An abattoir study of the prevalence of foot lesions and claw measurements in water buffalo in Egypt.

BACKGROUND: Lameness has been associated with compromised animal welfare and reduced productivity in dairy cattle herds worldwide. However, little is known about the prevalence of claw lesions in the dairy buffalo population in Egypt. Furthermore, the optimum measurements for claw trimming in buffalo are unknown. A cross-sectional cadaver study was conducted where 135 pair buffalo hind feet were collected from 4 slaughterhouses and examined for the presence of claw lesions. The proportion and associated 95% confidence interval (CI) of each type of lesion were calculated. A separate set of healthy claws (n = 26) underwent ultrasonography (US) and computed tomography (CT). The agreement between US and CT measurements was assessed using Passing-Bablok regression and intraclass correlation coefficient. The CT measurements were used to calculate trimming recommendations. RESULTS: At least one lesion was identified in 242 claws (89.6%, 95% CI = 85.4-93.0). In healthy claws, poor to moderate agreement was identified between US and CT measurements which could be due a sample size of the study. The average ± standard deviation (SD) minimum recommended external wall length of the lateral and medial claws in heifers was 7.1 ± 0.36 cm and 7.5 ± 0.35 cm, respectively. The average ± SD minimum recommended external wall length in buffaloes over five years of age was 8.2 ± 0.27 cm and 8.4 ± 0.39 cm for the lateral and medial claws, respectively. CONCLUSIONS: The study found a high prevalence of claw lesions in buffalo in Egypt, the clinical significance of which requires further elucidation. Recommended measurements will help guide claw trimming in buffalo to minimise lesions.

A cross-sectional study of the prevalence of lameness and digital dermatitis in dairy cattle herds in Egypt.

BACKGROUND: Lameness is a significant problem for the dairy industry worldwide. No previous studies have evaluated the prevalence of lameness or digital dermatitis (DD) in dairy cattle herds in Egypt. A total of 16,098 dairy cows from 55 dairy herds in 11 Egyptian governorates underwent visual locomotion scoring using a 4-point scoring system. Cows that had a lameness score ≥ 2 were considered clinically lame. Following manure removal with water and using a flashlight, the cows' hind feet were examined in the milking parlour to identify DD lesions and classify with M-score. Furthermore, each cow was assigned a hock score (a 3-point scale) and a hygiene score (a 4-point scale). The cow-, within-and between-herd prevalence of lameness and DD and associated 95% confidence intervals (CI) were calculated. The prevalence of hock lesions and poor cow hygiene was also calculated. RESULTS: Of the examined cows, 6,883 were found to be clinically lame (42.8%, 95% CI = 42.0-43.5%). The average within-herd prevalence of lameness was 43.1% (95% CI = 35.9-50.3%). None of the dairy herds recruited into the study were found to be free from clinical lameness. The average within-herd prevalence of DD was 6.4% (95% CI = 4.9-8.0%). The herd-level prevalence of DD was 92.7% (95% CI = 85.9-99.6%). Active DD lesions (M1, M2, M4.1) were identified in 464 cows (2.9%) while inactive lesions (M3, M4) were identified in 559 cows (3.5%). The within-herd prevalence of hock lesions (score 2 or 3) was 12.6% (95% CI = 4.03-21.1%) while a severe hock lesion had within-herd prevalence of 0.31% (95% CI = 0.12-0.51%). Cow-level prevalence of hock lesions was 6.2% (n = 847, 95% CI = 5.8-6.2%). The majority of examined cows had a hygiene score of 4 (n = 10,814, prevalence = 70.3%, 95% CI = 69.5-71%). CONCLUSIONS: The prevalence of lameness was higher than prevalence estimates reported for other countries which could be due to differing management and/or environmental factors. DD was identified at lower prevalence in most herds but with high herd-level prevalence. Poor cow hygiene was notable in most herds. Measures to reduce the prevalence of lameness and to improve cow hygiene in dairy cattle herds in Egypt are therefore needed.

L-Cysteine improves bovine oocyte developmental competence in vitro via activation of oocyte-derived growth factors BMP-15 and GDF-9.

This study was designed to investigate the effect of different concentrations of L-cysteine supplementation into the maturation medium on the oocyte nuclear maturation, cumulus cell expansion, ultrastructure of the oocytes and the expression of oocyte-derived growth factors BMP-15, GDF-9 and CB-1 genes. Cumulus oocyte complexes (COCs) were collected from cow's ovaries obtained from abattoir and incubated at 38.5°C in maturation media supplemented with 0, 0.6, 0.8 or 1 mM L-cysteine in 5% CO2 under humidified air for 24 hr. We found that a significantly higher percentage of oocytes progressed to metaphase II stage in the in vitro maturation (IVM) medium supplemented with L-cysteine, particularly 0.8 mM group, compared with untreated control oocytes. Additionally, L-cysteine treatment significantly increased the number of expanded COCs and the degree of expansion of individual COCs. Results of RT-qPCR showed significant increase in expression levels of BMP-15 and GDF-9 in L-cysteine-treated groups compared with control one. Electron microgram showed improvement of cytoplasmic maturation regarding ultrastructure of the oocytes and oocyte-cumulus cell gap junction communication in all L-cysteine-treated groups especially 0.8 mM L-cysteine-treated one. In conclusion, supplementation of IVM medium with a potential anti-oxidant, L-cysteine can effectively improve in vitro oocytes cytoplasmic and nuclear maturation via activation of oocyte maturation related BMP-15 and GDF-9 genes in bovine oocytes, benefiting the extended researches about the potential applications of L-cysteine in mammalian breeding technologies.

Effects of Donor Cell Types on the Development of Bovine Embryos Using Cytoplasm Injection Cloning Technology.

Cytoplasm injection cloning technology (CICT) is an efficient technique for evaluating the developmental potential of cloned embryos. In this study, we investigated the effects of donor cell type on the developmental potential and quality of cloned bovine embryos. Adult fibroblasts (AFs) and embryonic cells (ECs) were used as donor cells to clone bovine embryos using CICT. We initially used AF cells to develop cloned embryos and then cultured the cloned day-8 blastocysts for 10 days to obtain ECs as donor cells for second embryo cloning. We found that the bovine blastocysts cloned using AF cells had significantly reduced developmental rates, embryo quality, and ratios of inner cell mass (ICM) to the total number of cells compared to those using ECs as donor cells. Furthermore, there were significant differences in the DNA methyltransferase-, histone deacetylation-, apoptosis-, and development-related genes at the blastocyst stage in embryos cloned from AFs compared to those in embryos cloned from ECs. Our results suggest that using ECs as donor cells for nuclear transfer enhances the quantity and quality of cloned embryos. However, further investigation is required in terms of determining pregnancy rates and developing cloned embryos from different donor cell types.

Effect of nicotinamide supplementation in in vitro fertilization medium on bovine embryo development.

Increased oxidative stress is one of the main causes of poorly developed embryos in assisted reproductive technologies. Nicotinamide (NAM) has been shown to suppress reactive oxygen species (ROS) production through its potent antioxidative and anti-senescent effects. In the present study, we explored the effects of short-term NAM-treatment (3 and 5 h) during in vitro fertilization (IVF) on the development of bovine embryos. Treatment with 10 mM NAM for 3 h significantly increased the blastocyst formation but extending the treatment to 5 h did not enhance the benefits any further. Immunofluorescence analysis demonstrated that treatment with 10 mM NAM for 3 h decreased the expression of intracellular ROS, 8-oxo-7,8-dihydroguanine, caspase-3, and increased the expression of Sirt1, and incorporation of bromodeoxyuridine in one-cell stage embryos. Similarly, the level of H3K56ac significantly increased in the NAM-treated (3 and 5 h) one-cell stage embryos. Contrastingly, the treatment with 10 mM NAM for 5 h increased the caspase-9 level in blastocysts. Collectively, these findings suggest that NAM possesses antioxidant activity and supplementation of IVF medium with 10 mM NAM for 3 h improves the in vitro developmental competence of bovine embryos.

Nicotinamide-induced mouse embryo developmental defect rescued by resveratrol and I-CBP112.

Cell cycle of mouse embryo could be delayed by nicotinamide (NAM). Histone H3 lysine 56 (H3K56ac) acetylation plays an important role in mammalian genomic stability and the function of this modification in mouse embryos is not known. Hence, we designed to study the effects of NAM-induced oxidative stress on the developmental ability of mouse embryos, on the acetylation of H3K56ac and the possible functions of this modification related to mouse embryo development. Treatment with NAM (10, 20, or 40 mmol/L for 24 or 48 hr) during in vitro culture significantly decreased developmental rate of blastocyst (24 hr: 90.2 vs. 81.2, 43.2, and 18.2, with p > .05, p < .01, respectively; 48 hr: 89.3 vs. 53.2%, 12.1%, and 0% with p < .05, respectively). NAM treatment (20 mmol/L) for 6 and 31 hr resulted in increased intracellular reactive oxygen species levels in two-cell embryos, and apoptotic cell numbers in blastocysts. Resveratrol (RSV) and I-CBP112 rescued the 20 mmol/L NAM-induced embryo developmental defects. RSV and I-CBP112 increased the level of Sirt1 and decreased the level of H3K56ac induced by NAM in two-cell embryos (p < .05). These data suggest that NAM treatment decreases the expression of Sirt1, which induces high levels of H3K56 acetylation that may be involved in oxidative stress-induced mouse embryo defects, which can be rescued by RSV and I-CBP112.

Induction of Oxidative Stress and Mitochondrial Dysfunction by Juglone Affects the Development of Bovine Oocytes.

Juglone, a major naphthalenedione component of walnut trees, has long been used in traditional medicine as an antimicrobial and antitumor agent. Nonetheless, its impact on oocyte and preimplantation embryo development has not been entirely clarified. Using the bovine model, we sought to elucidate the impact of juglone treatment during the in vitro maturation (IVM) of oocytes on their maturation and development of embryos. Results showed a severe reduction in oocyte nuclear maturation and cumulus expansion and a significant increase in mitochondrial dysfunction and reactive oxygen species (ROS) levels in cumulus-oocyte complexes (COCs) treated with juglone (12.5, 25.0, and 50.0 µM). In addition, RT-qPCR showed downregulation of the expansion-related (HAS2, TNFAIP6, PTX3, and PTGS2) and mitochondrial (ATPase6 and ATP5F1E) genes in juglone-treated COCs. Moreover, the development rates of day 4 total cleavage and 8-16 cell stage embryos, as well as day 8 blastocysts, were significantly reduced following exposure to juglone. Using immunofluorescence, the apoptotic marker caspase-9 was overexpressed in oocytes exposed to juglone (25.0 µM) compared to the untreated control. In conclusion, our study reports that exposing bovine oocytes to 12.5-50.0 µM of juglone can reduce their development through the direct induction of ROS accumulation, apoptosis, and mitochondrial dysfunction.

A combination of bovine serum albumin with insulin-transferrin-sodium selenite and/or epidermal growth factor as alternatives to fetal bovine serum in culture medium improves bovine embryo quality and trophoblast invasion by induction of matrix metalloproteinases.

This study investigated the use of bovine serum albumin (BSA) plus insulin-transferrin-sodium selenite (ITS) and/or epidermal growth factor (EGF) as alternatives to fetal bovine serum (FBS) in embryo culture medium. The developmental ability and quality of bovine embryos were determined by assessing their cell number, lipid content, gene expression and cryotolerance, as well as the invasion ability of trophoblasts. The percentage of embryos that underwent cleavage and formed a blastocyst was higher (P<0.01) in medium containing ITS plus EGF and BSA than in medium containing FBS. Culture with ITS plus EGF and BSA also increased the hatching ability of blastocysts and the total cell number per blastocyst. Furthermore, the beneficial effects of BAS plus ITS and EGF on embryos were associated with a significantly reduced intracellular lipid content, which increased their cryotolerance. An invasion assay confirmed that culture with ITS plus EGF and BSA significantly improved the invasion ability of trophoblasts. Real-time quantitative polymerase chain reaction analysis showed that the mRNA levels of matrix metalloproteinase-2 (MMP2) and MMP9, acyl-CoA synthetase long-chain family member 3, acyl-coenzyme A dehydrogenase long-chain and hydroxymethylglutaryl-CoA reductase significantly increased upon culture with ITS plus EGF and BSA. Moreover, protein expression levels of matrix metalloproteinase-2 and -9 increased (P<0.01) in medium supplemented with ITS plus EGF and BSA compared with medium supplemented with FBS. Taken together, these data suggest that supplementation of medium with ITS plus EGF and BSA improves invitro bovine embryo production, cryotolerance and invasion ability of trophoblasts.

Melatonin Abrogates the Anti-Developmental Effect of the AKT Inhibitor SH6 in Bovine Oocytes and Embryos.

Melatonin, a nighttime-secreted antioxidant hormone produced by the pineal gland, and AKT, a serine/threonine-specific protein kinase, have been identified as regulators for several cellular processes essential for reproduction. The current study aimed to investigate the potential interplay between melatonin and AKT in bovine oocytes in the context of embryo development. Results showed that the inclusion of SH6, a specific AKT inhibitor, during in vitro maturation (IVM) significantly reduced oocyte maturation, cumulus cell expansion, cleavage, and blastocyst development that were rescued upon addition of melatonin. Oocytes treated with SH6 in the presence of melatonin showed lower levels of reactive oxygen species (ROS) and blastocysts developed exhibited low apoptosis while the mitochondrial profile was significantly improved compared to the SH6-treated group. The RT-qPCR results showed up-regulation of the mRNA of maturation-, mitochondrial-, and cumulus expansion-related genes including GDF-9, BMP-15, MARF1, ATPase, ATP5F1E, POLG2, HAS2, TNFAIP6, and PTGS2 and down-regulation of Bcl-2 associated X apoptosis regulator (BAX), caspase 3, and p21 involved in apoptosis and cell cycle arrest in melatonin-SH6 co-treated group compared to SH6 sole treatment. The immunofluorescence showed high levels of caspase 3 and caspase 9, and low AKT phosphorylation in the SH6-treated group compared to the control and melatonin-SH6 co-treatment. Taken together, our results showed the importance of both melatonin and AKT for overall embryonic developmental processes and, for the first time, we report that melatonin could neutralize the deleterious consequences of AKT inhibition, suggesting a potential role in regulation of AKT signaling in bovine oocytes.

Improved developmental competence in embryos treated with lycopene during in vitro culture system.

In vitro embryo development remains suboptimal compared to in vivo development due to the challenge from various stressors associated with in vitro culturing of oocytes. When 0.2 µM lycopene was added to oocyte in vitro maturation and embryo culture media, to assess its antioxidant effects on embryo development, we observed a significant (p < 0.05) increase in cleavage and blastocyst development rates compared to the corresponding controls (84.3 ± 0.6% vs. 73.1 ± 1.9% and 41.0 ± 1.4% vs. 33.4 ± 0.7%, respectively). Lycopene also significantly reduced (p < 0.05) intracellular reactive oxygen species concentrations in oocytes and blastocysts, whereas lipid peroxidation and mitochondrial activity increased compared to control conditions. The number of apoptotic nuclei was significantly reduced in the lycopene-treated compared to the control group (1.7 ± 0.1 vs. 4.7 ± 0.3), and the quantity of cells in the trophectoderm (207.1 ± 1.6 vs. 171.3 ± 1.0, respectively) and inner cell mass (41.9 ± 0.4 vs. 36.7 ± 0.4, respectively) was higher following treatment-although the inner cell mass-to-trophectoderm ratio was unchanged (1:3.3 vs. 1:3.4 for lycopene vs. control, respectively). Lycopene supplementation also significantly (p < 0.05) attenuated expression of IKBKB (Inhibitor of nuclear factor kappa B kinase, subunit beta) and reduced Caspase 9 and Caspase 3 protein abundance, while up-regulating GDF9 (Growth and differentiation factor 9), BMP15 (Bone morphogenetic protein 15), SOD2 (Superoxide dismutase 2), NDUFA2 (NADH dehydrogenase), ACADL (Acyl-CoA dehydrogenase, long chain), and ACSL3 (Acyl-CoA synthetase 3, long-chain membrane 3) transcription compared to control. Therefore, co-culturing with lycopene during oocyte maturation improved bovine embryo developmental potential during in vitro culture by improving embryonic resilience to stress.

Improvement of in vitro-produced bovine embryo treated with coagulansin-A under heat-stressed condition.

Heat stress has large effects on reproduction including conception rate in cattle. In this study, we examined the effects of coagulansin-A (coa-A), a steroidal lactone, on acquired thermo tolerance during in vitro production of bovine embryos. Oocytes were incubated in in vitro maturation (IVM) media with or without coa-A at two different temperatures, 40.5˚C and 42˚C, for 20 h. The treatment of coa-A significantly improved blastocyst development only at 40.5˚C (P < 0.05). Interestingly, immunofluorescence analysis demonstrated that coa-A induced heat shock protein 70 (HSP70) and phosphatidylinositol-3-kinase (PI3K), but significantly attenuated nuclear factor kappa B (NF-κB) and cyclooxygenase-2 (COX2). To determine the expression patterns of related genes at the transcription level, qRT-PCR was performed. Expression of HSP70 and PI3K was elevated, whereas expression of NF-κB, COX2 and inducible nitric oxide synthase (iNOS) was significantly (P < 0.05) downregulated in the coa-A-treated group compared with the control group. Moreover, pro-apoptotic genes were downregulated, and antiapoptic genes were upregulated in the coa-A group. We also counted the total cell number and apoptotic nuclei at the blastocyst and found that more cell numbers (143.1 ± 1.5) and less apoptotic damages (6.4 ± 0.5) in the coa-A treatment group comparing to control group (131.4 ± 2.0 and 10.8 ± 0.5), indicating the enhanced embryo quality. In conclusion, our results demonstrate that the coa-A not only improved the blastocyst development in vitro but also increased their resistance to heat stress condition through induction of HSP70/PI3K.

Polydatin improves the developmental competence of bovine embryos in vitro via induction of sirtuin 1 (Sirt1).

The aim of the present study was to investigate the beneficial effect of polydatin (PD), the glycoside form of resveratrol, on embryo development in vitro. Oocytes were aspirated from ovaries of Korean Hanwoo cows and cultured until Day 8 in a humidified atmosphere of 5% CO2 in air at 38.5°C. Protein and gene expression levels were determined through confocal microscopy and reverse transcription-polymerase chain reaction respectively, whereas the number of total and apoptotic cells in Day 8 blastocysts was determined using Hoechst 33342 staining and terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling. Of the different concentrations of PD (0.5, 1.0 and 2.0µM) added to the IVM medium, only 1.0µM PD significantly improved blastocyst development. Immunofluorescence analysis confirmed that protein levels of sirtuin 1 (Sirt1) increased significantly (P<0.05) after PD treatment, whereas levels of reactive oxygen species (ROS) were significantly (P<0.05) decreased, as evidenced by reductions in 8-oxoguanine immunoreactivity. Similarly, protein levels of nuclear factor (NF)-κB and cyclo-oxygenase (COX)-2 were significantly (P<0.05) lower in the PD-treated group than in the control group. Treatment with 1.0µM PD reduced gene expression of BCL2-associated X protein, inducible nitric oxide synthase, COX2 and Nfkb, but increased the expression of Sirt1, supporting the immunofluorescence data. PD possesses antioxidant activity and is useful for embryo development in vitro. We conclude that supplementation of IVM medium with PD improves embryo developmental competence via Sirt1.

Efficacy of different treatment protocols for endometritis in Camelus dromedarius.

Endometritis is considered a significant cause of infertility problems in dromedary camels. This study aimed to compare the efficacy of different treatment protocols for endometritis in dromedary camels under Abu Dhabi Emirates' conditions. A total of 112 dromedary she-camels with uterine infection were subjected to uterine swabbing for bacterial culture and received one of the following treatments: (i) uterine douching with lotagen every other day for three doses, (ii) single parenteral oxytetracycline injection, (iii) subcutaneous injection with ceftiofur for 5 days, or (vi) combined oxytetracycline-ceftiofur injection. The results showed that Escherichia coli was the most isolated bacteria, followed by Streptococcus species. Treatment efficacy was (P < 0.05) higher in ceftiofur and oxytetracycline-ceftiofur protocols compared with lotagen and oxytetracycline protocols. The fertility indexes, services per conception and pregnancy rate, were improved in ceftiofur and mixed oxytetracycline plus ceftiofur protocols as the pregnancy rate was (P < 0.05) higher in those protocols compared with lotagen and oxytetracycline protocols (71.4 and 67.9% vs. 39.3 and 42.9%, respectively). On the other hand, the number of services per conception was significantly lower in ceftiofur and oxytetracycline-ceftiofur protocols (1.2 for each protocol) than in lotagen and oxytetracycline protocols (1.8 and 1.7, respectively). In conclusion, subcutaneous injection of 1 ml ceftiofur per 50 kg body mass for 5 days can be used as an efficient treatment for uterine infection in female dromedary camels caused by E. coli and Streptococcus species for improving their fertility indexes.

Heat Stress as a Barrier to Successful Reproduction and Potential Alleviation Strategies in Cattle.

In recent decades, the adverse effects of global warming on all living beings have been unanimously recognized across the world. A high environmental temperature that increases the respiration and rectal temperature of cattle is called heat stress (HS), and it can affect both male and female reproductive functions. For successful reproduction and fertilization, mature and healthy oocytes are crucial; however, HS reduces the developmental competence of oocytes, which compromises reproduction. HS disturbs the hormonal balance that plays a crucial role in successful reproduction, particularly in reducing the luteinizing hormone and progesterone levels, which leads to severe problems such as poor follicle development with a poor-quality oocyte and problems related to maturity, silent estrus, abnormal or weak embryo development, and pregnancy loss, resulting in a declining reproduction rate and losses for the cattle industry. Lactating cattle are particularly susceptible to HS and, hence, their reproduction rate is substantially reduced. Additionally, bulls are also affected by HS; during summer, semen quality and sperm motility decline, leading to compromised reproduction. In summer, the conception rate is reduced by 20-30% worldwide. Although various techniques, such as the provision of water sprinklers, shade, and air conditioning, are used during summer, these methods are insufficient to recover the normal reproduction rate and, therefore, special attention is needed to improve reproductive efficiency and minimize the detrimental effect of HS on cattle during summer. The application of advanced reproductive technologies such as the production of embryos in vitro, cryopreservation during the hot season, embryo transfer, and timed artificial insemination may minimize the detrimental effects of HS on livestock reproduction and recover the losses in the cattle industry.

Attenuation of Oxidative Stress and Regulation of AKT Signaling by Vanillic Acid during Bovine Pre-Implantation Embryo Development.

Vanillic acid (VA) has shown antioxidant and anti-inflammatory activities in different cell types, but its biological effects in the context of early embryo development have not yet been clarified. In the current study, the impact of VA supplementation during in vitro maturation (IVM) and/or post-fertilization (in vitro culture; IVC) on redox homeostasis, mitochondrial function, AKT signaling, developmental competence, and the quality of bovine pre-implantation embryos was investigated. The results showed that dual exposure to VA during IVM and late embryo culture (IVC3) significantly improved the blastocyst development rate, reduced oxidative stress, and promoted fatty acid oxidation as well as mitochondrial activity. Additionally, the total numbers of cells and trophectoderm cells per blastocyst were higher in the VA-treated group compared to control (p < 0.05). The RT-qPCR results showed down-regulation of the mRNA of the apoptosis-specific markers and up-regulation of AKT2 and the redox homeostasis-related gene TXN in the treated group. Additionally, the immunofluorescence analysis showed high levels of pAKT-Ser473 and the fatty acid metabolism marker CPT1A in embryos developed following VA treatment. In conclusion, the study reports, for the first time, the embryotrophic effects of VA, and the potential linkage to AKT signaling pathway that could be used as an efficacious protocol in assisted reproductive technologies (ART) to improve human fertility.

Downregulation of PI3K/AKT/mTOR Pathway in Juglone-Treated Bovine Oocytes.

We have previously reported that juglone, a natural compound found in Juglandaceae with a wide range of biological activities, can reduces the developmental competence of bovine oocytes. In the current study, we investigated the possible mechanisms behind the toxicity of juglone and the relationship with PI3K/AKT/mTOR signaling during the in vitro maturation (IVM) of oocytes. Results show that oocyte exposure to juglone was associated with a significant decrease in filamentous actin (F-actin) accumulation. The RT-qPCR showed downregulation of the meiosis progression indicator GSK-3A, oocyte development marker BMP15, mitochondria fusion controlling MFN1, oxidative stress-related OGG1, and histone methylation-related EZH1, EZH2, SUZ12, G9a, and SUV39H2 genes in juglone-treated oocytes. In addition, glycolysis- (PFK1 and GLUT1), ATP synthesis- (ATPase8 and ATP5F1B), and OXPHOS-specific markers (SDHA and SDHD), as well as the oocyte survival regulators (SOD2, VEGF, and MAPK1) significantly decreased upon juglone treatment. Moreover, lower expression of PI3K, AKT, and mTOR was observed at the transcriptional and/or translational level(s). The autophagy markers LC3B and beclin-1 as well as the DNA damage-specific marker 8-OxoG displayed overexpression in juglone-exposed oocytes. Taken together, our results show that administration of juglone during the IVM can reduce the quality and developmental health of bovine oocytes through downregulation of the PI3K/AKT/mTOR pathway and its downstream signaling cascades.

Vascular endothelial growth factor: A novel marker for torsion-induced incomplete cervical dilatation in Egyptian buffaloes.

Background: Uterine torsion, the most complicated cause of maternal dystocia recorded in bovine, usually followed by incomplete cervical dilatation after successful detorsion, which interfere with vaginal fetal delivery. Aim: This study aimed to evaluate the histopathological changes, variable collagen concentrations, and vascular endothelial growth factor expressions in uterine and cervical tissues following successful detorsion. Methods: Animals were classified into four groups; normally calved cases (group A), cases that respond successfully to detorsion and followed by vaginal fetal delivery without (group B) or with (group C) cervical laceration, and cases that suffered from failure of complete cervical dilatation after successful detorsion (group D). Results: Histopathological findings revealed variable changes in all uterine torsion affected groups, which were characteristic and marked in animals that suffered from failure of complete cervical dilatation following successful detorsion. Moreover, failure of cervical dilatation was associated with the highest collagen concentrations as shown by Masson trichrome stain. On the other hand, immunohistochemical findings showed that the normally calved cases have the highest vascular endothelial growth factor expression compared with animals that suffered from failure of complete cervical dilatation. Conclusion: Our results showed that the vascular endothelial growth factor is essential for cervical dilatation and its lower expression is accompanied by incomplete cervical dilatation following successful detorsion.

Hematological and biochemical indices, growth performance, and puberty of goats fed with Mombasa and blue panic as salt-tolerant alternatives to alfalfa under arid conditions.

The objective of this study was to evaluate the impact of Mombasa or blue panic as a salt-tolerant alternative to alfalfa on growth performance, puberty, blood hematology, serum metabolites, and serum mineral profile in growing goats. Twenty-four growing goats of 4 months old age with 14.45 ± 0.6 kg average body weight were assigned to three treatment diets with 8 animals per treatment. Weights of each animal were measured at the onset of the trial and subsequently on a weekly basis until the end of the trial duration. A pair of blood samples were collected from each goat via a jugular vein puncture and were subjected to either hematological or biochemical analysis. The results showed that treatment diets had no significant effects (P > 0.05) on the final body weight and total body weight gain. However, blue panic had significantly increased (P < 0.05) neutral detergent fiber and crude protein digestibility. The diet-influenced MCV was significantly higher (P < 0.05) in the Alfalfa group. The serum concentration of glucose was significantly increased (P < 0.05) in the blue panic-fed group, while the urea was increased in the Mombasa-fed group. Additionally, the serum concentrations of P, Na, and Cl were significantly increased (P < 0.05) in the blue panic-fed group, but Mombasa significantly increased (P < 0.05) the K concentration. In conclusion, the study indicated that blue panic ranked the best among salt-tolerant alternatives to replace alfalfa, resulting in better feed utilization, serum metabolites, and serum minerals with no adverse effects on growth performance and puberty. This study provides new insight into the shift to the cultivation of salt-tolerant plants with a high level of crude protein in arid areas as a potential approach for the sustainability of the livestock industry.

Graphene Oxide-Silver Nanoparticle Nanocomposites Induce Oxidative Stress and Aberrant Methylation in Caprine Fetal Fibroblast Cells.

Graphene oxide-silver nanoparticle (GO-AgNPs) nanocomposites have drawn much attention for their potential in biomedical uses. However, the potential toxicity of GO-AgNPs in animals and humans remains unknown, particularly in the developing fetus. Here, we reported the GO-AgNP-mediated cytotoxicity and epigenetic alteration status in caprine fetal fibroblast cells (CFFCs). In brief, the proliferation and apoptosis rate of GO-AgNP-treated CFFCs (4 and 8 µg/mL of GO-AgNPs) were measured using the cell-counting kit (CCK-8) assay and the annexin V/propidium iodide (PI) assay, respectively. In addition, the oxidative stress induced by GO-AgNPs and detailed mechanisms were studied by evaluating the generation of reactive oxygen species (ROS), superoxide dismutase (SOD), lactate dehydrogenase (LDH), malondialdehyde (MDA), and caspase-3 and abnormal methylation. The expression of pro- and anti-apoptotic genes and DNA methyltransferases was measured using reverse transcription followed by RT-qPCR. Our data indicated that GO-AgNPs cause cytotoxicity in a dose-dependent manner. GO-AgNPs induced significant cytotoxicity by the loss of cell viability, production of ROS, increasing leakage of LDH and level of MDA, increasing expression of pro-apoptotic genes, and decreasing expression of anti-apoptotic genes. GO-AgNPs incited DNA hypomethylation and the decreased expression of DNMT3A. Taken together, this study showed that GO-AgNPs increase the generation of ROS and cause apoptosis and DNA hypomethylation in CFFCs. Therefore, the potential applications of GO-AgNPs in biomedicine should be re-evaluated.

Fibronectin protected bovine preantral follicles from the deleterious effects of kisspeptin.

Kisspeptin (Kp), a multifunctional neuropeptide critical for initiating puberty and regulating ovulation, was reported to be expressed in mammalian ovaries. Fibronectin (FN), a major secretory product of granulosa cells, provided the extracellular environment for the cumulus cells during maturation. In the current study, we aimed to investigate the potential interplay between FN and Kp in bovine preantral follicles in the context of follicular development and quality. The results showed that Kp significantly reduced the follicular diameters after 14 days in culture, and this was prevented by the addition of FN. Follicles treated with Kp in the presence of FN showed lower levels of apoptotic cells compared to the Kp-treated group. The immunofluorescence analysis showed high levels of cyclooxygenase-2 (COX2), nuclear factor kappa B (NF-κB), and caspase 3, and low levels of sirtuin 1 (Sirt1) and Poly ADP-Ribose Polymerase 1 (PARP1) in the Kp-treated group compared to the control and FN-Kp co-treated groups. The protein expression levels of phosphoinositide 3 kinase (PI3K) increased significantly in the FN and FN-Kp combination treatment groups. Finally, we examined the signal pathway affecting the follicular development after Kp treatment. We detected a significant decrease in the mRNA levels of B-cell lymphoma 2 (BCL2), Sirt1, and PI3K, but the mRNA levels of NF-κB, Caspase3, COX2, P21, and P53 were significantly higher than in the control. Taken together, our results showed the importance of FN for preantral follicle developmental, and, for the first time, we reported that FN could neutralize the deleterious consequences of Kp, suggesting a potential role in the regulation of PI3K/Sirt1 signaling in bovine preantral follicle development.