Journal of Advanced Veterinary Research

Cathepsin L is required for completion of oocyte meiotic maturation in mammals

Eman Awad

Infertility is a major concern affecting a huge population worldwide. Aneuploidy is one of the leading causes of the infertility, usually originated from errors that happens in meiosis I as chromosomal misalignment and abnormal chromosomal segregation. Recent research showed that cathepsins family play an important role in affecting the oocytes developmental competence in many species. Cathepsin B inhibition improved bovine developmental competence of the oocytes, especially in the low-quality oocytes. Here, we are the first to introduce the significance of another member of this family, cathepsin L (CTSL) in the process of oocyte maturation using mouse oocyte model. In this study we evaluated the expression of CTSL during oocyte meiotic maturation and the effect of CTSL inhibition using specific inhibitor “SCP110” on the subsequent maturation process. We found, CTSL is expressed in all stages of Meiosis I. In addition, inhibition of CTSL resulted in delaying the maturation time by elongation the time required for extrusion of the polar body (PBE). Moreover, CTSL inhibition reduced the maturation rate compared to the control group as we found a significant decrease in PBE percent. Trying to understand the reason of the lower maturation rates, we stained the chromosomes and spindles at metaphase I stage, we found a significant increase in chromosomal misalignment at metaphase plate. Finally, we evaluated matured oocytes quality after CTSL inhibition, importantly, aneuploidy incidence was increased significantly after CTSL inhibition. In summary, CTSL is required for normal meiosis completion in oocytes and production of healthy euploid egg.

Effects of kisspeptin-10 on the steroidogenic capacity and metabolic aspects of bovine granulosa cells in vitro

Nasser Sayed Abou khalil

Kisspeptin (Kp) potently stimulates the reproductive hormone secretion, modulates the cellular metabolic machinery, and induces the antioxidant defense mechanism in vivo. However, the data regarding steroidogenic, metabolic and antioxidant effects of Kp on granulosa cells on the level of in vitro studies are quiet rare leaving a wide gap in literature and giving a strong driving force for the present study. Thus, we aimed to clarify the effects of human kisspeptin-10 (Kp10) on these biological features in small and large bovine granulosa cells (BGCs). Upon preparation of the monolayer-BGCs, they were allocated to eight groups; two untreated-control groups, and six Kp10-treated groups according to the follicular size; three groups for each follicle size, supplemented with Kp10 at three different doses; 10^-8, 10^-7 and 10^-6M [KP(I-III)_S and KP(I-III)_L, respectively]. Spent media and BGCs were pooled after 24 hours from addition of Kp10. Kisspeptin stimulated the glucose consumption in media by BGCs obtained from the small sized follicles for production of estradiol 17-β and progesterone. In the small-sized follicles, the TC levels were significantly decreased in response to KP(I, II)_S, but not the KP(III)_S compared to their control (C_S). On the other hand, the KP(I-III)_L significantly increased the TC levels compared to their control (C_L). When Kp10 was used at the highest two doses, less glucose was consumed by BGCs collected from large sized follicles leading to low production of P₄ and preservation of cellular TC content. Improvement in the metabolic efficacy of BGCs in response to Kp10-treatment was evidenced by increased glucose utilization and decreased lactate production. Increased total antioxidant capacity versus decreased lipid peroxides in Kp10-treated groups could indicate that Kp10 induces cryoprotection by restoring the favorable redox status in BGCs. Those findings suggest that Kp10 causes a size- and dose- dependent physiological changes in the BGCs.

Genetic assessment of Shiga toxin and antibiotic resistance of E. coli isolated from milk of cows infected with sub-clinical mastitis

Khaled A.S. El-Khabaz

Bovine subclinical mastitis was one of the most important health problems facing dairy industry, its impact exceeded the economic aspects and extended to potential negative effects on human health. The current study aimed to determine the prevalence of E. coli as an important mastitic pathogen and identify some of its most important virulence gene as well as their antimicrobial resistance profile. In the present study E. coli was isolated and biochemically identified whereas out of 100 subclinically mastitic milk samples was nine samples were positive for E. coli with 9% prevalence rate. Serotyping of these isolates declared that 3 isolates were serotype O26:H11, 2 isolates in serotype O91:H21 and 1 isolate in each of serotypes O55:H7, O128:H2, O146:H21 and O124. Antimicrobial resistance profile of the obtained isolates showing that all the isolates were 100% resistant to both erythromycin and streptomycin, while 88.9% (8/9) were sensitive to gentamicin. The presence of 3 important virulence factors including shiga toxin1(stx₁), shiga toxin 2 (stx₂) and intimin (eae) genes, among the obtained isolates was reported using PCR. Molecular investigation revealing that 2 isolates contain the all studied virulence genes (stx₁, stx₂ and eae), 3 isolates contain (stx₁ and stx₂), while stx₁ was detected solely in 2 isolates, also 1 isolate contain only stx₂ and lastly 1 isolate was negative for any of the studied virulence factors. In a conclusion, there was a 9% prevalence rate of E. coli in subclinically mastitic milk samples in the current study, indicating its importance as a mastitic pathogen. The shiga toxin genes (stx₁ & stx₂) are widely distributed among E. coli isolates, while the intimin (eae) gene is less prevalent in comparison to shiga toxin genes. Also the recorded high multidrug resistance rate among the isolates posing threat to human health though entrance of these strains into the human being food chain whereas the isolated E. coli strains had the highest resistance to erythromycin and Streptomycin (100%), followed by Clindamycin (77.8%), Nalidixic acid (66.7%), and Gentamicin (11.1%) was the lowest.

Investigation of antibacterial efficiency of various lytic bacteriophages isolated from chickens against characterized multidrug-resistant pathogenic bacterial strains

Marwa Fathy

This study targeted isolation and characterization of potential bacteriophages (phages) infecting MDR pathogenic bacteria recovered from chickens and analyzed their efficacy as bio-control agents. A total of 45 different bacterial isolates (18 E. coli, 16 Salmonellae spp., 5 Staphylococcus spp., 2 Pseudomonas spp., 1 Proteus mirbalis, 1 Citrobacter spp., 1 Enterobacter aerogenes and 1 Klebsiella pneumonia) were obtained from chickens in the current study and previous studies. The identified isolates were investigated for the presence of virulence genes and MDR using PCR and disc diffusion method, respectively. Nine purified phages classified morphologically into 3 families (Myoviridae, Siphoviridae and Podoviridae) using Transmission Electron Microscope were recovered from chicken intestinal contents and showed viability at wide pH range, resistance to organic solvents and thermostability at high temperatures (up to 80ºC). The potential phages exhibited various bacterial host ranges using the spot test and the efficiency of plating (EOP) assay. The results revealed the prevalent of pathogenic E. coli and salmonella serovars among the recovered isolates with different virulence and genotypic patterns. The lytic phages were highly stable and have the capacity to infect different pathogenic MDR bacterial strains. This study demonstrated that these promising phages of avian origin could be used to control the pathogenic MDR E. coli and Salmonella serovars which possess public concerns on human health and poultry industry.

The effect of hot dry environment in the western Egyptian desert on the corpuscular oxidation in sheep

Mostafa A. Saleh

Nowadays, climate changes and heat stress (HS) are the main challenges facing livestock health. The present work aimed to study the response of some physiological, hematological and erythrocytic redox status in desert Barki sheep during the hot dry period in the western Egyptian desert (El-Kharga oasis). Thirty healthy male Barki sheep (4-5 months) were selected in this study. Fifteen of them were selected during July (hot dry period, HS-group) and the remaining 15 animals were selected during December (thermoneutral period, TN-group) as a control group. The calculated temperature humidity index (THI) regestered 65.4 (satisfactory) in December and 89.6 (risky HS) in July. Increased rectal temperature by 1.2 ˚C (3.15%, P < 0.001), doubled respiration rate (100%, P < 0.001) accompanied by reduction in red blood cells count (-10.83%, P = 0.0002), packed cell volume (-10.69%, P = 0.001) and hemoglobin concentration (-11.54, P = 0.0005) were noticed in HS group compared with NS group. Erythrocytes showed increased total oxidant status (60.6%, P=0.004), malondialdehyde as an indicator of lipid peroxidation (41.1 %, P=0.028) and decreased total antioxidant capacity (-21.3%, P=0.012) and superoxide dismutase (-33.9%, P=0.015) accompanied by increased erythrocytic oxidative stress index (OSI), which was doubled (101.4 %, P < 0.001) in HS group compared with NS group. Linear regression analysis (r²) and Pearson’s correlation (r) revealed a negative correlation of OSI with hemoglobin concentration (r² = 0.34, r = - 0.58, P < 0.001). In conclusion, HS is associated with corpuscular redox imbalance and oxidative stress, which may be linked to the pathogenesis of anemia in heat stressed sheep under the tropical conditions.