Nrf2 is a redox sensitive transcription factor regulating the expression of antioxidant genes as defense mechanism against various stressors. The aim of this study is to investigate the potential role of noncoding miRNAs as endogenous and quercetin as exogenous regulators of Nrf2 pathway in bovine granulosa cells. For this cultured granulosa cells were used for modulation of miRNAs (miR-28, 153 and miR-708) targeting the bovine Nrf2 and supplementation of quercentin to investigate the regulatory mechanisms of the Nrf2 antioxidant system. Moreover, cultured cells were treated with hydrogen peroxide to induce oxidative stress in those cells. Our results showed that, oxidative stress activated the expression of Nrf2 as a defense mechanism, while suppressing the expression of those miRNAs. Overexpression of those miRNAs resulted in downregulation of Nrf2 expression resulted in higher ROS accumulation, reduced mitochondrial activity and cellular proliferation. Quercetin supplementation showed its protective role against oxidative stress induced by H2O2 by inducing the expression of antioxidant enzymes. In conclusion, this study highlighted the involvement of miR-153, miR-28 and miR-708 in regulatory network of Nrf2 mediated antioxidant system in bovine granulosa cells function. Furthermore, quercetin at a low dose played a protective role in bovine granulosa cells against oxidative stress damage.

The present study aimed to determine the effects of zinc oxide nanoparticles (ZnO-NPs), thyme oil (THO), or their combination on the nutrient digestibility coefficients, reproductive parameters, and some blood metabolites of male Californian rabbits. One hundred rabbits, 29-weeks of age (initial body weight 3.48 ± 0.08 kg) were randomly distributed into four groups, 25 rabbits each. Treatment groups were fed a control diet, a control diet supplemented with ZnO-NPs (100 mg/kg), THO (500 mg/kg), or combination of ZnO-NPs (100 mg/kg) and THO (500 mg/kg). The feeding trial lasted for 35 days. Results showed improvements in dry matter, crude protein, ether extract, and crude fiber in ZnO-NPs, THO, and their combination treated groups compared to those of control. Furthermore, semen volume, sperm motility, vitality, and morphology were significantly improved (p < 0.01) in ZnO-NPs and THO groups rather than the control. Both ZnO-NPs and THO, as either individual or combined treatments significantly improved the serum alanine amino-transferase (ALT), aspartate amino-transferase (AST), urea, and creatinine compared to the control. Moreover, serum concentrations of testosterone were significantly increased in rabbits supplemented with ZnO-NPs, THO, or their combination compared to those of control (p < 0.05). In conclusion, ZnO-NPs, THO, or their combination improved the digestibility of nutrients, liver/ kidney functions, semen characteristics, and testosterone concentration in male rabbits.

Background Extracellular vesicles (EVs) are a promising biomarker and play a vital role in cell–cell communication. This study aimed (I) to identify and characterize EVs from low volume uterine lavage (LVL) and serum in mares with endometritis, compared to healthy controls and (II) to measure serum levels of interleukin 6 (IL-6), and prostaglandins (PGF2α and PGE2). Mares were divided into 30 sub-fertile (endometritis) and 20 fertile (controls). Serum and LVL was collected for EV isolation, and determination of serum levels of inflammatory mediators. Characterization and visualization of EVs were done by electron microscopy, dynamic light scattering and flow cytometry. Results Serial ultracentrifugation of LVL and use of a commercial kit for serum were strategies for EVs isolation. Mares with endometritis released higher amounts of larger size EVs. The EVs from mares with endometritis differentially expressed CD9 and CD63, compared to controls. Mares suffering from endometritis evoked higher levels of inflammatory mediators. Conclusions Thus, EVs could be used for a better understanding the regulatory mechanisms associated with developing endometritis in mares.

Transcription factors (TFs) are known to be involved in regulating the expression of several classes of genes during folliculogenesis. However, the regulatory role of TFs during oxidative stress (OS) is not fully understood. The current study was aimed to investigate the regulation of the TFs in bovine granulosa cells (bGCs) during exposure to OS induced by H2O2 in vitro. For this, bGCs derived from ovarian follicles were cultured in vitro till their confluency and then treated with H2O2 for 40 min. Twenty-four hours later, cells were subjected to various phenotypic and gene expression analyses for genes related to TFs, endoplasmic reticulum stress, apoptosis, cell proliferation, and differentiation markers. The bGCs exhibited higher reactive oxygen species accumulation, DNA fragmentation, and endoplasmic reticulum stress accompanied by reduction of mitochondrial activity after exposure to OS. In addition, higher lipid accumulation and lower cell proliferation were noticed in H2O2-challenged cells. The mRNA level of TFs including NRF2, E2F1, KLF6, KLF9, FOS, SREBF1, SREBF2, and NOTCH1 was increased in H2O2-treated cells compared with non-treated controls. However, the expression level of KLF4 and its downstream gene, CCNB1, were downregulated in the H2O2-challenged group. Moreover, targeted inhibition of NRF2 using small interference RNA resulted in reduced expression of KLF9, FOS, SREBF2, and NOTCH1 genes, while the expression of KLF4 was upregulated. Taken together, bovine granulosa cells exposed to OS exhibited differential expression of various transcription factors, which are mediated by the NRF2 signaling pathway.

Sexually dimorphic differences in genome activity, which is orchestrated by transcription factors (TFs), could explain the differential response of male and female embryos to environmental stressors. To proof this hypothesis, the expression of cellular and extracellular TFs was investigated in male and female bovine embryos in vitro cultured either under low (5%) or high (20%) oxygen levels. The intracellular reactive oxygen species (ROS), total cell number, expression of nuclear factor (erythroid‐derived 2) factor 2 (NFE2L2), Krüppel‐like factor 4 (KLF4), notch receptor 1 (NOTCH1), E2F transcription factor 1 (E2F1), and SREBF2 along with extracellular vesicles (EVs) biogenesis genes were assessed at the blastocyst stage and their released EVs. Low blastocyst rate in both sexes due to oxidative stress (OS) was accompanied by increased ROS accumulation and reduced cell number in female embryos. The messenger RNA and protein levels of NFE2L2, as well as KLF4 expression, were higher in male embryos exposed to OS compared with female embryos. However, the expression of NOTCH1 and E2F1 was higher in female embryos cultured in high oxygen level. Male embryos exposed to OS released more EVs enriched with NFE2L2, superoxide dismutase 1, and NOTCH1 accompanied by elevated expression of EVs biogenesis genes. Accordingly, differential expression of TFs and their release into spent media could partially explain the sexual dimorphic response of bovine embryos to environmental stresses.

Background No doubt that the corpus luteum (CL) plays a vital role in the regulation of female cyclicity in mammals. The scenarios among microRNAs (miRNAs) and their target genes and steroid hormones {estradiol (E2) and progesterone (P4)} are required for better understanding the molecular regulation of CL during its formation, maturation, and regression. We aimed to (I) study the changes in the relative abundance of miR-205, miR-26a-5p, miR-17-5p, and let-7b-5p and their target genes: LHCGR, CASP3, PCNA, AMH, and PLA2G3, during different stages of corpus luteum in Egyptian buffaloes, and (II) and to address different scenarios between steroid concentrations in the serum and the expression pattern of selected miRNAs and their targets. Methods The paired ovaries and blood samples were collected from apparently healthy 50 buffalo cows at a private abattoir. The ovaries bearing CL were macroscopically divided according to their morphological structure and color into hemorrhagic (CLH), developing (CLD), mature (CLM), regressed (CLR), and albicans (CLA). Small pieces from different stages of CL (CLH, CLD, CLM, CLR, and CLA) were cut and immediately kept at − 80 °C for total RNA isolation and qRT-PCR. The serum was separated for steroid level estimation. Results The LHCGR was expressed during different stages of CL, and the peak of expression was at the mid-luteal stage. The CASP3 revealed a stage-specific response at different stages of CL. The PCNA has an essential role in cellular proliferation in buffaloes CL. Both expression patterns of PLA2G3 and AMH were found over the various developmental and regression stages. It was noticed that miR-205 is conserved to target LHCGR and CASP3 transcripts. Moreover, CASP3 and AMH were targeted via miR-26a-5p. Additionally, the CASP3 and PLA2G3 were targeted via let-7b-5p. The P4 level reached its peak during CLM. There were positive and negative strong correlations between miRNAs (miR-26a-5p and miR-205), target genes (LHCGR and CASP3) during different stages of CL, and steroid hormones in the serum. Conclusions Taken together, the orchestrated pattern among miRNAs, target genes, and steroid hormones is essential for maintaining the proper development and function of CL in buffalo cows.

So far the intimate link between serum microRNA (miRNA) and uterine inflammation in mares is unknown. We aimed (I) to investigate expression profile of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 (II) and to measure concentrations of interleukin 6 (IL-6), and prostaglandins (PGF2α and PGE2) in serum of mares with healthy and abnormal uterine status (endometritis). This study was conducted on 80 Arabian mares: young (4–7 years), and old (8–14 years). Mares were divided into 48 sub-fertile (endometritis) and 32 fertile (control) at stud farms. Serum was collected for measuring IL-6, PGF2α, and PGE2, as well as miRNA isolation and qRT-PCR. Concentrations of IL-6, PGE2, and PGF2α were higher in mares with endometritis compared to control. Age of mares had a remarkable effect on IL-6, PGE2, and PGF2α concentrations. Relative abundance of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 was higher in both young and old mares with endometritis. We noticed that eca-miR-155, eca-miR-223, eca-miR-200a, and eca-miR-205 revealed higher expression level in old than young mares with endometritis. This is the first study that has revealed the changes in cell free miRNA and serum inflammatory mediators during endometritis, and these findings could be used for a better understanding the pathophysiology mechanisms of endometritis in equine.

Background: So far the intimate link between serum microRNA (miRNA) and uterine inflammation in mares is unknown. We aimed (I) to investigate the expression profile of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 (II) and to measure the concentrations of interleukin 6 (IL-6), and prostaglandins (PGF2α& PGE2) in serum of Arabian mares with healthy and abnormal uterine status (endometritis).Methods and Results: This study was conducted on 80 Arabian mares; young (4-7 years), and old (8-14 years). These animals were divided into 48 sub-fertile including 16 young and 32 old mares suspected of endometritis and 32 fertile as control (24 young and 8 old) at stud farms. Serum samples were collected for measuring IL-6, PGF2α, and PGE2 concentrations, as well as serum miRNA isolation and qRT-PCR. Serum concentrations of IL-6, PGE2, and PGF2α were higher (P≤0.001) in mares with endometritis (young and old) compared to the control ones. Age of mares had a remarkable effect(0.001≤P≤0.01) onIL-6, PGE2, and PGF2αconcentrations. The relative abundance of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 was higher (P≤0.001) in both young and old mares with endometritis. We noticed that eca-miR-155, eca-miR-223, eca-miR-200a, and eca-miR-205 revealed higher (0.001≤P≤0.01) expression level in old than young mares with endometritis. Conclusions: To the best of our knowledge, this is the first study revealed that serum miRNA and serum inflammatory mediators (IL-6, PGE2, and PGF2α) could be used as non-invasive gold standard biomarkers, and therefore might be served as an important additional diagnostic tool for endometritis in Arabian mares.