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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Nicotinamide Supplementation during the In Vitro Maturation of Oocytes Improves the Developmental Competence of Preimplantation Embryos: Potential Link to SIRT1/AKT Signaling.

Nicotinamide (NAM), the amide form of vitamin B3, plays pivotal roles in regulating various cellular processes including energy production and maintenance of genomic stability. The current study aimed at deciphering the effect of NAM, when administered during in vitro maturation (IVM), on the developmental competence of bovine preimplantation embryos. Our results showed that low NAM concentrations reduced the oxidative stress and improved mitochondrial profile, total cleavage and 8-16 cell stage embryo development whereas the opposite profile was observed upon exposure to high NAM concentrations (10 mM onward). Remarkably, the hatching rates of day-7 and day-8 blastocysts were significantly improved under 0.1 mM NAM treatment. Using RT-qPCR and immunofluorescence, the autophagy-related (Beclin-1 (BECN1), LC3B, and ATG5) and the apoptotic (Caspases; CASP3 and 9) markers were upregulated in oocytes exposed to high NAM concentration (40 mM), whereas only CASP3 was affected, downregulated, following 0.1 mM treatment. Additionally, the number of cells per blastocyst and the levels of SIRT1, PI3K, AKT, and mTOR were higher, while the inner cell mass-specific transcription factors GATA6, SOX2, and OCT4 were more abundant, in day-8 embryos of NAM-treated group. Taken together, to our knowledge, this is the first study reporting that administration of low NAM concentrations during IVM can ameliorate the developmental competence of embryos through the potential regulation of oxidative stress, apoptosis, and SIRT1/AKT signaling.

Supplementation of insulin-transferrin-sodium selenite in culture medium improves the hypothermic storage of bovine embryos produced in vitro.

Hypothermic storage of gametes and embryos at 4 °C can be used as an alternative to cryopreservation, but hypothermic preservation can maintain embryo viability for a short duration only. This study investigated the effect of insulin-transferrin-sodium selenite (ITS) in embryo culture medium on hypothermic storage of bovine embryos at 4 °C. Day 7 bovine embryos were subjected to hypothermic storage in tissue culture medium 199 supplemented with 50% fetal bovine serum and 25 mM HEPES for different time durations. After recovery, the embryos were assessed for survival and hatching rate and gene and protein expression levels. Supplementation of embryo culture medium with ITS significantly increased (P < 0.05) the survival and hatching ability of blastocysts stored at 4 °C for 72 h compared to the control group (100% and 76.3% vs 68.5% and 40.5%, respectively). Furthermore, the beneficial effects of ITS on embryos were associated with greater (P < 0.05) total cell number per blastocyst and lesser apoptotic cells number. Moreover, embryos cultured in ITS had lower intracellular lipid content. The protein expression of sirt1 was greater (P < 0.05) in the ITS group, however, caspase3 protein expression was significantly lesser (P < 0.05) in the ITS group. Quantitative reverse transcription PCR indicated that the mRNA levels of SIRT1 and HSP70 were (P < 0.05) increased upon culture with ITS; however, the mRNA levels of the pro-apoptotic genes BAX and CASP3 were reduced (P < 0.05). Taken together, these data suggest that supplementation of embryo culture medium with ITS improves in vitro bovine embryo quality and survival following hypothermic storage.

Matrix metalloproteinases improves trophoblast invasion and pregnancy potential in mice.

Successful implantation is closely linked to the expression of MMP-2 and MMP-9, which greatly influence the ability of an embryo to degrade the basement membrane of the uterine epithelium, mainly composed of type IV collagen, and invade the uterine stroma. The objective of this study was to determine the effect of MMP-2 and MMP-9 co-transfer with embryos on reproductive performance in mice. Using invasion assay, we tested the effect of MMP-2 and MMP-9 for their ability to support trophoblastic invasion in vitro. We performed co-transfer of MMP-2 and MMP-9 with mouse embryos to 2.5 days post-coitum (dpc) pseudo-pregnant uteri using nonsurgical embryo transfer (NSET) technique and evaluated the pregnancy outcomes. Uterine tissue samples were collected to determine collagen content by Masson's trichrome staining. Our results showed that in vitro treatment of MMP-2 and MMP-9 significantly promoted both spreading and invasion of mouse trophoblastic cells compared to the non-treated blastocysts. Moreover, embryo transfer results showed that MMP-9 co-transfer enhanced pregnancy outcome inform of live pup rate by degrading the extracellular matrix, collagen, and facilitate embryo implantation. Taken together our findings imply that MMP-9 can regulate trophoblastic cell invasion during preimplantation, which may have important consequences on embryo implantation, and shed the light on new strategies to avoid miscarriage and provides a platform for successful human embryo transfer technologies.

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.

Polydatin and I-CBP112 protects early bovine embryo against nicotinamide-induced mitochondrial dysfunction.

The mammalian Sirtuin family of seven enzymes, members of the NAD+-dependent histone deacetylase family that modify histones via direct deacetylation, is involved in the regulation of many antioxidant and oxidative stresses. In the present study, we explored the effects of nicotinamide (NAM)-induced oxidative stress on the in vitro development of bovine embryos, on the acetylation of histone H3 lysine 56 (H3K56ac) and on expression of apoptosis-related genes. Treatment with NAM (10, 20 or 40 mM for 24, 48 or 196 h) during IVC resulted in significantly decreased blastocyst formation (24 h: 38.8 vs. 33.1, 27.3 and 10.2%, with P > 0.05, P < 0.05 and P < 0.01, respectively; 48 h: 37.5 vs. 28.2, 13.4 and 0%, with P < 0.05 and P < 0.01, respectively; 196 h: 35.8 vs. 23.4, 0 and 0%, with P < 0.05, respectively). Treatment with NAM (20 and 40 mM for 24 h) resulted in increased intracellular reactive oxygen species (ROS) levels in 2-cell and blastocysts, and apoptotic cell numbers in blastocysts and decreased mitochondrial membrane potential (ΔΨ) in 2-cell embryos (P < 0.05). Polydatin (PD) and I-CBP112 rescued the 20 mM NAM-induced embryo developmental defects and reduced ROS levels and apoptotic cell numbers in blastocysts (P < 0.05). The gene expression of NF-κB, COX2 and p53 was significantly increased in the NAM-treated group. Immunofluorescence analysis confirmed that the protein levels of nuclear factor-kappa B (NF-κB) decreased significantly after PD and I-CBP112 treatment compared with the control (P < 0.05). High level of H3K56ac induced by NAM was decreased after PD and I-CBP112 treatment (P < 0.05). These findings suggest that NAM treatment induces high levels of H3K56 acetylation that may be involved in oxidative stress-induced bovine developmental defects, which can be tolerated by PD and I-CBP112 treatment.