Expression profile of viability and stress response genes as a result of resveratrol supplementation in vitrified and in vitro produced cattle embryos.

BACKGROUND: Resveratrol, a potent antioxidant, is known to induce the up-regulation of the internal antioxidant system. Therefore, it holds promise as a method to mitigate cryopreservation-induced injuries in bovine oocytes and embryos. This study aimed to (i) assess the enhancement in the quality of in vitro produced bovine embryos following resveratrol supplementation and (ii) monitor changes in the expression of genes associated with oxidative stress (GPX4, SOD, CPT2, NFE2L2), mitochondrial function (ATP5ME), endoplasmic reticulum function (ATF6), and embryo quality (OCT4, DNMT1, CASP3, ELOVL5). METHODS AND RESULTS: Three groups of in vitro bovine embryos were cultured with varying concentrations of resveratrol (0.01, 0.001, and 0.0001 µM), with a fourth group serving as a control. Following the vitrification process, embryos were categorized as either good or poor quality. Blastocysts were then preserved at - 80 °C for RNA isolation, followed by qRT-PCR analysis of selected genes. The low concentrations of resveratrol (0.001 µM, P < 0.05 and 0.0001 µM, P < 0.01) significantly improved the blastocyst rate compared to the control group. Moreover, the proportion of good quality vitrified embryos increased significantly (P < 0.05) in the groups treated with 0.001 and 0.0001 µM resveratrol compared to the control group. Analysis of gene expression showed a significant increase in OCT4 and DNMT1 transcripts in both good and poor-quality embryos treated with resveratrol compared to untreated embryos. Additionally, CASP3 expression was decreased in treated good embryos compared to control embryos. Furthermore, ELOVL5 and ATF6 transcripts were down-regulated in treated good embryos compared to the control group. Regarding antioxidant-related genes, GPX4, SOD, and CPT2 transcripts increased in the treated embryos, while NFE2L2 mRNA decreased in treated good embryos compared to the control group. CONCLUSIONS: Resveratrol supplementation at low concentrations effectively mitigated oxidative stress and enhanced the cryotolerance of embryos by modulating the expression of genes involved in oxidative stress response.

Abundance of selected genes implicated in testicular functions in Camelus dromedarius with high and low epididymal semen quality.

Studying testicular genes' expression may give key insights into precise regulation of its functions that influence epididymal sperm quality. The current study aimed to investigate the abundance of candidate genes involved in the regulation of testicular functions specially those regulate sperm function (PLA2G4D, SPP1, and CLUAP1), testicular steroidogenic function (ESR1 and AR), materials transport (AQP12B and LCN15), and defense mechanisms (DEFB110, GPX5, SOCS3, and IL6). Therefore, blood samples and testes with epididymis were collected from mature middle-aged (5-10 years) dromedary camels (n = 45) directly prior and after their slaughtering, respectively, during breeding season. Sera were evaluated for testosterone level and testicular biometry was measured with caliper. The epididymal tail semen was evaluated manually. Samples were distinguished based on testosterone level, testicular biometry, as well as epididymal semen features into high and low fertile groups. Total RNA was isolated from testicular tissues and gene expression was done using Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR). Results revealed that testosterone levels were significantly (P < 0.005) higher in camels with good semen quality than those of low quality. There was a significant (P < 0.0001) increase in testicular weight, length, width, thickness, and volume in high fertile than low fertile camels. PLA2G4D, SPP1, CLUAP1, ESR1, AR, AQP12B, LCN15, DEFB110, GPX5, and SOCS3 genes were upregulated (P < 0.001), and IL6 gene was downregulated (P < 0.01) in the testes of high fertile camels compared to the low fertile one. Thus, it could be concluded that examined genes might be valuable monitors of testicular functional status and fertility in dromedary camels.

In vitro effect of zearalenone on sperm parameters, oocyte maturation and embryonic development in buffalo.

The negative impact of zearalenone (ZEN; potent estrogenic mycotoxin) exposure on buffalo embryo production has not yet been determined. In the current study, buffalo sperm and oocytes were exposed to ZEN at different concentrations during maturation. Sperms (with and without ZEN exposure) were incubated for 2 h and evaluated for motility, viability, acrosome integrity, normality, and ultrastructure. Matured oocytes exposed to ZEN were stained to determine their nuclear maturation. Further, their developmental ability was evaluated after in vitro fertilization. Our results showed the toxic effects of ZEN at high concentrations (2000 ng/mL) on different buffalo sperm parameters. The number of acrosome-intact sperm was reduced at 0 h after exposure to a concentration of ≥ 100 ng/mL. Furthermore, the maturation rate of buffalo oocytes (telophase I + metaphase II) was significantly decreased in ZEN-treated oocytes with a higher degeneration rate. Oocytes matured in 1000 ng/mL ZEN and subsequently exhibited considerable reduction in cleavage rate and blastocyst formation compared with control oocytes (2.6% vs. 13.1%). Moreover, the morula rate was decreased (p < 0.001) in ZEN-treated oocytes at concentrations of ≥ 10 ng/mL. Overall, the adverse effects of in vitro ZEN exposure on buffalo sperm parameters and oocyte meiotic progression with a notable reduction in cleavage, morula, and blastocyst rates were defined by these results. Altogether, buffaloes should be considered sensitive to ZEN exposure with respect to their reproductive function.

Spatiotemporal expression pattern of miR-205, miR-26a-5p, miR-17-5p, let-7b-5p, and their target genes during different stages of corpus luteum in Egyptian buffaloes.

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.

Extracellular vesicles in low volume uterine lavage and serum: novel and promising biomarker for endometritis in Arabian mares.

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.

Dynamic patterns of expressed genes in granulosa cells during follicular and luteal stages in Egyptian buffaloes.

A better understanding of the molecular mechanisms in granulosa cells (GC) is warranted, during different follicular and luteal developmental stages in buffalo cows. We aimed to (I) study the expression of selected genes in GC during follicular and luteal phases, (II) evaluate correlations between GC gene expression and steroid concentrations {17-beta estradiol (E2) and progesterone (P4)} in follicular fluid (FF), and (III) study effect of ovarian status on follicular population as well as follicular size frequency. Ovaries were collected in pairs from buffaloes (n = 178). Ovaries bearing corpus luteum (CL) were subdivided into hemorrhagic, developing, mature, and albicans. Follicles from luteal groups were classified only into small (< 4 mm) and large (9-20 mm), while follicles from follicular groups were classified into three subgroups: small (< 4 mm), medium (5-8 mm), and large (9-20 mm). The FF and GC were collected for steroid concentrations measurement and gene expression, respectively. In the follicular phase, luteinizing hormone/choriogonadotropin receptor (LHCGR) and cytochrome P450 aromatase (CYP19) in small follicles decreased compared to medium ones. Large follicle showed an increase in LHCGR and CYP19 compared to medium ones. Follicle-stimulating hormone receptor (FSHR) decreased in large compared to medium size follicles. Proliferating cell nuclear antigen (PCNA) increased in small and large follicles. Meanwhile, anti-Mullerian hormone (AMH) and phospholipase A2 group III (PLA2G3) decreased in small and large follicles. The different stages of luteal phase had a profound impact on GC gene expression. There were strong (positive and/or negative) correlations between gene expression and steroid hormones. The different scenarios between expressed genes in GC and steroid concentrations are required for the proper growth and development of follicles and CL.

Alterations in the Expression Profile of Serum miR-155, miR-223, miR-17, miR-200a, miR-205, as well as Levels of Interleukin 6, and Prostaglandins during Endometritis in Arabian Mares.

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.

Impact of L-carnitine supplementation on the in vitro developmental competence and cryotolerance of buffalo embryos.

BACKGROUND AND AIM: Despite many trials, buffalo embryos have poor cryosurvivability because of their high lipid content. L-carnitine was found to be a lipid-reducing agent when added to oocyte and embryo culture media. The study aimed to determine the most effective concentration of L-carnitine to improve the oocyte developmental competence and cryotolerance of buffalo embryos. MATERIALS AND METHODS: In vitro maturation and embryo culture media were supplemented with four concentrations of L-carnitine: 0 (control), 0.25, 0.5, and 1 mM. Good-quality embryos on 7 days were vitrified using mixtures of dimethyl sulfoxide and ethylene glycol at two concentrations (3.5 and 7 M). RESULTS: The result showed that the cleavage and morula rates were significantly (p<0.05) higher in the 0.5 mM group. Blastocyst rates were significantly (p<0.05) higher at both 0.5 and 1 mM. The rates of viable embryos directly after thawing were significantly (p<0.05) increased in the 0.5 mM group. No significant difference was found in embryos cultured for 24 h after warming among all the groups. CONCLUSION: The addition of L-carnitine at a concentration of 0.5 mM to the culture media improves the oocyte developmental competence and cryotolerance of buffalo embryos directly after warming but not after 24 h of culture. Nevertheless, further studies must identify how L-carnitine exerts its beneficial micromechanisms.

Impact of nano-selenium on nuclear maturation and genes expression profile of buffalo oocytes matured in vitro.

Supplementation of maturation media with antioxidant (bulk form) improves oocyte maturation. However, the influence of adding antioxidant (nano-particles) on oocyte maturation is not well known. We aimed to evaluate the effect of selenium nano-particles (SeNP) and bulk selenium (Se) on buffalo oocytes maturation, in terms nuclear maturation and molecular level. Oocytes were distributed into four groups; 1st group was control, 2nd group was supplied with Se (10 ng/ml), 3rd and 4th groups were supplied with 1 µg/ml SeNP (67 nm), and SeNP (40 nm), respectively. Matured oocytes were fixed and stained to determine nuclear maturation. Oocytes and COC after IVM were stored at - 80 °C, for RNA isolation and qRT-PCR for selected genes. The Se and seNP (40 nm) had a positive effect on oocytes nuclear maturation rates. Apoptosis-related cysteine peptidase (CASP3) was reduced in all supplemented groups. Anti-Mullerian hormone (AMH) was up-regulated in oocytes supplemented with SeNP (40 nm). In COC, AMH increased in group supplemented with SeNP (67 nm). In oocytes, phospholipase A2 group III (PLA2G3) decreased in all supplemented groups. While in COC, PLA2G3increased in group supplied with Se. In COC, luteinizing hormone/choriogonadotropin receptor (LHCGR) increased in groups supplied with Se or SeNP (40 nm).Glutathione peroxidase 4 (GPX4) increased in all supplemented groups, in oocytes and COC. In oocytes, superoxide dismutase (SOD) was up-regulated in supplemented groups {Se and SeNP (67 nm)}.The DNA methyltransferase (DNMT) in oocytes was reduced in supplemented groups. In oocytes, the POU class 5 homeobox 1 (OCT4) increased in all supplemented groups. In COC, the OCT4 was over-expressed in group supplemented with SeNP (40 nm). Selenium supplementation in bulk or nano-particle improved in vitro buffalo oocytes maturation, viaup-regulation of antioxidant defense and development competence genes. SeNP (smaller size, 40 nm) induced higher expression of antioxidant gene.

Expression profiling of primary cultured buffalo granulosa cells from different follicular size in comparison with their in vivo counterpart.

This study aimed to: (i) characterize cultured granulosa cells (GCs) from different follicle sizes morphologically and molecularly; and (ii) select a suitable model according to follicular size that maintained GC function during culture. Buffalo ovaries were collected from a slaughterhouse and follicles were classified morphologically into: first group ≤ 4 mm, second group 5-8 mm, third group 9-15 mm and fourth group 16-20 mm diameter. GC pellets were divided into two portions. The first portion served as the control fresh pellet, and the secondwas used for 1 week for GC culture. Total RNA was isolated, and qRT-PCR was performed to test for follicle-stimulating hormone receptor (FSHR), cytochrome P450 19 (CYP19), luteinizing hormone/choriogonadotropin receptor (LHCGR), proliferating cell nuclear antigen (PCNA), apoptosis-related cysteine peptidase (CASP3), anti-Müllerian hormone (AMH), and phospholipase A2 group III (PLA2G3) mRNAs. Estradiol (E2) and progesterone (P4) levels in the culture supernatant and in follicular fluids were measured using enzyme-linked immunosorbent assay (ELISA). Basic DMEM-F12 medium maintained the morphological appearance of cultured GCs. The relative abundance of FSHR, CYP19, and LHCGR mRNAs was 0.001 ≤ P ≤ 0.01 and decreased at the end of culture compared with the fresh pellet. There was a fine balance between expression patterns of the proliferation marker gene (PCNA) and the proapoptotic marker gene (CASP3). AMH mRNA was significantly increased (P < 0.001) in cultured GCs from small follicles, while cultured GCs from other three categories (5-8 mm, 9-15 mm and 16-20 mm) showed a clear reduction (P < 0.001). Interestingly, the relative abundance of PLA2G3 mRNA was significantly (P < 0.001) increased in all cultured GCs. E2 and P4 concentrations were significantly (P < 0.001) decreased in all cultured groups. Primary cultured GCs from small follicles could be a good model for better understanding follicular development in Egyptian buffaloes.

Establishment a protocol for total RNA isolation from buffalo fresh and frozen semen for molecular applications.

To date, there is no an established protocol for total RNA isolation in Egyptian buffalo spermatozoa. The present study aimed (I) to establish a defined protocol for total RNA isolation from fresh and frozen spermatozoa, (II) to evaluate RNA quality and quantity from different extraction methods and studying gene expression. Warm and standard room temperature modified QIAzol Lysis Reagents were used for total RNA extraction. The quality and quantity of extracted RNA were checked, and subsequently qRT-PCR was performed using androgen receptor-like and three reference gene primers (GAPDH, ACTB and 18S). The warm modified QIAzol Lysis Reagents resulted higher yield of good quality RNA from fresh (569.54 ± 18.83 ng/µl) and frozen spermatozoa (110.59 ± 4.43 ng/µl), compared to standard room temperature modified QIAzol (421.26 ± 7.18 ng/µl) and (29.07 ± 5.25 ng/µl), for fresh and frozen semen samples respectively. The 260/280 ratio was 1.90 and 1.89 for fresh and frozen isolated semen by warm method respectively. The integrity of RNA was good and appeared as a sharp band on 2% agarose gel. The most stable reference gene was 18S. Reliable extraction method of high quality RNA yield could be a step forward for understanding mechanisms of spermatogenesis for improving male fertility.

PCR-RFLP of bone morphogenetic protein 15 (BMP15/FecX) gene as a candidate for prolificacy in sheep.

Bone morphogenetic protein 15 (BMP15/FecX) gene is considered one of the major genes and a candidate marker for the reproduction in farm animals, especially sheep. The present study aimed to detect the genetic polymorphisms of BMP15 gene in sheep using PCR-RFLP technique. In the present study, 115 ewes were assigned into high and low prolificacy categories according to their reproductive history. In high prolific group (n = 20), ewes produced twins more than single births. In the low prolific type (n = 95), the ewes produced single births more than twins. DNA was extracted from blood samples of all ewes, subjected to PCR-RFLP analysis and confirmed by sequence analysis. The PCR products of 356 bp size were cut with HinƒI restriction enzyme. Three digested fragments of 70, 117 and 169 bp were obtained in both types of sheep. All animals were homozygous with CC genotype. In conclusion, the accessible findings did not detect any mutation in FecX gene in sheep, regardless their prolificacy. Therefore, further attempts are necessary to detect other SNP for BMP-15 gene in Egyptian sheep breeds.

Impact of pregnancy and nutrition on oxidant/antioxidant balance in sheep and goats reared in South Sinai, Egypt.

AIM: To monitor the effect of nutrition and pregnancy on oxidative status of animals under the arid condition of South Sinai. MATERIALS AND METHODS: Blood samples were taken from two groups of animals: The first group retained in farm and fed on concentrate (high diet) and another group grazing natural forage (low diet). Each group was subdivided into pregnant and non-pregnant animals. Blood samples were assayed for their content of malondialdehyde (MDA), total antioxidant capacity (TAC), catalase (CAT), and superoxide dismutase (SOD) enzymes. RESULTS: MDA level significantly increased in pregnant animals fed either concentrate or grazing low-quality forage and accompanied by a low level of TAC in pregnant grazing animals fed low-quality forage. The activity of CAT decreased in pregnant fed either concentrate or grazing and SOD significant decrease in the pregnant grazing group. These data suggested that the animals might have experienced some degree of oxidative stress and lipid peroxidation and indicating that redox homeostasis was impaired in those pregnant and specially fed on forage rations. CONCLUSION: Pregnancy constituted the most oxidative stress facing the grazing and concentrated diet feed sheep and goats under arid and saline conditions of Southern Sinai, Egypt.