model. Female mice were administered fluoxetine via drinking water, and their reproductive outcomes were compared to those of control mice. Oocyte quantity and quality were assessed following superovulation, including the analysis of spindle morphology, chromatin configuration, and maturation markers. In vitro maturation assays were conducted to evaluate the developmental competence of oocytes exposed to fluoxetine. Finally, the impact of fluoxetine on blastocyst formation, litter size, offspring growth, and ovarian reserve was examined. The results show that fluoxetine treatment reduced the number of ovulated oocytes but did not significantly affect oocyte quality or meiotic spindle formation. Fluoxetine exposure impaired cytoplasmic maturation at the germinal vesicle stage, resulting in a lower proportion of fully mature oocytes and reduced in vitro maturation efficiency. While blastocyst numbers were modestly reduced in fluoxetine-treated mice, litter size and offspring ovarian reserve were unaffected. Unexpectedly, offspring of fluoxetine-treated mothers exhibited increased body weight. These findings suggest that while fluoxetine may impair oocyte developmental competence through disruptions in cytoplasmic maturation, it does not severely compromise overall reproductive outcomes or offspring fertility. Full article

Embryo transfer technology is extensively utilized in cattle breeding, with superovulation serving as its foundation. Superovulation currently relies on the administration of exogenous hormones to stimulate follicular development and maturation in the ovaries, with porcine pituitary-derived follicle-stimulating hormone (FSH) being the most commonly used. However, pituitary-derived FSH requires multiple injections, contains luteinizing hormone (LH) and other proteins, and carries a risk of disease transmission. The development of recombinant FSH proteins through protein recombinant technology represents a significant research direction for addressing the aforementioned challenges. Over the past 30 years, extensive studies have been conducted on the development of recombinant bovine, ovine, or porcine FSH. Nevertheless, to date, no commercially recombinant FSH has been widely applied in the superovulation of cattle. This review provides an overview of the molecular design, selection of expression systems, and biological activity of recombinant bovine, ovine, or porcine FSH. This review also summarizes the results of utilizing recombinant FSH in superovulation protocols for cattle. The utilization of recombinant proteins as an alternative to traditionally extracted products in livestock production represents an irreversible trend. The livestock industry is highly sensitive to costs; therefore, it is essential to develop high-quality and cost-effective recombinant FSH products through appropriate molecular design and the use of suitable expression systems. Full article

Superovulation and embryo transfer are key technologies to improve the reproductive ability of female animals and enhance the efficiency of livestock production. However, poor-quality oocytes or abnormal fluctuations of hormone levels caused by superovulation affect the embryonic development environment, which may lead to a significant decline in the number and quality of transferable embryos, thus reducing the efficiency of superovulation. In this study, nicotinamide adenine dinucleotide (NAD⁺) was injected into Huaxi cows during the superovulation period to observe the proliferation and apoptosis of transplanted embryos. We examined the proliferation, apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential of cumulus cells and oocytes directly treated with NAD⁺ and investigated the potential mechanism of NAD⁺ to improve the superovulation efficiency by serum metabolomics and single-cell RNA sequencing. The results show that the addition of NAD⁺ significantly increased the quantity and quality of transferable embryos after superovulation. Differential metabolites during estrus synchronization and embryo flushing are enriched in glycerophospholipid metabolic pathways, suggesting that NAD⁺ can regulate lipid metabolic pathways. We found that NAD⁺ optimized the secretion levels of the steroid hormone estradiol (E2) and progesterone (P4) during superovulation by regulating the activity of cumulus cells. In oocytes, we found that NAD⁺ can inhibit apoptosis, scavenge ROS, and enhance mitochondrial function, thereby promoting oocyte maturation and enhancing embryo developmental potential. In conclusion, NAD⁺ significantly improved the superovulation ability of Huaxi cattle and provides an effective way for animal husbandry to improve the yield of high-quality embryos. Full article

In small ruminants, laparotomy for ovarian exploration followed by oocyte collection has been progressively replaced by laparoscopic puncture of follicles, which has become an important method for obtaining oocytes in vivo. However, the superovulation protocols and collection frequency used for laparoscopic ovum pick-up (LOPU) in sheep still require further investigation. This study explored the factors influencing LOPU efficiency in sheep, including Controlled Internal Drug Release (CIDR) for estrus synchronization, FSH source and dose, and recovery intervals. The optimal superovulation protocol (using the CIDR device, a total of 16 mg of long-acting recombinant ovine FSH (LR-FSH) administered in two doses, and a one-month interval between LOPU sessions) was subsequently identified. Ovarian follicles were collected via LOPU from Hu sheep and Altay sheep for transcriptomic and metabolomic sequencing to explore interbreed differences in follicular development. The results indicated that LOPU efficiency was significantly higher in the CIDR group (p < 0.05) and with a 30-day recovery interval (p < 0.05). No significant differences in LOPU efficiency were observed between FSH sources or hormone doses. Furthermore, Hu sheep exhibited significantly higher LOPU efficiency and more antral follicles than Altay sheep. Transcriptomic analysis of follicular contents and metabolomic profiling of follicular fluid revealed that differentially expressed genes and metabolites were primarily enriched in pathways related to steroidogenesis, amino acid metabolism, and fatty acid metabolism. This study provides an optimized treatment protocol to enhance LOPU efficiency and integrates multi-omics analyses to elucidate the molecular mechanisms underlying follicular development differences among various breeds. Full article

In biomedical research, ovulation induction is a critical step in the reproductive biology of laboratory animals. This study evaluates the efficacy of peforelin, a synthetic gonadotropin-releasing hormone (GnRH) analog, in comparison to pregnant mare serum gonadotropin (PMSG, synonym: eCG), traditionally used for ovulation induction in mice. PMSG is derived from the serum of pregnant horses, and its production is becoming increasingly problematic due to animal welfare concerns and regulatory restrictions. The aim of this study was, therefore, to evaluate an ethically acceptable and less invasive alternative to PMSG. Female C57BL/6J mice, aged 3–4 weeks, were divided into two groups to receive either peforelin at three different concentrations or PMSG, followed by an injection of human chorionic gonadotropin (hCG) to induce ovulation. Key outcomes included the number and quality of oocytes collected, fertilization rates, ovary morphology, and follicular differentiation. Although the number of oocytes was significantly lower in the peforelin cohort, the fertilization rate was high. Ovarian morphology was not significantly altered compared to the PMSG cohort. This study showed that peforelin is suitable for superovulation in mice. These results suggest that peforelin could be an ethically acceptable alternative to PMSG stimulation for inducing superovulation in mice. Full article

The application of laparoscopic ovum pick-up (LOPU) and in vitro production of embryos (IVEP) technologies has opened up a new path for purebred breeding and breed improvement in goats. However, due to the complexity of the procedures and multiple influencing factors, these technologies have not been widely adopted in goat production. This study explores factors affecting the efficiency of LOPU in goats by comparing the use of controlled internal drug release (CIDR) for estrus synchronization, conventional FSH versus long-acting recombinant ovine FSH (R-FSH) for superovulation, and the timing of LOPU at 48 h, 60 h, and 72 h of follicular development. The metrics evaluated included the recovery rate of cumulus–oocyte complexes (COCs), the average number of ovarian follicles, the average number of COCs, and the average number of available COCs. The results demonstrated that the efficiency of LOPU was significantly higher with two doses of R-FSH compared to the conventional FSH superovulation protocol and the control group (p < 0.05), with two doses of R-FSH providing a higher LOPU efficiency than one dose (p < 0.05). Using CIDR for estrus synchronization showed no significant difference in LOPU efficiency compared to the non-CIDR group. Similarly, the efficiency of LOPU showed no significant difference between the hormone treatments at 48 h, 60 h, and 72 h. By exploring and optimizing the factors influencing LOPU, we ultimately established a LOPU technology system for goats that meets the production needs of small ruminants. Full article

Superovulation is a crucial step in assisted reproductive technology that involves the administration of gonadotrophins. Repeated superovulations result in severe ovarian damage. The present study investigated the effect of in vivo administration of lycopene on ovarian damage induced by four successive cycles of superovulation. Superovulated mice were simultaneously administered intraperitoneally with saline (R4) or 5 mg/kg lycopene (R4-Lyc). The evaluated parameters were the count of different types of follicles, expression of ovarian antioxidant- and apoptosis-related genes, and serum concentrations of estradiol, progesterone, and inhibin-B. Increased numbers of healthy follicles and a decreased count of atretic follicles were observed in mice of the R4-Lyc group compared to those of the R4 group. Moreover, significantly higher mRNA levels of Sod3, Cat, and Nrf2 and lower mRNA levels of Keap1, Tnf, Nfkb, and Casp3, together with decreased H₂O₂ concentrations and increased total antioxidant capacity, were detected in the ovaries of lycopene-treated mice. Regarding serum reproductive hormones, elevated concentrations of estradiol, progesterone, and inhibin-B were evident in lycopene-administered mice. The present study reports a significant role of lycopene in alleviating the ovarian damage induced by multiple hormonal superstimulations, which could help to improve the outcomes of in vitro embryo production. Full article

Superovulation is a technique used to increase the number of oocytes released for fertilization. This study investigated the effects of short-term differences in concentrate feed intake on in vivo embryo production through superovulation in indigenous Korean (Hanwoo) cows. The cows were given fresh water and hay ad libitum and randomly divided into three groups (control (CON, n = 9): 2.0 kg/day (unchanged diet); low concentrate (LC, n = 10): 0 kg/day; and high concentrate (HC, n = 8): 4.0 kg/day) according to the amount of formula they were fed. This feeding treatment began seven days before the start of the hormonal treatment for superovulation. From the results, the LC group had the greatest weight change and the lowest body condition score at harvest, followed by the CON and HC groups (p < 0.05). The LC group had the highest number of harvesting embryos, followed by the HC and CON groups (p < 0.05). Estradiol, progesterone, glucose, total cholesterol, high-density lipoprotein, low-density lipoprotein, total protein, and blood urea nitrogen concentrations did not differ between the groups, except for a temporary increase in the HC group on day 0. These findings suggest that more embryos may be harvested when short-term changes in concentrate intake are made during superovulatory responses in Hanwoo cows. Full article

In vitro embryonic technology is crucial for improving farm animal reproduction but is hampered by the poor quality of oocytes and insufficient development potential. This study investigated the relationships among changes in the gut microbiota and metabolism, serum features, and the follicular fluid metabolome atlas. Correlation network maps were constructed to reveal how the metabolites affect follicular development by regulating gene expression in granulosa cells. The superovulation synchronization results showed that the number of follicle diameters from 4 to 8 mm, qualified oocyte number, cleavage, and blastocyst rates were improved in the dairy heifers (DH) compared with the non-lactating multiparous dairy cows (NDC) groups. The gut microbiota was decreased in Rikenellaceae_RC9_gut_group, Alistipes, and Bifidobacterium, but increased in Firmicutes, Cyanobacteria, Fibrobacterota, Desulfobacterota, and Verrucomicrobiota in the NDC group, which was highly associated with phospholipid-related metabolites of gut microbiota and serum. Metabolomic profiling of the gut microbiota, serum, and follicular fluid further demonstrated that the co-metabolites were phosphocholine and linoleic acid. Moreover, the expression of genes related to arachidonic acid metabolism in granulosa cells was significantly correlated with phosphocholine and linoleic acid. The results in granulosa cells showed that the levels of PLCB1 and COX2, participating in arachidonic acid metabolism, were increased in the DH group, which improved the concentrations of PGD₂ and PGF_2α in the follicular fluid. Finally, the expression levels of apoptosis-related proteins, cytokines, and steroidogenesis-related genes in granulosa cells and the concentrations of steroid hormones in follicular fluid were determinants of follicular development. According to our results, gut microbiota-related phosphocholine and linoleic acid participate in arachidonic acid metabolism in granulosa cells through the gut–follicle axis, which regulates follicular development. These findings hold promise for enhancing follicular development and optimizing oocyte quality in subfertile dairy cows. Full article

Chronic stress has become a major problem that endangers people’s physical and mental health. Studies have shown that chronic stress impairs female reproduction. However, the related mechanism is not fully understood. P2X7 receptor (P2X7R) is involved in a variety of pathological changes induced by chronic stress. Whether P2X7R is involved in the effect of chronic stress on female reproduction has not been studied. In this study, we established a chronic restraint stress mouse model and chronic cold stress mouse model. We found that the number of corpora lutea was significantly reduced in the two chronic stress models. The number of corpora lutea indirectly reflects the ovulation, suggesting that chronic stress influences ovulation. P2X7R expression was significantly increased in ovaries of the two chronic stress models. A superovulation experiment showed that P2X7R inhibitor A-438079 HCL partially rescued the ovulation rate of the two chronic stress models. Further studies showed that activation of P2X7R signaling inhibited the cumulus expansion and promoted the expression of NPPC in granulosa cells, one key negative factor of cumulus expansion. Moreover, sirius red staining showed that the ovarian fibrosis was increased in the two chronic stress models. For the fibrosis-related factors, TGF-β1 was increased and MMP2 was decreased. In vitro studies also showed that activation of P2X7R signaling upregulated the expression of TGF-β1 and downregulated the expression of MMP2 in granulosa cells. In conclusion, P2X7R expression was increased in the ovaries of the chronic restraint-stress and chronic cold-stress mouse models. Activation of P2X7R signaling promoted NPPC expression and cumulus expansion disorder, which contributed to the abnormal ovulation of the chronic stress model. Activation of P2X7R signaling is also associated with the ovarian fibrosis changes in the chronic stress model. Full article

Insulin receptor signaling promotes cell differentiation, proliferation, and growth which are essential for oocyte maturation, embryo implantation, endometrial decidualization, and placentation. The dysregulation of insulin signaling in women with metabolic syndromes including diabetes exhibits poor pregnancy outcomes that are poorly understood. We utilized the Cre/LoxP system to target the tissue-specific conditional ablation of insulin receptor (Insr) and insulin-like growth factor-1 receptor (Igf1r) using an anti-Mullerian hormone receptor 2 (Amhr2) Cre-driver which is active in ovarian granulosa and uterine stromal cells. Our long-term goal is to examine insulin-dependent molecular mechanisms that underlie diabetic pregnancy complications, and our conditional knockout models allow for such investigation without confounding effects of ligand identity, source and cross-reactivity, or global metabolic status within dams. Puberty occurred with normal timing in all conditional knockout models. Estrous cycles progressed normally in Insr^d/d females but were briefly stalled in diestrus in Igf1r^d/d and double receptor (DKO) mice. The expression of vital ovulatory genes (Lhcgr, Pgr, Ptgs2) was not significantly different in 12 h post-hCG superovulated ovaries in knockout mice. Antral follicles exhibited an elevated apoptosis of granulosa cells in Igf1r^d/d and DKO mice. However, the distribution of ovarian follicle subtypes and subsequent ovulations was normal in all insulin receptor mutants compared to littermate controls. While ovulation was normal, all knockout lines were subfertile suggesting that the loss of insulin receptor signaling in the uterine stroma elicits implantation and decidualization defects responsible for subfertility in Amhr2-Cre-derived insulin receptor mutants. Full article

Rat animal models are widely used owing to their relatively superior cognitive abilities and higher similarity compared with mouse models to human physiological characteristics. However, their use is limited because of difficulties in establishing embryonic stem cells and performing genetic modifications, and insufficient embryological research. In this study, we established optimal superovulation and fertilized–egg transfer conditions, including optimal hormone injection concentration (≥150 IU/kg of PMSG and hCG) and culture medium (mR1ECM), to obtain high-quality zygotes and establish in vitro fertilization conditions for rats. Next, sgRNA with optimal targeting activity was selected by performing PCR analysis and the T7E1 assay, and the CRISPR/Cas9 system was used to construct a rat model for muscular dystrophy by inducing a deficiency in the fukutin gene without any off-target effect detected. The production of fukutin knockout rats was phenotypically confirmed by observing a drop-in body weight to one-third of that of the control group. In summary, we succeeded in constructing the first muscular dystrophy disease rat model using the CRISPR/CAS9 system for increasing future prospects of producing various animal disease models and encouraging disease research using rats. Full article

Ovulation and artificial insemination rates have been observed to decrease in sheep and cows when exposed to dietary phytoestrogens at concentrations greater than 25 mg/kg DM. A grazing trial was undertaken to investigate the effects of coumestrol and other key phytoestrogens on the superovulatory response, embryo numbers and quality in beef cows grazing legume pastures. A 7-week controlled grazing trial was conducted with legume and ryegrass pasture treatments, with cows exposed to legumes at two timed treatments, 4 and 7 weeks. Twenty Angus cows were subjected to a conventional estrus synchronization and superovulation protocol. Embryos were recovered via conventional uterine body flushing 7 days post artificial insemination (AI). Numerous phytoestrogens were identified in both pasture and plasma samples, including coumestrol and formononetin. Concentrations of phytoestrogens in the pasture ranged from 0.001 to 47.5 mg/kg DM and 0 to 2.6 ng/mL in plasma. Approximately 50% of cows produced viable embryos 7 days post AI. A significant interaction between the effect of treatment groups on the embryo stage was observed (p < 0.05). The results suggest that concentrations of >25 mg/kg DM of phytoestrogens less than 20 days preceding AI may negatively affect oocyte developmental competence, reduce progesterone production and thus contribute to early embryonic loss. Full article