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.

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.

Effect of different combinations of cryoprotectants on in vitro maturation of immature buffalo (Bubalus bubalis) oocytes vitrified by straw and open-pulled straw methods.

This study was designed to evaluate effects of different combinations of cryoprotectants on the ability of vitrified immature buffalo oocytes to undergo in vitro maturation. Straw and open-pulled straw (OPS) methods for vitrification of oocytes at the germinal vesicle stage also were compared. The immature oocytes were harvested from ovaries of slaughtered animals and were divided into three groups: (i) untreated (control); (ii) exposed to cryoprotectant agents (CPAs); or (iii) cryopreserved by straw and OPS vitrification methods. The vitrification solution (VS) consisted of 6 m ethylene glycol (EG) as the standard, control vitrification treatment, and this was compared with 3 m EG + 3 m dimethyl sulfoxide (DMSO), 3 m EG + 3 m glycerol, and 3 m DMSO + 3 m glycerol. Cryoprotectants were added in two steps, with the first step concentration half that of the second (and final) step concentration. After warming, oocyte samples were matured by standard methods and then fixed and stained for nuclear evaluation. Rates of MII oocytes exposed to CPAs without vitrification were lower (54.3 +/- 1.9% in EG, 47.5 +/- 3.4% in EG + DMSO, 36.8 +/- 1.2% in EG + glycerol and 29.9 +/- 1.0% in DMSO + glycerol; p < 0.05) than for the control group (79.8 +/- 1.3%). For all treatments in each vitrification experiment, results were nearly identical for straws and OPS, so all results presented are the average of these two containers. The percentages of oocytes reaching telophase-I or metaphase-II stages were lower in oocytes cryopreserved using all treatments when compared with control. However, among the vitrified oocytes, the highest maturation rate was seen in oocytes vitrified in EG + DMSO (41.5 +/- 0.6%). Oocytes cryopreserved in all groups with glycerol had an overall low maturation rate 19.0 +/- 0.6% for EG + glycerol and 17.0 +/- 1.1% for DMSO + glycerol. We conclude that the function of oocytes was severely affected by both vitrification and exposure to cryoprotectants without vitrification; the best combination of cryoprotectants was EG + DMSO for vitrification of immature buffalo oocytes using either straw or OPS methods.

Influence of Phoxim on testicular and seminal vesicle organs, testosterone and cholinesterase level and its tissue residues in male rats.

The effect of Phoxim (Volaton) at two dosage levels (23 and 46 mg/kg b.wt.) on male reproduction tissues and their residues in rats were studied. The tested doses were given orally to male rats for 60 consecutive days. Sex organs weight analysis, semen picture, testosterone and cholinestrase enzyme (ChE) levels, histochemistry, histopathological changes and mating trials were the criteria used to evaluate the reproductive efficiency of the treated rats. There was a dose-related decrease in the weights of testicles and sperm motility associated with an increase in the percentages of dead and morphologically abnormal spermatozoa of treated rats. A decrease in plasma testosterone levels was observed in the treated groups. Histopathological examination revealed that phoxim caused testicular lesions characterized by moderate to severe degenerative changes of spermatogonial cells and by partial arrest of spermatogenesis. Plasma, brain and testicular ChE levels were reduced in treated rats. Phoxim and its oxygen analog concentrations were progressively increased by the time of exposure and represented double fold in liver as compared to that in skeletal muscles and testicles. The histochemical examination of testicles of treated rats showed a marked decreament in the ChE activity in tunica albuginea and sperms. A decrease in this enzyme was also noticed in liver hepatocytes, granular layer of the cerebral cortex and medulla of suprarenal gland.

Toxicological potential of malathion residues in stored soybean seeds.

Succinate-14C-malathion penetrates readily into soybean seeds. The total internal residues inside the seeds amounted to 58-65% of the applied dose after 30 weeks, of which 8-9% were in the form of bound residues. The major part of the internal methanol extractables are chloroform soluble metabolites which include malathion (about 60%), monocarboxylic acid (15%) and its decarboxylation product (8%). The water soluble metabolites contained only one radioactive substance, namely malathion dicarboxylic acid. The toxicological potential of the total internal residues was studied by feeding mice with the washed seeds for about 2.5 months. Treated mice suffered from deterioration of hepatic and renal function as indicated by the observed increased level of blood serum esterases and blood urea nitrogen. The results of blood biochemistry are supported by the histopathological changes observed in the liver, kidney, stomach and intestine. The organs showed degenerative changes including leucocytic aggregation, congestion and dilatation of blood vessels. Other adverse effects caused by malathion residues are indicated from cytogenetic studies on bone marrow of treated mice. Studies showed an initial bone marrow toxicity as indicated by increase in percentage of polychromatic erythrocytes over controls. This effect diminished upon prolongation of feeding period over one month. Feeding with malathion residues affected a gradual increase, with feeding period, in the percentage of polychromatic erythrocytes with micronuclei, a parameter recommended for detecting chemical mutagenes in animal test systems.