Targeting GNAQ in hypothalamic nerve cells to regulate seasonal estrus in sheep.

Kazakh sheep are typical seasonal estrus animals. Their reproductive system regulation mainly involves the complex regulation of the hypothalamic-pituitary-gonadal axis (HPGA), which is also closely related to reproductive hormone secretion. Gonadotropin-releasing hormone (GnRH), synthesized and secreted by the hypothalamus, is the key to controlling sheep reproductive activity. We studied how GNAQ (G protein subunit alpha q) regulates estrus in sheep by controlling GnRH expression and secretion. We used hypothalamic nerve cells as the research model. GNAQ overexpression and RNA interference vectors were constructed and transfected into the hypothalamic nerve cells of fetal Kazakh sheep. qPCR, western blotting, and enzyme-linked immunosorbent assay were used to detect GNAQ gene expression in Kazakh ewe tissues and analyze its regulatory effect on GnRH expression in the hypothalamic nerve cells. The fetal sheep hypothalamic nerve cells were successfully isolated and cultured. qPCR and cell immunofluorescence showed that the purity of positive cells was >95%. The tissue expression profile showed that there were different degrees of GNAQ gene expression in the Kazakh ewe tissue. Expression levels were relatively higher in the hypothalamus, pituitary, brain, and uterine tissues. When GNAQ expression was downregulated in the hypothalamic nerve cells, the upstream genes KISS1 (kisspeptin), GPR54 (KISS1 receptor), and ER (estrogen receptor) were all upregulated, as were the downstream genes PLCB1 (phospholipase C beta 1), PRKCB (protein kinase C beta), and GNRH. At the same time, GnRH secretion levels were also upregulated. GNAQ regulated its downstream gene PLCB1 in the hypothalamic nerve cells, and directly regulated GnRH expression and secretion through the calcium and PRKC signaling pathways. GNAQ also regulated kisspeptin expression, subsequently regulating GnRH expression and secretion indirectly through the kisspeptin-GPR54 signaling pathway. Our results are of great importance for improving the reproductive performance of seasonal-estrus sheep.

Associations among Mammary Ultrasound Measurements, Milk Yield of Non-Dairy Ewe Lambs and the Growth of Their Single Lambs.

Mammary cistern size was positively correlated with milk yield of mature dairy ewes, but the association in ewe lambs is unknown. This experiment aimed to examine the associations between mammary ultrasound measurements and the milk yield of ewe lambs at one year of age and to determine the accuracy of using maternal mammary ultrasound to predict single lamb growth rates. Single-bearing ewe lambs (n = 45) were randomly selected and 30 were milked once at weeks three (W3), five (W5), and seven (W7) of lactation. Mammary ultrasound scans were performed at day 110 of pregnancy, W3, W5, W7, and weaning (L69). Single lambs (n = 30) were weighed at birth and at each mammary scanning event. Udder measurements explained 26.8%, 21.4%, and 38.4% of the variation in milk yield at W3, W5 and W7, respectively, and 63.5% and 36.4% of the variation in single lamb growth to W3 and to L69. This ultrasound technique was more accurate in predicting single lamb growth to W3 than milk yield and may enable the identification of pregnant ewe lambs whose progeny would have greater growth rates. More research is needed to identify accurate indicators of superior milk yield and determine whether ultrasound could be used to select ewe lambs.

miR-509-5p anti-infection response for mycoplasma pneumonia in sheep by targeting NF-κB pathway.

MicroRNAs play a key role in Mannan-binding lectin-mediated resistance to Mycoplasma ovipneumoniae pneumonia, by regulating the translation of mRNAs of target genes, thereby regulating the immune response. Additionally, TRAF6 is a key molecule in Toll-like receptor signal transduction, which mediates inflammation and apoptosis signaling pathways and is widely involved in inflammation and immune response. While the molecular regulation mechanism has not been reported. In this study, we screened differentially expressed miRNAs and genes of Anti-infection for M. pneumonia on Sheep, through relevant bioinformatics analysis. Further, the effect of differential expression of NF-κB signaling pathway related genes on the molecular mechanism of M. pneumonia was detected. We used miRNA-mRNA integrated analysed, the target gene TRAF6 of miR-509-5p was selected. TRAF6 dual luciferase reporter vector was co-transfected into HEK 293T cells and primary sheep respiratory mucosal epithelial cells to detect changes in luciferase activity. qRT-PCR was used to analyze the effect of miR-509-5p on the expression and regulation of TRAF6 and other genes related to the NF-κB signaling pathway. The result confirmed that TRAF6 was a target gene of miR-509-5p. Compared with miR-509-5p-NC group, the luciferase activity of miR-509-5p group was significantly down-regulated (P < 0.01). Further, in sheep respiratory mucosal epithelial cells, miR-509-5p mimic could significantly down-regulate the fold change value of TRAF6 (P < 0.01). On the contrary, miR-509-5p-inhibitor up-regulated the fold change value of TRAF6 (P < 0.05). Interestingly, the expression levels of other genes were different. Among them, miR-509-5p mimic significantly up-regulated TLR4 and IRAK4 (P < 0.05), significantly down-regulated TAK1 (P < 0.05) and NF-κB (P < 0.01). miR-509-5p-inhibitor significantly up-regulated NF-κB (P < 0.05) and TAK1 (P < 0.01). miR-509-5p targets TRAF6 to affect the expression of downstream genes, which negatively regulates the NF-κB pathway, thereby affecting the inflammatory response.

Mammary Gland Structures Are Not Affected by an Increased Growth Rate of Yearling Ewes Post-Weaning but Are Associated with Growth Rates of Singletons.

The experiment aimed to examine the impacts of an increased growth rate of ewes between three and seven months of age on udder development using ultrasound and to establish whether ultrasonography could be used to identify ewe mammary structures that may be indirect indicators of singleton growth to weaning. Udder dimensions, depths of gland cistern (GC), parenchyma (PAR) and fat pad (FP) were measured in late pregnancy (P107), early lactation (L29), and at weaning (L100) in 59 single-bearing yearling ewes selected from two treatments. The 'heavy' group (n = 31) was preferentially fed prior to breeding achieving an average breeding live-weight of 47.9 ± 0.38 kg at seven months of age. The 'control' group (n = 28) had an average breeding live-weight of 44.9 ± 0.49 kg. Udder dimensions, GC, PAR and FP did not differ between treatments. Lamb growth to L100 was positively associated (p < 0.05) with PAR at P107 and GC at L29. There was no evidence of negative effects of the live-weight gain treatments on udder development of yearling ewes as measured by ultrasonography. The results suggest that this ultrasound method has the potential to identify pregnant yearling ewes which would wean heavier singletons.

Effect of adding crude glycerine to diets with feed additives on the feed intake, ruminal degradability, volatile fatty acid concentrations and in vitro gas production of feedlot Nellore cattle.

The effects of adding crude glycerine with sodium monensin or essential oils to beef cattle diets on the intake, degradability of DM and nutrients, rumen concentration of volatile fatty acids (VFA) and in vitro gas production were evaluated. Five ruminally cannulated Nellore steers were randomly assigned to a 5 × 5 Latin square design. The treatments were as follows: CONT, without crude glycerine and additives; EO, with essential oils and without crude glycerine; MON, with sodium monensin and without crude glycerine; EOG, with essential oils and crude glycerine; MONG, with sodium monensin and crude glycerine. Treatments with essential oil and sodium monensin increased the NDF and STC intake and the DM degradability. When crude glycerine was combined with either sodium monensin or essential oil, there was a reduction in DM, NDF and STC intake and an increase in DM and CP degradability of the diets. The adding crude glycerine to essential oil diets reduced the CH4 production. Sodium monensin treatments reduced DM and NDF intake and the production of total gas, CH4 , total VFA and acetic acid concentration. In conclusion, the adding crude glycerine (200 g/kg DM) with either sodium monensin (0.03 g/kg DM) or essential oil (0.5 g/kg DM) can be utilized in diets for Nellore cattle without causing detrimental effects on feed intake and improving the DM degradability.

Timing of exogenous progesterone administration is critical for embryo development and uterine gene expression in an ovine model of maternal constraint.

Progesterone (P4) administration in early pregnancy enhances embryo growth in sheep but is associated with decreased embryo survival. This study examined the effects of exogenous P4 administered during specific time periods between pregnancy Day 0 and Day 6 to determine the critical time point for advancement of embryo growth without pregnancy loss and to examine Day 6 and Day 19 endometrial gene expression. Suffolk (S) embryos were transferred into Cheviot (C) ewes that received exogenous P4 (CP4) on Days 0-3 (CP40-3), Days 0-6 (CP40-6), Days 2-4 (CP42-4) or Days 3-6 (CP43-6). Additionally, S embryos were transferred to C and S ewes that did not receive P4 (CnP4 and SnP4). Day 19 embryos from CP4 ewes were longer (P<0.05) than those from CnP4 ewes. CP42-4 ewes had embryos of similar size to those of CP40-3 and CP40-6 ewes but had higher pregnancy rates. There was altered expression of genes associated with embryo implantation and histotroph production: diacylglycerol-O-acyltransferase (DGAT2), hepatocyte growth factor (HGF) and prostaglandin endoperoxide synthase 2 (PTSG2) on Day 6 and endometrial galectin 15 (LGALS15) and mucin glycoprotein 1 (MUC1) on Day 19. This suggests that specific timing of P4 administration is critical to the enhanced embryo growth and survival observed. These findings provide a platform for further investigation aimed at advancing embryo development and survival.

Effect of exogenous progesterone on embryo size and ewe uterine gene expression in an ovine 'dam size' model of maternal constraint.

Progesterone (P4), acting via its receptor, regulates uterine function and histotroph production, which are crucial to embryo growth. This study aimed to examine exogenous P4 effects on embryo size and differential endometrial gene expression at Day 19 of gestation using a 'dam size' sheep model of maternal constraint. Purebred Suffolk (S, genotypically large) embryos were transferred into recipient groups of Cheviot (C, genotypically small) or Suffolk ewes that had, or had not, been pre-treated with P4 from Days 0 to 6 of pregnancy. At Day 19S embryos were collected from four experimental groups: P4 pretreated S ewes (SP4; n=5), untreated S ewes (SnP4; n=15), P4 pretreated C ewes (CP4; n=7) and untreated C ewes (CnP4; n=21). Day-19 embryos from CP4 ewes were larger (P<0.05) than those from CnP4 ewes and similar in size (P>0.05) to embryos from SnP4 and SP4 ewes. Expression of mucin 1 (MUC1) and prostaglandin-endoperoxide synthase 2 (PTGS2) was upregulated in uterine horns ipsilateral to the corpus luteum from CP4 ewes. Prostaglandin receptor (PGR), MUC1 and PTGS2 expression was upregulated, whilst cathepsin L (CTSL) and radical S-adenosyl methionine domain-containing 2 (RSAD2) expression was downregulated in the ipsilateral horn of SP4 ewes. This suggests that pretreating ewes with exogenous P4 may alleviate early pregnancy maternal constraint via mechanisms that alter uterine function. However, further research is required to investigate the timing of P4 administration and its impact on conception rates.

Electrical synapses control hippocampal contributions to fear learning and memory.

The role of electrical synapses in synchronizing neuronal assemblies in the adult mammalian brain is well documented. However, their role in learning and memory processes remains unclear. By combining Pavlovian fear conditioning, activity-dependent immediate early gene expression, and in vivo electrophysiology, we discovered that blocking neuronal gap junctions within the dorsal hippocampus impaired context-dependent fear learning, memory, and extinction. Theta rhythms in freely moving rats were also disrupted. Our results show that gap junction-mediated neuronal transmission is a prominent feature underlying emotional memories.