Overexpression of KAT8 induces a failure in early embryonic development in mice.

Embryo quality is strongly associated with subsequent embryonic developmental efficiency. However, the detailed function of lysine acetyltransferase 8 (KAT8) during early embryonic development in mice remains elusive. In this study, we reported that KAT8 played a pivotal role in the first cleavage of mouse embryos. Immunostaining results revealed that KAT8 predominantly accumulated in the nucleus throughout the entire embryonic developmental process. Kat8 overexpression (Kat8-OE) was correlated with early developmental potential of embryos to the blastocyst stage. We also found that Kat8-OE embryos showed spindle-assembly defects and chromosomal misalignment, and that Kat8-OE in embryos led to increased levels of reactive oxygen species (ROS), accumulation of phosphorylated γH2AX by affecting the expression of critical genes related to mitochondrial respiratory chain and antioxidation pathways. Subsequently, cellular apoptosis was activated as confirmed by TUNEL (Terminal Deoxynucleotidyl Transferase mediated dUTP Nick-End Labeling) assay. Furthermore, we revealed that KAT8 was related to regulating the acetylation status of H4K16 in mouse embryos, and Kat8-OE induced the hyperacetylation of H4K16, which might be a key factor for the defective spindle/chromosome apparatus. Collectively, our data suggest that KAT8 constitutes an important regulator of spindle assembly and redox homeostasis during early embryonic development in mice.

NET1 is a critical regulator of spindle assembly and actin dynamics in mouse oocytes.

BACKGROUND: Neuroepithelial transforming gene 1 (NET1) is a RhoA subfamily guanine nucleotide exchange factor that governs a wide array of biological processes. However, its roles in meiotic oocyte remain unclear. We herein demonstrated that the NET1-HACE1-RAC1 pathway mediates meiotic defects in the progression of oocyte maturation. METHODS: NET1 was reduced using a specific small interfering RNA in mouse oocytes. Spindle assembly, chromosomal alignment, the actin cap, and chromosomal spreads were visualized by immunostaining and analyzed under confocal microscopy. We also applied mass spectroscopy, and western blot analysis for this investigation. RESULTS: Our results revealed that NET1 was localized to the nucleus at the GV stage, and that after GVBD, NET1 was localized to the cytoplasm and predominantly distributed around the chromosomes, commensurate with meiotic progression. NET1 resided in the cytoplasm and significantly accumulated on the spindle at the MI and MII stages. Mouse oocytes depleted of Net1 exhibited aberrant first polar body extrusion and asymmetric division defects. We also determined that Net1 depletion resulted in reduced RAC1 protein expression in mouse oocytes, and that NET1 protected RAC1 from degradation by HACE1, and it was essential for actin dynamics and meiotic spindle formation. Importantly, exogenous RAC1 expression in Net1-depleted oocytes significantly rescued these defects. CONCLUSIONS: Our results suggest that NET1 exhibits multiple roles in spindle stability and actin dynamics during mouse oocyte meiosis.

KAT8 functions in redox homeostasis and mitochondrial dynamics during mouse oocyte meiosis progression.

As a histone acetyltransferase, lysine acetyltransferase 8 (KAT8) participates in diverse biological processes. However, the effect of KAT8 on oocyte maturation in mice remains unclear. In this study, we found that mouse oocytes overexpressing Kat8-OE induced maturation failure manifested reduced rates of GVBD and first polar body emission. In addition, immunostaining results revealed that Kat8 overexpressing oocytes showed inappropriate mitochondrial distribution patterns, overproduction of reactive oxygen species (ROS), accumulation of phosphorylated γH2AX, hyperacetylation of α-tubulin, and severely disrupted spindle/chromosome organization. Moreover, we revealed that Kat8 overexpression induced a decline in SOD1 proteins and KAT8's interaction with SOD1 in mouse ovaries via immunoprecipitation. Western blotting data confirmed that Kat8-OE induced downregulation of SOD1 expression, which is a key factor for the decline of oocyte quality in advanced maternal age. Also, the injection of Myc-Sod1 cRNA could partially rescue maternal age-induced meiotic defects in oocytes. In conclusion, our data demonstrated that high level of KAT8 inhibited SOD1 activity, which in turn induced defects of mitochondrial dynamics, imbalance of redox homeostasis, and spindle/chromosome disorganization during mouse oocyte maturation.

3-Acetyldeoxynivalenol induces apoptosis, barrier dysfunction and endoplasmic reticulum stress by inhibiting mTORC1-dependent autophagy in porcine enterocytes.

3-Acetyldeoxynivalenol (3-Ac-DON), an acetylated form of deoxynivalenol, is widely present in mycotoxin-contaminated food, feed as well as in other natural sources. Ingestion of 3-Ac-DON may result in intestinal dysfunction, leading to gut diseases in humans and animals. Nevertheless, the molecular mechanism of 3-Ac-DON in intestinal epithelial cytotoxicity remains unclear. In this study, intestinal porcine epithelial cell line 1 (IPEC-1) cells were treated with different concentrations of 3-Ac-DON for 12 h or 24 h, respectively. The results showed that 3-Ac-DON caused decreased cell viability, cell cycle arrest in G1 phase and depolarization of mitochondrial membrane potential. Western blotting analysis showed that 3-Ac-DON significantly decreased the expression of tight junction proteins, inhibited autophagy and activated endoplasmic reticulum (ER) stress in IPEC-1 cells (P < 0.05). Further investigation demonstrated that 3-Ac-DON caused apoptosis, ER stress and barrier dysfunction were reversed after co-treatment with the autophagy activator rapamycin (100 nM), indicating that autophagy plays a key role in the process of 3-Ac-DON-induced cell damage. In addition, we demonstrated that 3-Ac-DON inhibits the occurrence of autophagy mediated by mTORC1 protein. In conclusion, our research indicated that the mTORC1 protein and autophagy played a key role in the 3-Ac-DON-induced cytotoxic in IPEC-1 cells, which would provide new therapeutic targets and ideas for 3-Ac-DON-mediated intestinal injury.

Chlorpyrifos induces placental oxidative stress and barrier dysfunction by inducing mitochondrial apoptosis through the ERK/MAPK signaling pathway: In vitro and in vivo studies.

Chlorpyrifos (CPF) is an organophosphorus pesticide that is widely used in agricultural production and residential environments worldwide. In this study, we determined the harmful effects and toxicological mechanism of CPF in porcine trophectoderm (pTr) cells and the placenta of female mice during pregnancy. The findings revealed that CPF significantly decreased cell viability and increased intracellular lactate dehydrogenase (LDH) release in pTr cells. Similarly, CPF induced reproductive toxicity in pregnant maternal mice, including decreased maternal, fetal, and placental weights. Moreover, following CPF treatment, pTr cells and the placenta of female mice showed significant apoptosis. JC-1 staining and flow cytometry analysis also revealed that the mitochondrial membrane potential (MMP) of pTr cells treated with CPF was significantly depolarized. Additionally, CPF can induce an increase in reactive oxygen species (ROS) and barrier dysfunction in pTr cells and the placenta of female mice. We further verified that CPF-induced mitochondrial apoptosis is mediated by the MAPK signaling pathway, as shown by using of small molecular inhibitors of related proteins. Also, CPF-induced oxidative stress, barrier dysfunction, and mitochondrial apoptosis in pTr cells were alleviated by U0126, an inhibitor of the ERK/MAPK signaling pathway. These findings suggested that exposure to CPF in early pregnancy might be a potential risk fator affecting placental formation and function in humans and animals.

Role of FBXL5 in redox homeostasis and spindle assembly during oocyte maturation in mice.

As an E3 ubiquitin ligase, F-box and leucine-rich repeat protein 5 (FBXL5) participates in diverse biologic processes. However, the role of Fbxl5 in mouse oocyte meiotic maturation has not yet been fully elucidated. The present study revealed that mouse oocytes depleted of Fbxl5 were unable to complete meiosis, as Fbxl5 silencing led to oocyte meiotic failure with reduced rates of GVBD and polar body extrusion. In addition, Fbxl5 depletion induced aberrant mitochondrial dynamics as we noted the overproduction of reactive oxygen species (ROS) and the accumulation of phosphorylated γH2AX with Fbxl5 knockdown. We also found that Fbxl5-KD led to the abnormal accumulation of CITED2 proteins in mouse oocytes. Our in vitro ubiquitination assay showed that FBXL5 interacted with CITED2 and that it mediated the degradation of CITED2 protein through the ubiquitination-proteasome pathway. Collectively, our data revealed critical functions of FBXL5 in redox hemostasis and spindle assembly during mouse oocyte maturation.

Effect of High Fat Diet on Disease Development of Polycystic Ovary Syndrome and Lifestyle Intervention Strategies.

Polycystic ovary syndrome (PCOS) is a prevalent endocrine and metabolic disorder that affects premenopausal women. The etiology of PCOS is multifaceted, involving various genetic and epigenetic factors, hypothalamic-pituitary-ovarian dysfunction, androgen excess, insulin resistance, and adipose-related mechanisms. High-fat diets (HFDs) has been linked to the development of metabolic disorders and weight gain, exacerbating obesity and impairing the function of the hypothalamic-pituitary-ovarian axis. This results in increased insulin resistance, hyperinsulinemia, and the release of inflammatory adipokines, leading to heightened fat synthesis and reduced fat breakdown, thereby worsening the metabolic and reproductive consequences of PCOS. Effective management of PCOS requires lifestyle interventions such as dietary modifications, weight loss, physical activity, and psychological well-being, as well as medical or surgical interventions in some cases. This article systematically examines the pathological basis of PCOS and the influence of HFDs on its development, with the aim of raising awareness of the connection between diet and reproductive health, providing a robust approach to lifestyle interventions, and serving as a reference for the development of targeted drug treatments.

Cinnamaldehyde alleviates zearalenone-induced LS174T cell apoptosis, barrier dysfunction and mucin reduction through JNK/NF-κB signaling pathway.

As a natural aldehyde organic compound, cinnamaldehyde (CA) is one of the main components of cinnamon essential oil with multiple bioactivities. In this study, we investigated the protective effects of CA on zearalenone (ZEA)-induced apoptosis, barrier dysfunction and mucin reduction, as well as underlying mechanisms in LS174T cells. In the present study, cells pre-treated with or without CA for 24 h were left untreated or subjected to ZEA for indicated time points Our results showed that 10 µM CA significantly prevented ZEA-induced cell viability decline, reversed ZEA-induced increase of the LDH level, cell cycle disruption and apoptosis in LS174T cells. Periodic acid-schiff (PAS) staining analysis showed that CA significantly alleviated the reduction of mucin secretion in LS174T cells caused by ZEA exposure. Western blot analysis showed that CA significantly reversed ZEA-induced reduction of the expression of mucin 2 (MUC2) and tight junction (TJ) proteins (claudin-1, claudin-3, ZO-1 and ZO-2) in LS174T cells. Notably, CA can significantly reduce the upregulation of the main effector of MAPK and NF-κB signaling pathways in LS174T cells. Further study showed that CA protects cells against ZEA-induced cellular damage through JNK/NF-κB signaling pathway in LS174T cells. Supplementation with CA might be an potential strategy to alleviate the damaging effect of ZEA on epithelial cells.

Effect of cloprostenol on luteolysis and comparison of different estrus synchronization protocols in jennies.

Estrus synchronization is necessary for intensive donkey farming. Studies on estrus synchronization in jennies are, however, scarce. We aimed to investigate the susceptibility of the donkey corpus luteum to cloprostenol and design a successful estrus synchronization protocol. Firstly, the effects of different cloprostenol doses and the timing effect of cloprostenol treatment on estrous cycle was investigated. The time from treatment to luteolysis, the ovulation interval, pre-ovulatory diameter, and ovulation rates were compared between groups. Secondly, to identify the best protocol, eight estrus synchronization protocols from three categories were examined. In the first category, jennies in groups A (n = 55) and B (n = 30) received a progesterone releasing intra-vaginal device (JVID®) and cloprostenol treatment. In the second category (group C to F), jennies were pretreated with deslorelin, and then treated with JVID and cloprostenol, including groups C (n = 50), D (n = 50), E (n = 70), and F (n = 65). In the third category, jennies were treated with deslorelin and cloprostenol, including groups G (n = 40) and H (n = 40). Comparisons were made among groups regarding the degree of synchronization, ovulation, and pregnancy rates. Treatment with 0.4 mg cloprostenol on the third day following ovulation minimized the length of the luteal phase and estrous cycle. Synchronization rate varied from 60.0% to 88.6% among groups and was highest in group E. Pregnancy rates did not differ among the eight protocols. In conclusion, cloprostenol effectively induced luteolysis in jennies and a treatment protocol combining deslorelin, cloprostenol, and JVID is efficient for estrus synchronization in donkeys.

Genome-wide association study to identify SNPs and candidate genes associated with body size traits in donkeys.

The Yangyuan donkey is a domestic animal breed mainly distributed in the northwest region of Hebei Province. Donkey body shape is the most direct production index, can fully reflect the donkey's growth status, and is closely related to important economic traits. As one of the main breeding selection criteria, body size traits have been widely used to monitor animal growth and evaluate the selection response. Molecular markers genetically linked to body size traits have the potential to accelerate the breeding process of animals via marker-assisted selection. However, the molecular markers of body size in Yangyuan donkeys have yet to be explored. In this study, we performed a genome-wide association study to identify the genomic variations associated with body size traits in a population of 120 Yangyuan donkeys. We screened 16 single nucleotide polymorphisms that were significantly associated with body size traits. Some genes distributed around these significant SNPs were considered candidates for body size traits, including SMPD4, RPS6KA6, LPAR4, GLP2R, BRWD3, MAGT1, ZDHHC15, and CYSLTR1. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses indicated that these genes were mainly involved in the P13K-Akt signaling pathway, Rap1 signaling pathway, regulation of actin cytoskeleton, calcium signaling pathway, phospholipase D signaling pathway, and neuroactive ligand-receptor interactions. Collectively, our study reported on a list of novel markers and candidate genes associated with body size traits in donkeys, providing useful information for functional gene studies and offering great potential for accelerating Yangyuan donkey breeding.

Comparative Proteome Analysis of Serum Uncovers Differential Expression of Proteins in Donkeys (Equus Asinus) With Endometritis Caused by Escherichia Coli.

Endometritis is a common disease in donkeys that causes economic losses to donkey farms and the common cause is bacterial infection. Uterine flush fluid proteomics has been used to study protein biomarkers associated with endometritis in mares. As a convenient diagnostic tool, serum proteomics has not been studied yet in equine species with endometritis. This study is aiming to evaluate the serum proteomics in jennies with and without endometritis and identify potential proteins as biomarker for endometritis diagnosis. Nine donkeys recruited into this study were diagnosed of bacterial (Escherichia coli) endometritis and nine healthy jennies were selected as control. Blood samples of each donkey was collected, and serum was separated from each sample. Peptides samples extracted from the serum were analyzed using nano-ultrahigh-performance liquid chromatography-tandem mass spectrometry in data-independent acquisition mode. Protein identification and quantification were performed followed by differential and functional analysis. Of 579 proteins identified in all jennies, 12 proteins were exclusively identified in jennies with endometritis (group E) including myeloperoxidase and Ras-related protein Rab-1B, which might be associated with bacterial infection. There were 11 differentially expressed proteins detected between the two groups of jennies with 4 downregulated proteins and 7 upregulated proteins in jennies with endometritis. Some upregulated proteins along with the GO and KEGG annotation indicated inflammatory response against uterine infection. Characteristic serum proteins identified in jennies with endometritis were associated with inflammation or bacterial infection. These proteins might be potential biomarkers for endometritis diagnosis in jennies.

Optimization of recovery and maturation methods for cumulus-oocyte complexes in jennies.

Embryo production in donkeys is inefficient compared with that in other livestock. Obtaining a sufficient number of MII oocytes is the first step to solving this problem. In this study, the number, morphology and maturation rates of cumulus-oocyte complexes (COCs) obtained from abattoir-derived ovaries or live jennies were compared. The diameter of follicles from abattoir-derived ovaries was measured and divided into group 1 (2-6 mm), group 2 (6-10 mm), group 3 (10-20 mm), group 4 (20-28 mm) and group 5 (>28 mm). The results showed that the number of follicles per ovary in group 2 (3.6 ± 0.28) and 3 (4.2 ± 0.90) was higher than that in the other groups (p < .05). The recovery rate in group 3 was higher than group 1 (48.8% vs. 26.8%, p = .00), but lower than group 5 (48.8% vs. 76.5%, p = .025). The percentage of grade A COCs in group 3 was higher than group 2 (59.3% vs. 39.5%, p = .00) and group 1 (59.3% vs. 26.7%, p = .00). Moreover, the percentage of grade A COCs in group 4 (55.0%, p = .710) and group 5 (46.2%, p = .351) was reduced compared with that in group 3. From the above results, the developing follicles (group ovum pick-up [OPU], 10-20 mm) and preovulation follicles (group OPU-Preov, >35 mm) were aspirated from live jennies using OPU. Although there was no difference in the recovery rates of COCs between group 3 and OPU (48.8% vs. 43.0%, p = .184), the percentage of grades A COCs in group OPU was higher than group 3 (72.5% vs. 59.3%, p = .036). There was no difference in the maturation rate between group 3 and OPU (60.3% vs. 69.3%, p = .171) after the COCs matured in vitro. The rates of recovery (72.2%) and maturation (92.3%) in group OPU-Preov were higher than those in other groups (p < .05). Moreover, the effects of maturation time and serum type on maturation rates were evaluated in groups B44 (44 h, FBS), B36 (36 h, FBS) and D44 (44 h, foetal donkey serum, FDS). These results indicated that the maturation rate in group B36 was lower than group B44 (13.1% vs. 47.0%, p = .00) and group D44 (13.1% vs. 53.3%, p = .00). In conclusion, the quality of donkey COCs from OPU was higher than that from abattoir-derived ovaries, the suitable time of donkey in vitro maturation (IVM) was 44 h, and FBS could be replaced with FDS in donkey IVM medium.

l-Proline improves the cytoplasmic maturation of mouse oocyte by regulating glutathione-related redox homeostasis.

Recent studies have shown that l-proline (proline) is an antioxidant to protect cells from oxidative stress in vivo and in vitro. Glutathione (GSH) is a major cellular redox regulator involved in controlling redox balance and is regarded as one of the key indices to predict the cytoplasmic maturation of oocytes. The objectives of this study are to investigate the effect of proline on the developmental potential of mouse oocytes and to determine the role of gap junctional communication (GJC) on intraoocyte GSH concentration during in vitro maturation (IVM). Compared with control (0 mmol/L), 0.5 mmol/L proline supplementation enhanced rates of activated oocytes, 2-cell and 4-cell embryos, and blastocysts. Furthermore, 0.25 and 0.5 mmol/L proline supplementation markedly upregulated mRNA expression of glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) in oocytes and cumulus cells, enhanced GSH concentration in oocytes, and reduced reactive oxygen species (ROS) level in oocytes. Interestingly, carbenoxolone disodium salt (CBX) treatment reduced GSH concentration in oocytes and the rate of early embryo development without proline incubation. Notably, CBX-triggered reduction in the rates of the number of 2-cell and 4-cell embryos and blastocysts were rescued by 0.5 mmol/L proline supplementation. Collectively, these results indicate a novel functional role of proline in oocyte cytoplasmic maturation and regulation of glutathione-related redox homeostasis.

Overexpression of Tfap2a in Mouse Oocytes Impaired Spindle and Chromosome Organization.

Transcription factor AP-2-alpha (Tfap2a) is an important sequence-specific DNA-binding protein that can regulate the transcription of multiple genes by collaborating with inducible viral and cellular enhancer elements. In this experiment, the expression, localization, and functions of Tfap2a were investigated in mouse oocytes during maturation. Overexpression via microinjection of Myc-Tfap2a mRNA into the ooplasm, immunofluorescence, and immunoblotting were used to study the role of Tfap2a in mouse oocyte meiosis. According to our results, Tfap2a plays a vital role in mouse oocyte maturation. Levels of Tfap2a in GV oocytes of mice suffering from type 2 diabetes increased considerably. Tfap2a was distributed in both the ooplasm and nucleoplasm, and its level gradually increased as meiosis resumption progressed. The overexpression of Tfap2a loosened the chromatin, accelerated germinal vesicle breakdown (GVBD), and blocked the first polar body extrusion 14 h after maturation in vitro. The width of the metaphase plate at metaphase I stage increased, and the spindle and chromosome organization at metaphase II stage were disrupted in the oocytes by overexpressed Tfap2a. Furthermore, Tfap2a overexpression dramatically boosted the expression of p300 in mouse GV oocytes. Additionally, the levels of pan histone lysine acetylation (Pan Kac), histone H4 lysine 12 acetylation (H4K12ac), and H4 lysine 16 acetylation (H4K16ac), as well as pan histone lysine lactylation (Pan Kla), histone H3 lysine18 lactylation (H3K18la), and H4 lysine12 lactylation (H4K12la), were all increased in GV oocytes after Tfap2a overexpression. Collectively, Tfap2a overexpression upregulated p300, increased the levels of histone acetylation and lactylation, impeded spindle assembly and chromosome alignment, and ultimately hindered mouse oocyte meiosis.

Integration analysis of metabolome and transcriptome reveals the effect of exogenous supplementation with mixtures of vitamins ADE, zinc, and selenium on follicular growth and granulosa cells molecular metabolism in donkeys (Equus asinus).

Vitamins and microelements play essential roles in mammalian ovarian physiology, including follicle development, ovulation, and synthesis and secretion of hormones and growth factors. However, it is nevertheless elusive to what extent exogenous supplementation with mixtures of vitamins ADE, zinc (Zn), and selenium (Se) affects follicular growth and granulosa cells (GCs) molecular function. We herein investigated their effect on follicular growth and GCs physiological function. We showed that follicular growth and ovulation time was accelerated and shortened with the increases of vitamins ADE, Zn, and Se doses by continually monitoring and recording (one estrus cycle of about 21 days) with an ultrasound scanner. Integrated omics analysis showed that there was a sophisticated network relationship, correlation expression, and enrichment pathways of the genes and metabolites highly related to organic acids and their derivatives and lipid-like molecules. Quantitative real-time PCR (qPCR) results showed that vitamin D receptor (VDR), transient receptor potential cation channel subfamily m member 6 (TRPM6), transient receptor potential cation channel subfamily v member 6 (TRPV6), solute carrier family 5 member 1 (SLC5A1), arachidonate 5-lipoxygenase (ALOX5), steroidogenic acute regulatory protein (STAR), prostaglandin-endoperoxide synthase 2 (PTGS2), and insulin like growth factor 1 (IGF-1) had a strong correlation between the transcriptome data. Combined multi-omics analysis revealed that the protein digestion and absorption, ABC transporters, biosynthesis of amino acids, aminoacyl-tRNA biosynthesis, mineral absorption, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, arginine biosynthesis, and ovarian steroidogenesis were significantly enriched. We focused on the gene-metabolite interactions in ovarian steroidogenesis, founding that insulin receptor (INSR), phospholipase a2 group IVA (PLA2G4A), adenylate cyclase 6 (ADCY6), cytochrome p450 family 1 subfamily b member 1 (CYP1B1), protein kinase camp-activated catalytic subunit beta (PRKACB), cytochrome p450 family 17 subfamily a member 1 (CYP17A1), and phospholipase a2 group IVF (PLA2G4F) were negatively correlated with ß-estradiol (E2), progesterone (P4), and testosterone (T) (P < 0.05). while ALOX5 was a positive correlation with E2, P4, and T (P < 0.05); cytochrome p450 family 19 subfamily a member 1 (CYP19A1) was a negative correlation with cholesterol (P < 0.01). In mineral absorption, our findings further demonstrated that there was a positive correlation between solute carrier family 26 member 6 (SLC26A6), SLC5A1, and solute carrier family 6 member 19 (SLC6A19) with Glycine and L-methionine. Solute carrier family 40 member 1 (SLC40A1) was a negative correlation with Glycine and L-methionine (P < 0.01). TRPV6 and ATPase Na+/K+ transporting subunit alpha 1 (ATP1A1) were positively associated with Glycine (P < 0.05); while ATPase Na+/K+ transporting subunit beta 3 (ATP1B3) and cytochrome b reductase 1 (CYBRD1) were negatively related to L-methionine (P < 0.05). These outcomes suggested that the vitamins ADE, Zn, and Se of mixtures play an important role in the synthesis and secretion of steroid hormones and mineral absorption metabolism pathway through effects on the expression of the key genes and metabolites in GCs. Meanwhile, these also are required for physiological function and metabolism of GCs. Collectively, our outcomes shed new light on the underlying mechanisms of their effect on follicular growth and GCs molecular physiological function, helping explore valuable biomarkers.

Endometrial and vaginal microbiome in donkeys with and without clinical endometritis.

Endometrial and vaginal microbiomes are critical in the study of endometritis, which is an important cause of infertility in donkeys. Our objective was to investigate the difference of the endometrial and vaginal microbiomes between healthy donkey jennies (group C) and jennies with endometritis (group E). Endometrial and vaginal swab samples were collected, and the 16 s rRNA gene amplicon high-throughput sequencing technique was applied to identify the microbial composition in the samples. A similar microbial composition pattern was found between endometrial and vaginal samples, which indicated the impact of the vaginal microbiome on the endometrial microbial environment and health. There was a significant difference of endometrial and vaginal swab samples between the two groups. Ruminococcaceae and Lachnospiraceae were significantly more abundant in endometrial and vaginal microbiomes of group E than in group C. Their dominance was consistent with increased anaerobic bacterial taxa in the functional analysis, which might be associated with the pathogenesis of endometritis in donkeys. Sphingomonadaceae, a bacterial family reported in bovine semen, was statistically more abundant in endometrial microbiome of group E than in group C, which might suggest an association between high abundance of Sphingomonadaceae possibly due to uncleared semen and donkey endometritis. Our study revealed the composition of the vaginal and endometrial microbiomes in healthy and endometritis donkeys. These findings will provide more insights into the pathogenesis of donkey endometritis.

Effects of Exercise on Donkey Semen Parameters and Some Physiological Indices.

To study the effects of exercise on donkey semen parameters, Dezhou donkey (Equus asinus) jackasses (n = 18) were assigned to three groups: control (CN, n = 6), 1-hour exercise (1 h EX, n = 6) and 2-hour exercise (2 h EX, n = 6) groups. They were exercised with a walking machine for 3 days/week for 8 weeks. Semen parameters and reproductive hormones were evaluated weekly. The heart rate, rectal temperature, blood haematology indexes, and the body condition score (BCS) were also measured. Results showed that the ejaculation volume increased while the sperm concentration decreased after exercise. However, other sperm parameters were unaffected. Reproductive hormones at resting period were rarely affected by exercise. The red blood cell number increased (P = .023), while the mean erythrocyte hemoglobin concentration decreased (P = .045) after the 8-week exercise in the 2 h EX group compared with the CN. After the 8-week exercise, the resting heart rate of the two exercise groups decreased significantly compared with the CN. The BCS in the CN group increased after the experiment (P = .024). In conclusion, we found that exercise had positive effects on some physiological indices but did not affect semen parameters expect for volume and concentration of the jackass.

Effects of IGF-1 on the Three-Dimensional Culture of Ovarian Preantral Follicles and Superovulation Rates in Mice.

Insulin-like growth factor-1 (IGF-1) plays a crucial role during folliculogenesis, which has been demonstrated by previous research. However, the optimal IGF-1 dosage in the three-dimensional (3D) culture system is unknown. Mouse secondary follicles (140−150 µm) were cultured for 6 days within an alginate bead in a medium supplemented with 0 (G0), 5 ng/mL (G5), 10 ng/mL (G10), or 50 ng/mL IGF-1 (G50). Secretions of 17ß-estradiol and progesterone were significantly increased in G10 and G50 (p < 0.05). However, G50 significantly inhibited follicular growth (p < 0.05), while G10 showed a higher oocyte maturation rate. Thus, the 10 ng/mL IGF-1 was used in subsequent experiments. IGF-1 enhanced the function of granulosa cells (GCs) by upregulating expressions of Star, Cyp19a1, Hsd3b1, Fshr, and Lhcgr. Oocyte secretory function was promoted by upregulating expressions of Bmp-15, Gdf-9, and Fgf-8. Addition of IGF-1 showed anti-apoptotic effect. However, G10 did not improve fertilization rate of MII oocytes compared to G0. In an intraperitoneal injection experiment in mice, IGF-1 significantly increased the number of ovulated oocytes (p < 0.05). In conclusion, 10 ng/mL IGF-1 can promote the production of mature oocytes in the 3D culture medium and injection of IGF-1 before superovulation increases the number of ovulated oocytes.

Antimicrobial Susceptibility of Bacterial Isolates from Donkey Uterine Infections, 2018-2021.

BACKGROUND: Endometritis is a common reproductive disease in equine animals. No investigation about the bacterial characteristics and antimicrobial susceptibility pattern of donkeys with endometritis has thus far been reported. OBJECTIVES: To determine the common uterine bacterial isolates from donkeys with endometritis and to evaluate their susceptibility to antimicrobials used for the treatment thereof. STUDY DESIGN: Retrospective case-series. METHODS: Medical records at an equine clinical diagnostic center were retrospectively reviewed to identify submissions from donkeys with bacterial endometritis between 2018 and 2021. Data were extracted and analyzed descriptively in terms of the frequency of bacterial species, susceptibility to antimicrobials and multidrug resistance. RESULTS: A total of 73 isolates were identified from 30 donkeys, of which 92% of the isolates were Gram-negative bacteria. Mixed cultures were found in 90% of the donkeys. The most common isolates were Escherichiacoli (31.5%) and Acinetobacter spp. (21.9%). Susceptibility testing revealed that amikacin (98%), cefoxitin (95%), trimethoprim-sulfamethoxazole (78%) and gentamicin (74%) were the most efficient agents for donkeys. Multidrug resistance (MDR) was found in 20% of all bacterial isolates, of which all Pseudomonas aeruginosa isolates showed a multidrug resistance profile. Main limitations: The sample size was relatively small, which means a bias of selection may exist. The antimicrobial resistance and MDR of agents without break points were not calculated, which means the relative results may be underestimated in our study. CONCLUSIONS: Severe infections were detected in donkeys with endometritis. Antimicrobial resistance and MDR bacteria are not rare in our study. This study demonstrated that bacteria identification and antimicrobial susceptibility testing are highly recommended before the treatment of uterine infections in donkeys. Further studies, including the epidemiological investigation of bacterial endometritis of donkeys, should be conducted to provide a better understanding of this critical problem.