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Endometritis is a common disease affecting fertility in cows during the perinatal period, which disturbs the molecular milieu of the uterine environment and impairs embryo development and implantation. Exosomes are important extracellular components that transmit a variety of micro RNAs (miRNAs), which perform key regulatory functions. In this study, we investigated plasma exosomal miRNAs from cows with endometritis and from cultured endometrial epithelial cells (EECs) challenged with lipopolysaccharide (LPS) to explore the role of EEC-derived exosomes and their miRNAs in bovine endometritis. Plasma exosomes were collected from nine healthy dairy cows and nine dairy cows with endometritis, and culture supernatant exosomes were isolated from EECs challenged with or without LPS. Exosomal RNA was extracted using commercial kits and miRNA profiles were generated using RNA-seq. We found that miR-218 was differentially expressed in EECs under conditions of endometrial inflammation. Inhibition studies suggested that reduced levels of miR-218 in EEC-derived exosomes when transferred into placental trophoblast cells impaired embryonic development and decreased placental trophoblast cell migration by targeting secreted frizzled related protein 2. We propose that exosomal miR-218 secreted from EECs acts as a driver of embryonic development and differentiation. In addition, exosomal miR-218 may provide a valuable diagnostic marker for bovine endometritis.

The present study evaluated the effects of natural astaxanthin (ASTA) from Haematococcus pluvialis on the antioxidant capacity, lipid metabolism, and ASTA accumulation in the egg yolk of laying hens. Hy-Line Brown layers (n = 288, 50 wk old) were randomly assigned to 1 of 4 dietary treatment groups. Each group had 6 replicates of 12 hens each. All birds were given a corn-soybean meal–based diet containing 0, 25, 50, or 100 mg/kg ASTA for 6 wk. The results showed that the total antioxidant capacity, superoxide dismutase level, and glutathione peroxidase level in the plasma, livers, and egg yolks were significantly increased in the ASTA groups compared with those of the control group (P

The objective of this study was to investigate differences in mRNA expression between fresh and frozen–thawed sperm in roosters. In trial 1, gene expression profiles were measured using microarray with Affymetrix GeneChip Chicken Genome Arrays. The results showed that 2,115 genes were differentially expressed between the 2 groups. Among these genes, 2,086 were significantly downregulated and 29 were significantly upregulated in the frozen–thawed sperm group. Gene Ontology (GO) analysis showed that more than 1,000 differentially expressed genes (DEG) of all significantly regulated genes were involved in GO terms including biological processes, molecular function, and cellular component. Kyoto Encyclopedia of Genes and Genomes analysis showed that DEG were significantly (P

Sperm motility is a crucial trait in chicken production, and the epididymis is an essential organ in the reproductive system. Currently, the molecular mechanisms underlying sperm motility in the epididymis are unclear. In this study, eight cDNA libraries and eight miRNA libraries were constructed from roosters (four chickens per group) with diverse sperm motility. After a comparative analysis of epididymal transcriptomes, we detected 84 differentially expressed genes (DEGs) using the edgeR package. Integrated interpretation of DEGs indicated that MMP9, SLN, WT1, PLIN1, and LRRIQ1 are the most promising candidate genes affecting sperm motility in the epididymis of roosters. MiR-146a, mir-135b, and mir-205 could play important regulatory roles in sperm maturation, capacitation, and motility. Additionally, a comprehensive analysis of the mRNA and miRNAs transcriptomes in silico identified a promising gene-miRNA pair miR-135b-HPS5, which may be a vital regulator of sperm motility in the epididymis. Our findings provide novel integrated information of miRNAs and genes that shed light on the regulatory mechanisms of fertility in roosters.

In vitro fertilization (IVF) for ovine embryos is strongly associated with low pregnancy rates and negative effects on embryonic and postnatal development, but the mechanisms remain unclear. We aimed to identify genes essential for successful implantation of IVF embryos in sheep by species-specific microarray analysis. Differential gene expression analysis between caruncles and intercaruncle sites of the ovine uterus with in vivo (IVV) -produced embryos on day 10 of pregnancy found 43 differentially expressed genes (DEGs) with at least a 2-fold change, such as coiled-coil domain-containing 152 (CCDC152) and pregnancy-associated plasma protein A (PAPPA). Gene ontology analysis revealed these DEGs were related to cell proliferation, the immune system process, localization and response to stimulus. Moreover, we identified 93 DEGs in endometrial caruncles with IVV- versus in vitro (IVT)-produced embryos on days 12, 14 and 16 of pregnancy, including prostaglandin-endoperoxide synthase 2 (PTGS2) and cubilin (CUBN). GO analysis revealed that these DEGs were related to cell adhesion, cell proliferation, embryo implantation, embryonic morphogenesis, the immune system process and localization. Two DEGs (PAPPA and RSAD2) in caruncle versus intercaruncle sites at day 10 were also differentially expressed between the IVV and IVT groups at the three implantation stages. Self-organizing feature map cluster analysis of transcript profiles during embryo implantation revealed different gene expression patterns between the IVV and IVT groups. In conclusion, this study identified many genes that may be associated with mechanisms underlying IVF-induced ovine embryo implantation failure during the peri-implantation period, and such genes provide potential candidates for further study.

Fatness traits are important in pigs because of their implications for fattening efficiency, meat quality, reproductive performance and immunity. Songliao black pigs and Landrace pigs show important differences in production and meat quality traits, including fatness and muscle growth. Therefore, we used a high-throughput massively parallel RNA-seq approach to identify genes differentially expressed in backfat tissue between these two breeds (six pigs in each). An average of 37.87 million reads were obtained from the 12 samples. After statistical analysis of gene expression data by edgeR, a total of 877 differentially expressed genes were detected between the two pig breeds, 205 with higher expression and 672 with lower expression in Songliao pigs. Candidate genes (LCN2, CES3, DGKB, OLR1, LEP, PGM1, PCK1, ACACB, FADS1, FADS2, MOGAT2, SREBF1, PPARGC1B) with known effects on fatness traits were included among the DEGs. A total of 1071 lncRNAs were identified, and 85 of these lncRNAs were differentially expressed, including 53 up-regulated and 32 down-regulated lncRNAs, respectively. The differentially expressed genes and lncRNAs involved in glucagon signaling pathway, glycolysis/gluconeogenesis, insulin signaling pathway, MAPK signaling pathway and so on. Integrated analysis potential trans-regulating or cis-regulating relation between DEGs and DE lncRNAs, suggested lncRNA MSTRG.2479.1 might regulate the expressed level of VLDLR affecting porcine fat metabolism. These results provide a number of candidate genes and lncRNAs potentially involved in porcine fat deposition and provide a basis for future research on the molecular mechanisms underlying in fat deposition.

Omega-3/n-3 polyunsaturated fatty acids (ω-3/n-3 PUFAs) play an important role in male reproductive function. The present study was designed to evaluate the effects of linseed oil (LO) as a source of α-linolenic acid (ALA, n-3 PUFA) on semen quality, plasma reproductive hormone and expression of key enzyme and protein related to steroidogenesis in aging layer breeder roosters. Ninety-six 57-wk-old Nongda No.3 layer breeder roosters were randomly assigned into one of four dietary treatments. All birds were fed a basal diet for 1wk and then assigned to a corn-soybean meal-based diet containing 0, 1, 2, 4% LO for 4 wk. After feeding trial, the roosters were slaughtered and investigated. The results showed that semen volume was dramatically increased relative to the other treatments in 2% LO group (P 0.05). With an increase in dietary LO, semen concentration, sperm viability, sperm motility and total sperm count increased linearly (P 0.05). Plasma follicle-stimulating hormone (FSH) level increased gradually and reached a maximum when 4% LO was fed (P 0.01). Plasma luteinizing hormone (LH) levels in 1% and 2% LO group were improved significantly (P 0.05) relative to the control group. Plasma testosterone (T) levels were remarkably improved compared with the control when birds were fed 2% and 4% LO (P 0.05). A significant increase of steroidogenic acute regulatory protein (StAR) mRNA expression in 2% and 4% LO group was observed relative to the control group (P 0.05). An increase in dietary LO supplementation from 1% to 4% markedly enhanced (P 0.05) the mRNA expression of cholesterol side-chain cleavage enzyme (P450scc) compared to the control. A significant increase (P 0.05) in the Steroidogenic Factor 1 (SF-1) mRNA levels was observed in the 2% and 4% LO-added groups. SF-1 protein expression was markedly increased by adding LO in diets (P 0.05), and reached a maximum in 2% LO group. In conclusion, the results above suggest that dietary LO may improve semen quality by increasing the T hormone secretion, which may be related to higher StAR and P450scc mRNA expression and SF-1 expression. These findings provide a potential for using LO to attenuate the age-related sub-fertility in commercial layer breeder roosters.

The objective of this study was to evaluate the effects of natural astaxanthin (ASTA) from Haematococcus pluvialis on production performance, egg quality, antioxidant enzyme activity, free radical scavenging ability, and gene expression of antioxidant enzymes in laying hens. Nongda No. 3 laying hens (n = 450) were randomly allocated to 1 of 5 dietary treatments. Each treatment had 6 replicates of 15 hens each. All birds were assigned to a corn-soybean meal-based diet containing 0, 20, 40, 80, or 160 mg/kg ASTA for 4 wk. With increasing dietary ASTA, no significant effects were observed on egg weight, feed consumption, feed efficiency, laying rate, Haugh unit, or eggshell strength. Yolk color darkened linearly with increasing dose of ASTA (P

Background: Natural astaxanthin (ASTA) has strong antioxidant properties and has been widely used as a health product to improve human health. However, the effects of ASTA on the reproductive performance of aging roosters have been poorly studied. We aimed to investigate the effects of dietary ASTA on semen quality and antioxidant capacity in aging roosters and to explore the potential mechanism of semen quality change via anti-oxidation defense system.Methods: In the present study, 96 53-week-old Jinghong No. 1 layer breeder roosters were fed a corn-soybean meal basal diet containing ASTA at 0, 25, 50, or 100 mg/kg for 6 week. Results: Semen quality in the ASTA groups remarkably improved as compared to those in the control group and antioxidant activities, the abilities to scavenge hydroxyl radicals and superoxide anions increased gradually with ASTA addition (P < 0.05). In addition, the mRNA levels of antioxidant enzymes, the mRNA and protein levels of the mitogen-activated protein kinase (MAPK), nuclear factor-erythroid 2-related factor 2 (Nrf2) were markedly increased in the 50-100 mg/kg ASTA group (P < 0.05). Conclusions: Collectively, these results demonstrate that dietary ASTA may improve semen quality by increasing antioxidant enzyme activities, and the ability to scavenge hydroxyl radicals, which may be related to up-regulation of the MAPK/Nrf2 pathway.

Skeletal muscle development is a complex biological process involving multiple key genes, signaling pathways and noncoding RNAs, including microRNAs and circular RNAs (circRNAs). However, the regulatory relationship among them is so complicated that it has not yet been fully elucidated. In this study, we found that miR-7 inhibited C2C12 cell proliferation and differentiation by targeting transcription factor 12 (TCF12). circHIPK3 acted as a competing endogenous RNA, and its overexpression effectively reversed the regulation of miR-7 on C2C12 cell proliferation and differentiation by increasing TCF12 expression. Taken together, our findings provide evidence that circHIPK3 regulates skeletal muscle development through the miR-7/TCF12 pathway. This study provides a scientific basis for further research on skeletal muscle development at the circRNA level.

Doublesex and mab-3-related transcription factor (dmrt) genes are widely distributed across various biological groups and play critical roles in sex determination and neural development. Here, we applied bioinformatics methods to exam cross-species changes in the dmrt family members and evolutionary relationships of the dmrt genes based on genomes of 17 fish species. All the examined fish species have dmrt1–5 while only five species contained dmrt6. Most fish harbored two dmrt2 paralogs (dmrt2a and dmrt2b), with dmrt2b being unique to fish. In the phylogenetic tree, 147 DMRT are categorized into eight groups (DMRT1–DMRT8) and then clustered in three main groups. Selective evolutionary pressure analysis indicated purifying selections on dmrt1–3 genes and the dmrt1–3–2(2a) gene cluster. Similar genomic conservation patterns of the dmrt1–dmrt3–dmrt2(2a) gene cluster with 20-kb upstream/downstream regions in fish with various sex-determination systems were observed except for three regions with remarkable diversity. Synteny analysis revealed that dmrt1, dmrt2a, dmrt2b, and dmrt3–5 were relatively conserved in fish during the evolutionary process. While dmrt6 was lost in most species during evolution. The high conservation of the dmrt1–dmrt3–dmrt2(2a) gene cluster in various fish genomes suggests their crucial biological functions while various dmrt family members and sequences across fish species suggest different biological roles during evolution. This study provides a molecular basis for fish dmrt functional analysis and may serve as a reference for in-depth phylogenomics.

The objective of this study was to evaluate the effects of different levels of dietary natural astaxanthin (ASTA) (from the microalga Haematococcus pluvialis) and storage at 4°C and 25°C on the quality of eggs from laying hens. Nongda No. 3 laying hens (n = 450) were randomly allocated to 1 of 5 dietary treatments. Each treatment had 6 replicates of 15 hens each. All birds were assigned to a corn-soybean meal-based diet containing 0, 20, 40, 80, or 160 mg/kg natural ASTA for 4 wk. A total of 540 eggs were collected at the end of the 4-week feeding trial. Sixty fresh eggs were collected and measured for egg quality within 24 h after collection. The other 480 eggs were used in a factorial arrangement with 5 dietary ASTA levels, 4 storage times, and 2 storage temperatures. During the 8-week storage period at 4°C and 25°C, egg quality measurements were performed every 2 wk on 12 eggs per treatment. No significant effects (P > 0.05) on yolk index, yolk pH, Haugh units, weight loss, or eggshell strength were observed with increasing concentrations of dietary ASTA. Yolk color darkened linearly with increasing dose of ASTA (P < 0.05). During storage of eggs, yolk index and Haugh units decreased significantly (P < 0.05), whereas yolk pH and weight loss increased (P < 0.05). An interaction was observed between dietary ASTA level and storage time on yolk index, yolk color, and Haugh units (P < 0.05). These results demonstrated that dietary ASTA from H. pluvialis delayed the decrease in yolk index and yolk color during storage at 4°C and 25°C. Therefore, we speculate that there may be a combined effect of dietary ASTA level and storage time on egg internal quality; this information may provide additional options by which to extend the storage time of eggs.

Uterine prostaglandins F2α (PGF2α) is essential for implantation, initiation of luteolysis and delivery. Previous studies have demonstrated that the expression of Cyclooxygenase-2 (COX-2), an enzyme limiting PGF2α rate, is regulated by steroid hormones, and also dihydrotestosterone (DHT) may be involved in regulating COX-2 expression both positively and negatively. However, it remains unclear how whether DHT regulates COX-2 expression and consequent PGF2α release in bovine endometrial epithelial cells (EECs). In this study, we evaluated the localization of the two isoforms of DHT synthetase 5α-reductase (5α-red1 and 5α-red2) and androgen receptor (AR) in bovine endometria by immunohistochemistry, and investigated 5α-red1, 5α-red2, AR, and DHT levels at the different stages of endometria (follicle, early-, mid-, and late-pregnancy phases). The results showed that 5α-red1, 5α-red2 and AR all were expressed in endometria, and their expressions and the level of DHT significantly increased in the late-pregnancy phase compared with the mid-pregnancy phase. Moreover, we cultured EECs from the mid-pregnancy phase and the in vitro study showed that DHT dose-dependently increased COX-2 expression and PGF2a release, but AR antagonist (flutamide) inhibited the stimulating effect via DHT. In addition, the DHT-induced COX-2 expression and PGF2α release were subjected to the regulation of both EGFR/PI3K/Akt/NFkB signaling as the inhibitors of EGFR (AG1478) and PI3K/Akt (LY294002) and NFkB (QNZ) attenuated the DHT mediated effect. Taken together, the results demonstrated that DHT-induced COX-2 expression and consequent PGF2α release in bovine EECs were mediated through AR-derived EGFR transactivation and PI3K/Akt cascade leading to NFkB activation.

The reproductive efficiency of roosters is an important trait in poultry production; however, the molecular mechanisms underlying this trait are not clearly understood. Here, we compared the mRNA and microRNA (miRNA) transcriptomes of testis from roosters with divergent sperm motility. A total of 302 differentially expressed genes (DEGs), including 182 upregulated genes and 120 downregulated genes, were identified in high sperm motility groups compared with low sperm motility groups. A subset of these DEGs related to steroid hormone biosynthesis and thus could be important for spermatogenesis. Additionally, we detected 13 differentially expressed miRNAs (DEMs) between two groups, and target gene prediction indicated seven of these could be associated with spermatogenesis. Based on a comprehensive analysis of these transcriptomes, miRNA-mRNA interaction networks were constructed. Six DEGs were predicted to be regulated by DEMs. Subsequently, we validated the negative regulation of family with sequence similarity 84, member A (FAM84A) by miR-215 using a dual-luciferase reporter system. These results provide new insights into the molecular profile of the testis and identify genes that may determine sperm motility in chickens.

As a common disease of cows occurring during their perinatal period, endometritis is known to affect fertility. At present, the studies on endometritis mainly focus on preventing microbial invasion. However, the mechanism that uterine inflammation affects embryo activity and implantation is unclear. Mainly containing lipids, proteins, mRNAs, and microRNAs, exosomes widely exist in various tissues and body fluids. Exosome extractions were used by commercial kits and confirmed through morphological examinations and Western blot. After exosomes’ mRNA profiles were generated using RNA sequencing, it was investigated how uterine cavity fluid exosomes affect the developmental competence of in vitro fertilization (IVF) embryos in case of endometritis. In this study, the isolated exosomes were spherical particles with a diameter of 30–150 nm according to the transmission electron microscopy. Identified with Western blotting, positive CD63 and CD9 expressions showed that the isolated exosomes could be used for the subsequent tests. We found 118 differentially expressed miRNAs in the exosomes of the uterine cavity fluid in healthy cows and those with endometritis, among which, 52 miRNAs were down regulated and 66 up regulated. Furthermore, the qRT-PCR results confirmed the up-regulation of three miRNAs and down-regulation of six miRNAs, which were consistent with the deep sequencing results. IVF embryos co-incubated with the endometritis exosomes significantly decreased the blastocyst formation rate in comparison with those co-incubated with the healthy exosomes (21.84 + 3.17 vs. 32.37 + 2.69). Therefore, exosome miRNAs may be a cause of infertility in cows with endometritis.

Fatty traits are very important in pig production. However, the role of microRNAs (miRNAs) in fat deposition is not clearly understood. In this study, we compared adipose miRNAs from three full-sibling pairs of female Landrace pigs, with high and low backfat thickness, to investigate the associated regulatory network. We obtained an average of 17.29 million raw reads from six libraries, 62.27% of which mapped to the pig reference genome. A total of 318 pig miRNAs were detected among the samples. Among them, 18 miRNAs were differentially expressed (p-value &lt, 0.05, |log2fold change| &ge, 1) between the high and low backfat groups, 6 were up-regulated and 12 were down-regulated. Functional enrichment of the predicted target genes of the differentially expressed miRNAs, indicated that these miRNAs were involved mainly in lipid and carbohydrate metabolism, and glycan biosynthesis and metabolism. Comprehensive analysis of the mRNA and miRNA transcriptomes revealed possible regulatory relationships for fat deposition. Negatively correlated mRNA&ndash, miRNA pairs included miR-137&ndash, PPARGC1A, miR-141&ndash, FASN, and miR-122-5p&ndash, PKM, indicating these interactions may be key regulators of fat deposition. Our findings provide important insights into miRNA expression patterns in the backfat tissue of pig and new insights into the regulatory mechanisms of fat deposition in pig.

Polydactyly is a commonly observed limb malformation in humans and other vertebrates. The Beijing-You chicken expressing the polydactyly phenotype provides an opportunity to investigate the potential cause for polydactyly. Here we extensively exploited genetic determinants of the chicken polydactyly in a genome wide association study using over 580,000 SNPs characterized in a Beijing-You × Lohmann F1 cross, consisting of 79 animals. A total of 10 loci clustered on the short arm of chromosome 2 were identified to be significantly associated with the trait. Among the 10 significant SNPs, 7 were located in a linkage disequilibrium block of 1737kb. The strongest association signal (rs317674023, P=5.48×10(-8)) residing nearby Bone Morphogenetic Protein Receptor-Associated Molecule 1 (BRAM1) was identified in the genomic region. Our results provide insights to the genetic basis underlying chicken polydactyly and may facilitate studies of the limb malformation in humans and other species.

Mucin 1 (MUC1), a cell surface glycoprotein, is expressed mainly in the endometrial luminal epithelium (LE) and glandular epithelium (GE) of the endometrium in many mammalian species including mice, rats, pigs, sheep, horses and humans during various stages of a menstrual or oestrous cycle, where it plays an important role in embryo implantation and placentation. However, the expression and function of MUC1 in uterine tissues during early pregnancy in sheep after artificially-induced oestrous is not known. Therefore, we investigated the expression and function of MUC1 in the early pregnant and non-pregnant uterine tissues of sheep with natural oestrous or artificially-induced oestrous on days 10, 12, 14, 16 and 18 of the cycle by in situ hybridization, quantitative real-time polymerase chain reaction, immunohistochemical staining and western blotting methods. According to our results, MUC1 mRNA and protein expression increased initially but then decreased from days 10-18, peaking on day 14 in the uterine tissues of non-pregnant ewes after both natural and artificially-induced oestrous. MUC1 protein localisation was observed in the LE on days 10, 12 and 14 and in the GE on days 16 and 18. In contrast, MUC1 mRNA and protein expression increased on days 10 and 12, decreased on day 14, but increased again on days 16 and 18 in the uterine tissues of pregnant ewes both in natural oestrous and in artificially-induced oestrous. Additionally, the MUC1 mRNA and protein expression levels in the uterine tissues of the early pregnant sheep were significantly lower than those in the non-pregnant sheep on days 10, 14, and 18, except on day 16(P 0.01). Enhancing MUC1 protein expression with oestrogen or/and progesterone decreased the blastocyst adhesion rate when blastocysts were co-cultured with endometrial epithelial cells (EECs), while inhibiting MUC1 protein expression with IFN-τ increased the blastocyst adhesion rate when the blastocysts were co-cultured with EECs. Compared with the ewes undergoing natural oestrus, the expression trend and regulation of MUC1 did not change, and the MUC1 expression levels only increased under artificial oestrus conditions. Our data provide important information for improving the conception rate in sheep undergoing artificially-induced oestrus and offer some reference points relating to embryo transfer, oestrus synchronisation and superovulation.

Embryonic cryopreservation has a relatively low survival rate because of cytoskeletal damage. However, molecular anti-freezing mechanisms have been largely unexplored. This study investigated the significance of RhoA, involved in embryonic development, and the Rho/RhoA-associated kinase (ROCK) signalling pathway in cryopreservation. The anti-freezing mechanism in murine dormant embryos, compared with normal blastocysts, was assessed by combining molecular, physiological and pharmacological approaches. Real-time PCR and western blotting experiments showed high RhoA expression in cryo-dormant and dormant embryos. RhoA GTPases were overexpressed on the surface of trophectoderm cells in dormant embryos. Treatment with Y-27632, a ROCK antagonist, decreased survival of both normal and dormant blastocysts, while recombinant RhoA protein remarkably increased survival, after freeze–thawing, of normal hatched blastocysts. Our findings elucidated the molecular mechanism of anti-freezing, involving RhoA phosphorylation, meditated by the Rho/ROCK signalling pathway, in hatched and diapaused murine blastocysts. In addition, evidence for a potentially protective additive suggests a new method for improving the anti-freezing potential of mammalian embryos, without protecting the zona pellucida.

Somatic cell nuclear transfer (SCNT) is frequently used to produce transgenic cloned livestock, but it is still associated with low success rates. To our knowledge, we are the first to report successful production of transgenic cattle that overexpress bovine adipocyte-type fatty acid binding proteins (A-FABPs) with the aid of SCNT. Intragenomic integration of additional A-FABP gene copies has been found to be positively correlated with the intramuscular fat content in different farm livestock species. First, we optimized the cloning parameters to produce bovine embryos integrated with A-FABP by SCNT, such as applied voltage field strength and pulse duration for electrofusion, morphology and size of donor cells, and number of donor cells passages. Then, bovine fibroblast cells from Qinchuan cattle were transfected with A-FABP and used as donor cells for SCNT. Hybrids of Simmental and Luxi local cattle were selected as the recipient females for A-FABP transgenic SCNT-derived embryos. The results showed that a field strength of 2.5 kV/cm with two 10-μs duration electrical pulses was ideal for electrofusion, and 4–6th generation circular smooth type donor cells with diameters of 15–25 μm were optimal for producing transgenic bovine embryos by SCNT, and resulted in higher fusion (80%), cleavage (73%), and blastocyst (27%) rates. In addition, we obtained two transgenic cloned calves that expressed additional bovine A-FABP gene copies, as detected by PCR-amplified cDNA sequencing. We proposed a set of optimal protocols to produce transgenic SCNT-derived cattle with intragenomic integration of ectopic A-FABP-inherited exon sequences.

Myostatin, a secreted growth factor highly expressed in skeletal muscle, negatively regulates skeletal muscle growth and differentiation. Recently, myostatin is emerged as a potential target for anti-atrophy and anti-fibrotic therapies. Therefore, to investigate the regulation of myostatin in sheep adult fibroblasts, we used the RNA interference mediated by lentiviral vector to gene silence myostatin. Simultaneously, we also had constructed the sheep myostatin overexpression vector to further explore the function of myostatin in fibroblasts. The results here demonstrated that the lentiviral vector could significantly reduce myostatin gene both at mRNA and protein level by 71% and 67%, respectively (P

Objective—To determine whether a mutation in the fibrillin 2 gene (FBN2) is associated with canine hip dysplasia (CHD) and osteoarthritis in dogs. Animals—-1,551 dogs. Procedures—Hip conformation was measured radiographically. The FBN2 was sequenced from genomic DNA of 21 Labrador Retrievers and 2 Greyhounds, and a haplotype in intron 30 of FBN2 was sequenced in 90 additional Labrador Retrievers and 143 dogs of 6 other breeds. Steady-state values of FBN2 mRNA and control genes were measured in hip joint tissues of fourteen 8-month-old Labrador Retriever–Greyhound crossbreeds. Results—The Labrador Retrievers homozygous for a 10-bp deletion haplotype in intron 30 of FBN2 had significantly worse CHD as measured via higher distraction index and extended-hip joint radiograph score and a lower Norberg angle and dorsolateral subluxation score. Among 143 dogs of 6 other breeds, those homozygous for the same deletion haplotype also had significantly worse radiographic CHD. Among the 14 crossbred dogs, as the dorsolateral subluxation score decreased, the capsular FBN2 mRNA increased significantly. Those dogs with incipient hip joint osteoarthritis had significantly increased capsular FBN2 mRNA, compared with those dogs without osteoarthritis. Dogs homozygous for the FBN2 deletion haplotype had significantly less FBN2 mRNA in their femoral head articular cartilage. Conclusions and Clinical Relevance—The FBN2 deletion haplotype was associated with CHD. Capsular gene expression of FBN2 was confounded by incipient secondary osteoarthritis in dysplastic hip joints. Genes influencing complex traits in dogs can be identified by genome-wide screening, fine mapping, and candidate gene screening.

In this study, the sheep CIB4 cDNA was cloned from the small tail Han sheep by RT-PCR and RACE (rapid amplification of cDNA ends), and CIB4 cDNA and amino acid sequence were analyzed. Our results showed that the sheep CIB4 gene expressed two alternatively spliced variants L-CIB4 (long CIB4) and S-CIB4 (short CIB4). Sequence analysis indicated that the sheep CIB4 cDNA cloned (L-CIB4) was 745-bp in length (GenBank accession number: FJ039532) with 185 amino acids residues. The sheep CIB4 cDNA showed more than 72% of sequence identity, at the nucleotide level, to its equivalents in cattle, horse, chimpanzee, humans, mice and rats, while at the deduced protein level, the value increased to 79.6%. Semi-quantitative RT-PCR using total RNA from different tissues showed that CIB4 has a strong tissue-specific expression pattern in sheep. L-CIB4 expression level was shown to be no different in small tail Han sheep and the Dorset ram, but both were significantly different from the Texel (P0.05). Surprisingly, the short spliced form, S-CIB4, could only be detected in small tail Han sheep, suggesting that CIB4 may be linked in some way to the high fecundity of this breed.

The molecular mechanism regulated by microRNAs (miRNAs) that underlies postnatal hypertrophy of skeletal muscle is complex and remains unclear. Here, the miRNAomes of longissimus dorsi muscle collected at five postnatal stages (60, 120, 150, 180, and 210 days after birth) from Large White (commercial breed) and Min pigs (indigenous breed of China) were analyzed by Illumina sequencing. We identified 734 miRNAs comprising 308 annotated miRNAs and 426 novel miRNAs, of which 307 could be considered pig-specific. Comparative analysis between two breeds suggested that 60 and 120 days after birth were important stages for skeletal muscle hypertrophy and intramuscular fat accumulation. A total of 263 miRNAs were significantly differentially expressed between two breeds at one or more developmental stages. In addition, the differentially expressed miRNAs between every two adjacent developmental stages in each breed were determined. Notably, ssc-miR-204 was significantly more highly expressed in Min pig skeletal muscle at all postnatal stages compared with its expression in Large White pig skeletal muscle. Based on gene ontology and KEGG pathway analyses of its predicted target genes, we concluded that ssc-miR-204 may exert an impact on postnatal hypertrophy of skeletal muscle by regulating myoblast proliferation. The results of this study will help in elucidating the mechanism underlying postnatal hypertrophy of skeletal muscle modulated by miRNAs, which could provide valuable information for improvement of pork quality and human myopathy.

Epidermal growth factor (EGF) has been shown to facilitate the in vitro maturation of sheep oocytes, and enhance embryo’s capability for further development. However, such kind of molecular mechanism has not yet been elucidated. In the present study, we investigated the effect of EGF-mediated mitogen-activated protein kinases 3 and 1 (MAPK3/1) pathway on in vitro maturation of sheep oocytes. U0126, a specific inhibitor of MEK (MAPK kinase), was added into the maturation culture medium to block the EGF-mediated MAPK3/1 pathway with different doses. Then, the nuclear maturation of sheep oocytes was examined. Additionally, the effect of EGF-mediated MAPK3/1 on cytoplasmic maturation was examined though in vitro fertilization and embryonic development. The rate of germinal vesicle breakdown (GVBD) after 6 h of culture with 10−4 mol/l of U0126 (50.4%) was significantly decreased compared with control (67.2%, p < 0.05), and the first polation body (PB1) extrusion rate after 22 h of culture in drug treatment was also significantly inhibited compared with control (28.6% vs. 48.4%, p < 0.05). However, 10−6 mol/l U0126 had slight effect on oocyte nuclear maturation. The normal distribution rate of α-tubulin in the oocytes after 22 h of in vitro maturation was significantly decreased in the 10−4 mol/l U0126 group (54%) compared with control (68%, p < 0.05). After in vitro fertilization, the cleavage rate in drug treatments (56.8% in 10−6 mol/l U0126 group and 42.6% in 10−4 mol/l U0126 group) was significantly decreased compared with control (72.3%, p < 0.01). The blastocyst rate in 10−4 mol/l U0126 group (17.6%) was also significantly decreased compared with control (29.9%, p < 0.05). Collectively, these results suggest that EGF-mediated MAPK3/1 pathway is conducive to in vitro maturation of sheep oocytes.

The deposition of intramuscular fat is an important factor affecting the beef quality, such as flavour and palatability. In this study, for further identifying the differential molecular mechanisms regulating the deposition of fat between intramuscular and external adipose tissues, particularly subcutaneous and perirenal adipose tissues, it was designed to obtain transcript sequence data and compare the transcriptomes among intramuscular, subcutaneous, and perirenal adipose tissues by RNA-Seq. A total of 66,206,912, 55,114,070 and 67,320,426 fragments were sequenced for the intramuscular (IAT), subcutaneous (SAT), and perirenal adipose tissue (PAT) respectively. Among them, total 953, 1,534, 2,026 genes showing differential expression between IAT and SAT, IAT and PAT, SAT and PAT, were identified respectively (FDR

The discovery and identification of Ovis aries (sheep) miRNAs will further promote the study of miRNA functions and gene regulatory mechanisms. To explore the microRNAome (miRNAome) of sheep in depth, samples were collected that included eight developmental stages: the longissimus dorsi muscles of Texel fetuses at 70, 85, 100, 120, and 135 days, and the longissimus dorsi muscles of Ujumqin fetuses at 70, 85, 100, 120, and 135 d, and lambs at 0 (birth), 35, and 70 d. These samples covered all of the representative periods of Ovis aries growth and development throughout gestation (about 150 d) and 70 d after birth. Texel and Ujumqin libraries were separately subjected to Solexa deep sequencing; 35,700,772 raw reads were obtained overall. We used ACGT101-miR v4.2 to analyze the sequence data. Following meticulous comparisons with mammalian mature miRNAs, precursor hairpins (pre-miRNAs), and the latest sheep genome, we substantially extended the Ovis aries miRNAome. The list of pre-miRNAs was extended to 2,319, expressing 2,914 mature miRNAs. Among those, 1,879 were genome mapped to unique miRNAs, representing 2,436 genome locations, and 1,754 pre-miRNAs were mapped to chromosomes. Furthermore, the Ovis aries miRNAome was processed using an elaborate bioinformatic analysis that examined multiple end sequence variation in miRNAs, precursors, chromosomal localizations, species-specific expressions, and conservative properties. Taken together, this study provides the most comprehensive and accurate exploration of the sheep miRNAome, and draws conclusions about numerous characteristics of Ovis aries miRNAs, including miRNAs and isomiRs.

BackgroundCanine hip dysplasia (HD) is a common polygenic trait characterized by hip malformation that results in osteoarthritis (OA). The condition in dogs is very similar to developmental dysplasia of the human hip which also leads to OA.Methodology/principal findingsA total of 721 dogs, including both an association and linkage population, were genotyped. The association population included 8 pure breeds (Labrador retriever, Greyhounds, German Shepherd, Newfoundland, Golden retriever, Rottweiler, Border Collie and Bernese Mountain Dog). The linkage population included Labrador retrievers, Greyhounds, and their crosses. Of these, 366 dogs were genotyped at ∼22,000 single nucleotide polymorphism (SNP) loci and a targeted screen across 8 chromosomes with ∼3,300 SNPs was performed on 551 dogs (196 dogs were common to both sets). A mixed linear model approach was used to perform an association study on this combined association and linkage population. The study identified 4 susceptibility SNPs associated with HD and 2 SNPs associated with hip OA.Conclusion/significanceThe identified SNPs included those near known genes (PTPRD, PARD3B, and COL15A1) reported to be associated with, or expressed in, OA in humans. This suggested that the canine model could provide a unique opportunity to identify genes underlying natural HD and hip OA, which are common and debilitating conditions in both dogs and humans.

Ovis aries is one of the most important agricultural livestock for meat production, and also is an ideal model organism for biological and comparative genomics studies. Many miRNAs have been reported for their important roles in developmental processes in various animals, but there is limited information about O. aries miRNAs. In this study, combining a computational method based on expressed sequence tag (EST) analysis with experimental identification based on small RNA cDNA library, we identified 31 miRNAs belong to 24 families in sheep, 2 of which were novel miRNAs which had never been previously identified in any species. Especially, we cloned 12 miRNAs from the sheep skeletal muscle, which were good candidate miRNAs to be studied about the miRNA-dependant regulated process of muscle development, and we identified four pairs of miRNA/miRNA* and one pair of miRNA-3p/miRNA-5p from sheep EST sequences. Expression analysis indicated that some miRNAs were expressed in a specific tissue, and the pair of miRNA-3p/miRNA-5p and one pair of miRNA/miRNA* had a similar relative expression pattern in some tissues, respectively. Further, we predicted 120 potential target genes of 31 oar-miRNAs on the 3′UTR of O. aries genes. Gene ontology analysis showed that most of these genes took part in the cellular process and metabolic process. Our results enriched the O. aries miRNA database and provided useful information for investigating biological functions of miRNAs and miRNA* in sheep.