After estrus, when mature follicles fail to ovulate, they may further develop to form follicular cysts, affecting the normal function of ovaries, reducing the reproductive efficiency of dairy cows and causing economic losses to cattle farms. However, the key points of ovarian follicular cysts pathogenesis remain largely unclear. The purpose of the current research was to analyze the formation mechanism of ovarian follicular cysts from hormone and gene expression profiles. The concentrations of progesterone (P₄), estradiol (E₂), insulin, insulin-like growth factor 1 (IGF1), leptin, adrenocorticotropic hormone (ACTH) and ghrelin in follicle fluid from bovine follicular cysts and normal follicles were examined using enzyme-linked immunosorbent assay (ELISA) or 125I-labeled radioimmunoassay (RIA); the corresponding receptors’ expression of theca interna cells was tested via quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the mRNA expression profiling was analyzed via RNA sequencing (RNA-seq). The results showed that the follicular cysts were characterized by significant lower E₂, insulin, IGF1 and leptin levels but elevated ACTH and ghrelin levels compared with normal follicles (p < 0.05). The mRNA expressions of corresponding receptors, PGR, ESR1, ESR2, IGF1R, LEPR, IGFBP6 and GHSR, were similarly altered significantly (p < 0.05). RNA-seq identified 2514 differential expressed genes between normal follicles and follicular cysts. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis linked the ovarian steroidogenesis pathway, especially the STAR, 3β-HSD, CYP11A1 and CYP17A1 genes, to the formation of follicular cysts (p < 0.01). These results indicated that hormone metabolic disorders and abnormal expression levels of hormone synthesis pathway genes are associated with the formation of bovine ovarian follicular cysts.

Objective: This study investigated the efficacy of different concentrations of cholesterolloaded cyclodextrin (CLC) on cryopreservation in boar sperm quality.Methods: In this study, we treated boar sperm with different concentrations of CLC before freezing and analyzed the sperm cholesterol concentration, plasma membrane, acrosome integrity rate and total motility rate before and after freeze-thawing. We also investigated the levels of reactive oxygen species (ROS), malondialdehyde (MDA), adenosine triphosphate (ATP), and structural- and oxidative-damage related proteins in all groups after thawing.Results: The results revealed that the cholesterol concentration of the CLC-treated groups was higher than that of the control group, both before freezing and after thawing (p<0.05). The plasma membrane integrity rate, acrosome integrity rate, and total motility rate of sperm were also enhanced after thawing in the CLC-treated group (all p<0.05). Moreover, ROS and MDA production and ATP loss were reduced in CLC-treated sperm during freezing and thawing (p<0.05). Finally, CLC pretreatment partially prevented the consumption of various proteins involved in metabolism including capping actin protein of muscle Z-line subunit beta (CAPZB), heat shock protein 90 alpha family class A member 1 (HSP90AA1) and phosphoglycerate mutase 2 (PGAM2) (p<0.05).Conclusion: The CLC treatment increased cholesterol concentration and decreased structural injury and oxidative damage during boar sperm freezing and thawing, improving the efficacy of sperm cryopreservation in boar.

Postovulatory aging is known to impair the oocyte quality and embryo development due to oxidative stress in many different animal models, which reduces the success rate or pregnancy rate in human assisted reproductive technology (ART) and livestock timed artificial insemination (TAI), respectively. Salidroside (SAL), a phenylpropanoid glycoside, has been shown to exert antioxidant and antitumor effects. This study aimed to investigate whether SAL supplementation could delay the postovulatory oocyte aging process by alleviating oxidative stress. Here, we show that SAL supplementation decreases the malformation rate and recovers mitochondrial dysfunction including mitochondrial distribution, mitochondrial membrane potential (ΔΨ) and ATP content in aged oocytes. In addition, SAL treatment alleviates postovulatory aging-caused oxidative stress such as higher reactive oxygen species (ROS) level, lower glutathione (GSH) content and a reduced expression of antioxidant-related genes. Moreover, the cytoplasmic calcium ([Ca²⁺]c) and mitochondrial calcium ([Ca²⁺]mt) of SAL-treated oocytes return to normal levels. Notably, SAL suppresses the aging-induced DNA damage, early apoptosis and improves spindle assembly in aged oocytes, ultimately elevating the embryo developmental rates and embryo quality. Finally, the RNA-seq and confirmatory experience showed that SAL promotes protective autophagy in aged oocytes by activating the MAPK pathway. Taken together, our research suggests that supplementing SAL is an effective and feasible method for preventing postovulatory aging and preserving the oocyte quality, which potentially contributes to improving the successful rate of ART or TAI.

(1) Background: Cortisol and melatonin (MT) act in regulating follicular development. We hypothesized that abnormal levels of cortisol, MT, and steroids in theca interna cells might be involved in the development of follicular cysts in sows. (2) Methods: To test this hypothesis, we measured the mRNA levels of enzymes involved in steroid hormone synthesis, the glucocorticoid receptor (GR), and melatonin receptors (MTRs) in theca interna cells of cystic and normal porcine follicles. (3) Results: The concentrations of estradiol, progesterone, and cortisol were greater in cystic follicles than in control ones (p = 0.034, p = 0.020, p = 0.000), but the concentration of MT was significantly lower (p = 0.045). The levels of GR, 11β-HSD1, and 11β-HSD2 were higher in cystic follicles than in control l follicles. MT types 1 and 2 were significantly lower in cystic follicles (p < 0.05). The mRNA expression levels of genes encoding the steroid hormone synthesis enzymes, steroidogenic acute regulatory protein (StAR), recombinant cytochrome P45011A1 (CYP11A1), and 3β-hydroxysteroid dehydrogenase (3β-HSD) in theca interna cells of cystic follicles were significantly higher than in control follicles. Thus, there was disruption of hormone secretion in the fluid of cystic follicles in sows. (4) Conclusions: The levels of steroid hormones, cortisol and MT are disrupted in porcine cystic follicles.

Cryopreserved semen is extensively utilized in the artificial insemination (AI) of domestic animals; however, suboptimal conception rates due to oxidative damage following AI continue to pose a challenge. The present study investigated the effects of Pyrroroquinoline Quinone (PQQ), a novel antioxidant, on the semen quality of Holstein bulls during cryopreservation, as well as its potential molecular mechanisms. Semen samples were diluted with varying concentrations of PQQ (0, 50 μmol/L, 100 μmol/L, 150 μmol/L) prior to cryopreservation. Following the freeze–thaw process, a comprehensive evaluation was conducted to assess sperm motility, plasma membrane integrity, acrosome integrity, and the levels of reactive oxygen species (ROS), malondialdehyde (MDA), and adenosine triphosphate (ATP). Western blot analysis was employed to examine the levels of proteins including PGAM2, CAPZB, CAT, SOD1, and GPX1. Notably, the inclusion of 100 μmol/L PQQ significantly enhanced sperm motility, membrane integrity, and acrosome integrity post freeze–thawing (p < 0.05). Furthermore, the group treated with 100 μmol/L PQQ exhibited reduced levels of MDA and ROS (p < 0.05), while ATP levels were significantly elevated (p < 0.05). Interestingly, treatment with 100 μmol/L PQQ resulted in decreased consumption of PGAM2, CAPZB, CAT, SOD1, and GPX1 proteins in sperm after freeze–thawing, compared to the control group (p < 0.05). These findings indicate that PQQ treatment enhances the quality of bull semen, mitigates oxidative stress damage, and ultimately improves the efficacy of sperm cryopreservation.

N-carbamylglutamate (NCG) supplementation during gestation improves reproductive performance in sows after conventional artificial insemination. However, whether NCG can improve reproductive performance and change fecal microbiota and serum metabolite levels during pregnancy in sows after fixed-time artificial insemination (FTAI) remains unclear. Two hundred multiparous sows were assigned a diet from mating until farrowing: control (corn–soybean meal) or NCG supplementation (0.05% NCG). At days 30, 70, and 110 of gestation and after farrowing, maternal microbial diversity and serum metabolites were studied. Supplementation of NCG increased the number of piglets born alive and the litter weight (all p < 0.05) and altered the fetal microbial community during gestation. Some genera were particularly abundant at different time points during gestation and after farrowing, but none were commonly abundant across all four time points. Metabolic analysis revealed that NCG supplementation significantly increased the serum concentrations of NCG, ferulic acid, cinnamoylglycine, 3-phenyllactic acid, and gamma-glutamylglutamic acid in the NCG group compared with levels in the control group. Our results reveal that NCG supplementation during gestation improves reproductive performance in sows after FTAI, exerting both direct (increased serum NCG levels) and indirect effects (altered intestinal microbiome and serum metabolites) on sow reproduction and, ultimately, improving placental and fetal development.