Effects of Low and High Maternal Protein Intake on Fetal Skeletal Muscle miRNAome in Sheep.

Prenatal maternal feeding plays an important role in fetal development and has the potential to induce long-lasting epigenetic modifications. MicroRNAs (miRNAs) are non-coding, single-stranded RNAs that serve as one epigenetic mechanism. Though miRNAs have crucial roles in fetal programming, growth, and development, there is limited data regarding the maternal diet and miRNA expression in sheep. Therefore, we analyzed high and low maternal dietary protein for miRNA expression in fetal longissimus dorsi. Pregnant ewes were fed an isoenergetic high-protein (HP, 160-270 g/day), low-protein (LP, 73-112 g/day), or standard-protein diet (SP, 119-198 g/day) during pregnancy. miRNA expression profiles were evaluated using the Affymetrix GeneChip miRNA 4.0 Array. Twelve up-regulated, differentially expressed miRNAs (DE miRNAs) were identified which are targeting 65 genes. The oar-3957-5p miRNA was highly up-regulated in the LP and SP compared to the HP. Previous transcriptome analysis identified that integrin and non-receptor protein tyrosine phosphatase genes targeted by miRNAs were detected in the current experiment. A total of 28 GO terms and 10 pathway-based gene sets were significantly (padj < 0.05) enriched in the target genes. Most genes targeted by the identified miRNAs are involved in immune and muscle disease pathways. Our study demonstrated that dietary protein intake during pregnancy affected fetal skeletal muscle epigenetics via miRNA expression.

Effects of urea supplementation on ruminal fermentation characteristics, nutrient intake, digestibility, and performance in sheep: A meta-analysis.

Background and Aim: As a non-protein nitrogen source, urea is a popular, low cost, and easily obtained protein supplement. The objective of the present study was to perform a meta-analysis of the effects of urea supplementation on rumen fermentation and sheep performance. Materials and Methods: A total of 32 experiments from 21 articles were compiled into a dataset. The levels of dietary urea varied from 0 to 31 g/kg of dry matter (DM). Parameters observed were rumen fermentation product, nutrient intake, nutrient digestibility, and sheep performance. This dataset was analyzed using a mixed model methodology, with urea supplementation levels as fixed effects and the different experiments as random effects. Results: Increasing levels of urea were associated with increases (p=0.008) in rumen pH, butyrate (C4) production, and ammonia (NH3-N) concentration. Urea supplementation had minor effects on total volatile fatty acids (p=0.242), total protozoa (p=0.429), and the microbial N supply (p=0.619), but tended to increase methane production (CH4; p<0.001). Supplementation of urea increased the intake of dry matter (DM; p=0.004) and crude protein (CP; p=0.001). Digestibility parameters, such as DM digestibility (DMD) and CP digestibility (CPD), also increased (p<0.01) as a result of urea supplementation. Retained N (p=0.042) and N intake (p<0.001) were higher with increasing levels of urea supplementation. In terms of animal performance, supplementation of urea increased average daily gain (ADG; p=0.024), but decreased the hot carcass weight percentage (p=0.017). Conclusion: This meta-analysis reports the positive effects of urea supplementation on rumen fermentation products (i.e., pH, C4, and NH3-N), intake (DM, CP, and N), digestibility (DMD and CPD), and ADG in sheep.

Differential protein input in the maternal diet alters the skeletal muscle transcriptome in fetal sheep.

Maternal nutrition during pregnancy is one of the major intrauterine environmental factors that influence fetal development by significantly altering the expression of genes that might have a consequence on the physiological, morphological, and metabolic performance of the offspring in the postnatal period. The impact of maternal dietary protein on the expression of genes in sheep fetal skeletal muscle development is not well understood. The current study aims to investigate the impact of high and low maternal dietary protein on the holistic mRNA expression in the sheep fetal skeletal muscle. Dams were exposed to an isoenergetic high-protein diet (HP, 160-270 g/day), low-protein diet (LP, 73-112 g/day), and standard protein (SP, 119-198 g/day) diets during pregnancy. Fetal skeletal muscles were obtained at the 105th day of pregnancy and mRNA expression profiles were evaluated using Affymetrix GeneChip™ Ovine Gene 1.0 ST Array. The transcriptional analysis revealed a total of 323, 354, and 14 genes were differentially regulated (fold change > 2 and false discovery rate ≤ 0.05) in HP vs. SP, LP vs. HP, and SP vs. LP, respectively. Several myogenic genes, including MYOD1, MYH2, MYH1, are significantly upregulated, while genes related to the immune system, such as CXCL11, HLA-E, CXCL10, CXCL9, TLRs, are significantly downregulated in the fetal muscle of the HP group compared to those of SP and LP group. Bioinformatic analysis revealed that the majority of these genes are involved in pathways related to the immune system and diseases. The results of our study demonstrate that both augmented and restricted dietary proteins in maternal diet during pregnancy alter the expression of genes as well as the offspring's genetic marks.

Effect of shearing on some physiological and hormonal parameters in Akkaraman sheep.

OBJECTIVE: Shearing is one of the practices that is applied periodically to fiber producing animals, which can also alter resistance of animals to high temperatures in especially summer months. This study aimed to investigate effects of shearing on some physiological and hormonal parameters in Akkaraman sheep during summer season. METHODS: This study was carried out on 39 non-pregnant Akkaraman ewes (aged 1.5 years at the beginning of experiment). The 39 ewes were chosen randomly from the flock belonging to the Erciyes University and they were assigned to two groups as follows: i) group A (n = 20) designed as the control group, they were shorn and group B (n = 19) designed as the experimental group, they were unshorn. Prior to the shearing (-1 day) and on days 1, 7, 15, 30, 45, 60, 75, and 90 following the shearing, blood samples were taken from the vena jugularis of each sheep. Cortisol, ß-endorphin, growth hormone (GH), thyroxine (T4), triiodothyronine (T3), and heat shock protein 70 (HSP-70) concentrations were determined using the enzyme immunoassay method. Body weight (BW), rectal temperature (RT), pulse rate (PR), and respiratory rate (RR) of each sheep were recorded at the same time. The data obtained were analyzed using two-way repeated measures analysis of variance. RESULTS: Statistical analysis showed a significant effect of shearing×period interaction (p<0.01) and a significant effect of period (p<0.01) on BW, HSP-70, cortisol, T4 and RT, PR, GH, ß-endorphin, T3, respectively. Also these analysis showed no significant effect of shearing× period interaction or period on RR. CONCLUSION: The results showed that the thermoregulation abilities of sheep were affected by shearing treatment and the shorn ewes were less affected by heat stress. In conclusion, based on the data of this study, shearing can be considered as a necessary management practice that requires protection for sheep from the effect of heat stress.

Effects of vitamin and mineral premix withdrawal from diets on carcass and meat quality of feedlot steers.

The primary objective of the present study was to assess the effects of vitamin and mineral premix (VMP) withdrawal from the diets 30 and 60 days ahead of slaughter on carcass and meat quality of Holstein Friesian steers. A total of 45 animals at 16 to 17 months of age were used and the selected animals were divided into three experimental groups: control group (fed with a diet with VMP), VMP withdrawal 30 days ahead of slaughter (VMP30 group), and VMP withdrawal 60 days ahead of slaughter (VMP60 group). Meat samples were taken at 24 h postmortem from the 13th rib section and meat quality was evaluated on the Longissimus dorsi thoracis (LT) muscle. After slaughter, carcass yield and meat drip loss, cooking loss, thawing loss, and shear force traits were determined. Meat pH and color parameters were measured at 24, 48, 72, 96, 120, and 144 h of postmortem. The fatty acid composition in 13th rib section' adipose tissue was determined. The hot and cold carcass weights, carcass yield and chilling loss were not affected by the withdrawal of VMP from the diet. Withdrawal of VMP from the diets 30 and 60 days ahead of slaughter did not have any significant effects on ultimate pH, drip loss, cooking loss, thawing loss, shear force, and meat color. Additionally, dry matter, crude protein, ash, fat contents, moisture-protein ratio of the meat samples, and fatty acid profiles were not affected by VMP30 and VMP60 treatments. It was concluded based on present finding VMP could be withdrawn safely from the diets 30 and 60 days ahead of slaughter without any negative effects on carcass and meat quality traits of feedlot steers. Withdrawal of VMP may reduce feeding costs and environmental damages generated by animal breeding systems.

Amebicidal effects of fenugreek (Trigonella foenum-graecum) against Acanthamoeba cysts.

Trigonella foenum-graecum L. (TF) is known to the public as a chest emollient, mucous expectorant, laxative and is used to prevent maturation of boils and diabetes since ancient times. In this study, we aimed to determine the amebicidal effects against Acanthamoeba cysts. Plant extracts were prepared at concentrations of 1, 2, 4, 8, 16, and 32 mg/ml and were placed in a hemocytometer with cell counts 22 × 106 cell/ml. The fatty acid profiles of TF seeds were determined. Standard Acanthamoeba cysts were added and incubated at 25°C. The viability of the parasite was checked and recorded at hours 3, 24, 48, 72, 96, and 102. The values of lethal concentration doses (LD50 and LD90) were calculated using probit analysis. This study revealed that T. foenum-graecum prevented proliferation of the parasite at certain times. However, further for in vivo and controlled experimental studies are needed in order to find out how to use this plant as medication.

Oxidative stress modulates the expression of apoptosis-associated microRNAs in bovine granulosa cells in vitro.

Despite its essential role in ovulation, oxidative stress (OS) has been found to be cytotoxic to cells, while microRNAs (miRNAs) are known as a major regulator of genes involved in cellular defense against cytotoxicity. However, a functional link between OS and miRNA expression changes in granulosa cells (GCs) remains to be investigated. Here, we investigate the OS modulation of apoptosis-associated miRNAs and their biological relevance in bovine GCs. Following the evaluation of cell viability, accumulation of reactive oxygen species (ROS), cytotoxicity and mitochondrial activity, we used a ready-to-use miRNA PCR array to identify differentially regulated miRNAs. The results showed that exposure to 150 µM H2O2 for 4 h creates remarkable signs of OS in GCs characterized by more than 50% loss of cell viability, higher nuclear factor erythroid 2-related factor 2 (NRF2) nuclear translocation, significantly (p < 0.05) higher abundance of antioxidant genes, significantly (p < 0.001) higher accumulation of ROS, lower mitochondrial activity and a higher (p < 0.001) number of apoptotic nuclei compared to that of the control group. miRNA expression analysis revealed that a total of 69 miRNAs were differentially regulated in which 47 and 22 miRNAs were up- and downregulated, respectively, in stressed GCs. By applying the 2-fold and p < 0.05 criteria, we found 16 miRNAs were upregulated and 10 miRNAs were downregulated. Target prediction revealed that up- and downregulated miRNAs potentially targeted a total of 6210 and 3575 genes, respectively. Pathway analysis showed that upregulated miRNAs are targeting the genes involved mostly in cell survival, intracellular communication and homeostasis, cellular migration and growth control and disease pathways. Our results showed that OS modulates the expression of apoptosis-associated miRNAs that might have effects on cellular or molecular damages.

Concentration dependent antioxidative and apoptotic effects of sulforaphane on bovine granulosa cells in vitro.

Sulforaphane (SFN) has received a great deal of research attention because of its ability to induce the production of a battery of antioxidant enzymes in certain concentrations through the activation of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling pathway, which may effectively neutralize reactive oxygen species (ROS) induced oxidative stress. This study was conducted to investigate the potential of different concentrations of SFN in inducing antioxidative and apoptotic effects in granulosa cells (GCs). For this purpose, bovine GCs were collected from preovulatory antral follicles and cultured with different concentrations of SFN (0-80 µM) and based on phenotypic evaluation three concentrations were selected: 2 µM (low), 10 µM (medium), and 20 µM (high) for further investigations. The results showed that there was a dramatic loss of cell viability and higher cytotoxic effects of SFN on GCs at higher concentrations (>15 µM). The expression of NRF2 increased significantly (p < 0.05) with fold change ranged 3-8 in SFN treated GCs, whereas Kelch Like ECH Associated Protein 1 (KEAP1) expression was either downregulated or similar as control group under the same conditions. Moreover, the relative expression of the genes (PRDX1, CAT, TXN1and SOD1) downstream to NRF2 activation was found to be highly expressed (fold change ranged from 2 to 5, p < 0.05) in SFN treated GCs compared to the untreated control. In addition, ROS accumulation was higher in GCs treated with 20 µM SFN which in turn results in a higher accumulation of lipid droplets. Compared to control, no changes in the mitochondrial activity was observed at 2 and 10 µM SFN concentrations; however, significantly lower mitochondrial activity was found at high concentration (20 µM). The results of this study clearly showed that 10 µM SFN concentration played a crucial role in activating Nrf2 pathway without inducing apoptotic characteristics and this concentration may have beneficial effects in boosting the production of phase II antioxidant enzymes in GCs. However, at high concentration (20 µM), SFN may generate excessive ROS that causes mitochondrial dysfunction and induces cellular stress and eventually leads to apoptosis. These data strongly suggest a concentration dependent antioxidative and apoptotic effects of SFN on GCs.

Effect of Hempseed (Cannabis sativa sp.) Inclusion to the Diet on Performance, Carcass and Antioxidative Activity in Japanese Quail (Coturnix coturnix japonica).

This study was conducted to determine the effects of hempseed (H) on performance, carcass traits, and antioxidant activity in Japanese quail (Coturnix coturnix japonica). A total of 192 quail with seven-days old were divided into four experimental groups with four replicates. The treatments were; i) Control diet (C, no hempseed); ii) 5% hempseed in diet (H5); iii) 10% hempseed in diet (H10); and iv) 20% hempseed in diet (H20). The body weight (BW) and feed intake (FI) of quail was determined at 7, 21 and 42 d of age. At 42 d of age four quail were slaughtered and the carcass and internal organ traits were determined. Malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), nitric oxide (NO) and total protein were determined in the blood serum end of the experiment. The BW of the groups were not significant at 7 and 21 d, however in the 20% hempseed group BW decreased at 42 d (p<0.05). The FI and feed conversion ratio were not significant among the treatment groups. The carcass, liver, intestine and heart weight and their percentage to carcass were significantly differ in treatment groups (p<0.05). The serum MDA and NO decreased in hempseed addition (p <0.001). The serum SOD, CAT and GSH-Px were increased by hempseed supplementation (p<0.001). In conclusion, hempseed supplementation to quail diets may not improve quail performance traits but increase antioxidant activity in blood.