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.

Expression profile of genes related to pregnancy maintenance in Dromedary Camel during the first trimester.

So far, few signals involved in embryo-maternal dialogue have been identified in pregnant she-camel. Our objective was to investigate expression profiles of genes relevant to uterine extracellular matrix remodeling (ITGB4, SLCO2A1, FOS, and JUN), uterine tissue vascularization, and placental formation (VEGFA, PGF, and PDGFA), embryonic growth and development (IGF1 and PTEN), plus cell death of uterine tissue (BCL2) in early pregnant versus non-pregnant she-camels. Forty genital tracts (20 pregnant and 20 non-pregnant) and blood samples were collected from abattoirs. Total RNA was extracted from uterine tissues and qRT-PCR was conducted for candidate genes. Serum concentrations of progesterone (P4) and estradiol17-ß (E2) were measured. Expression of ITGB4, FOS, and PGF genes increased (P < 0.001) in the right uterine horn of pregnant versus non-pregnant she-camels. Moreover, JUN, SLCO2A1, VEGFA, and PTEN mRNAs were up-regulated (P < 0.001) in various segments of uterine tissues in pregnant groups. The PDGFA transcript was over-expressed (P < 0.001) in both uterine horns of pregnant groups. Additionally, IGF1 was higher (P < 0.001) in the right horn and the uterine body of pregnant groups, and expression of BCL2 was increased (P < 0.001) in the pregnant uterine body. Moreover, serum concentrations of P4 were higher (P < 0.001) and E2 lower (P < 0.05) in pregnant she-camels. Taken together, the fine-tuning of genes related to implantation, matrix formation, vascularization, and placental formation is highly required for successful pregnancy in she-camels.

Effects of cysteamine supplementation on cryopreserved buffalo bull semen quality parameters.

This work aimed to determine the effect of cysteamine (25, 50, 100 and 200 µM) incorporated during dilution on frozen thawed buffalo semen quality. Semen was collected twice weekly for 7 consecutive weeks from three Egyptian buffalo bulls using an artificial vagina. Semen samples were pooled and extended with a Tris-based extender, cooled, equilibrated and finally frozen in liquid nitrogen. The diluted semen was evaluated for motility, viability, morphology, plasma membrane and DNA integrity, in addition to oxidative stress and in vitro fertilizing capability. The post thaw motility and velocity parameters noticeably increased with different concentrations of cysteamine (mainly 100 µM) during different incubation periods. The post thaw sperm viability and normality significantly (p < 0.05) improved with concentrations of 50 and 100 µM. Plasma membrane integrity substantially increased at all concentrations of cysteamine. Cysteamine reduced alanine aminotransferase (at all concentrations), aspartate aminotransferase (at 25-100 µM), and creatine kinase (at 100 and 200 µM). Cysteamine at a concentration of 100 µM noticeably enhanced the total antioxidant capacity and glutathione peroxidase and decreased nitric oxide production. Cysteamine, at concentrations of 100 and 200 µM, increased the DNA intensity in the comet head (%) and decreased the DNA % in the comet tail. The comet tail length and moment substantially decreased at concentrations of 50-200 µM. Cysteamine did not affect the in vitro fertilizing capability of sperm. In conclusion, cysteamine incorporation (mainly at a concentration of 100 µM) in buffalo semen extender showed varying protective effects on different sperm parameters against cryo-damage; however, it did not affect the in vitro fertilizing capacity of sperm.

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.

Differential molecular and hormonal changes in oocytes, granulosa cells and follicular fluid of pregnant and non-pregnant camels.

This study aimed to compare the expression of genes regulating follicles development, survival and steroid hormones secretion in oocytes and granulosa cells (GCs) and study the correlation between their expression and follicular fluid (FF) levels of progesterone (P4) in pregnant and non-pregnant camels. In total, 138 ovarian pairs from slaughtered camels were used. Gene expression and hormonal assay were determined using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. The obtained results revealed that the number of follicles (3-8 mm) was significantly (P < 0.05) lower in pregnant, compared with non-pregnant, camels. P4 level in the FF was significantly (P < 0.05) higher in pregnant, compared with non-pregnant, camels. However, no significant (P > 0.05) difference was noticed in the oestradiol (E2) level. STAR, PTEN, IGF1 and BCL2 mRNA levels were significantly higher in GCs and significantly lower in oocytes of pregnant, compared with non-pregnant, camels. However, follicle-stimulating hormone receptor (FSHR) mRNA level was significantly lower in GCs and oocytes, and the BMP15 mRNA level was significantly lower in oocytes of pregnant, compared with non-pregnant, camels. P4 level in FF was positively correlated with STAR, PTEN, IGF1 and BCL2 mRNA levels in GCs and negatively correlated with BMP15 mRNA levels in oocytes and FSHR mRNA levels in GCs and oocytes of pregnant camels. It could be concluded that pregnancy-induced variations in oocytes and GC expression of BMP15, IGF1, FSHR, STAR, BCL2, and PTEN genes might be associated with a decrease in the number of follicles and an increase in the FF level of P4.

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.

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.