ALAS1 associated with goat kidding number trait was regulated by the transcription factor ASCL2 to affect granulosa cell proliferation.

ALAS1 is a member of the α-oxoamine synthase family, which is the first rate-limiting enzyme for heme synthesis and is important for maintaining intracellular heme levels. In the ovary, ALAS1 is associated with the regulation of ovulation-related mitochondrial P450 cytochromes, steroid metabolism, and steroid hormone production. However, there are few studies on the relationship between ALAS1 and reproductive traits in goats. In this study, a mutation located in the promoter region of ALAS1 (g.48791372C>A) was found to be significantly (p < 0.05) associated with the kidding number of Yunshang black goats. Specifically, the mean kidding number in the first three litters and the kidding numbers of all three litters were significantly (p < 0.05) higher in individuals with the CA genotype or AA genotype than in those with the CC genotype. To further investigate the regulatory mechanism of ALAS1, the expression of ALAS1 in goat ovarian tissues with different genotypes was verified by real-time quantitative PCR. The results showed that the expression of ALAS1 was significantly higher in the ovaries of individuals with AA genotype than those with AC and CC genotypes (p < 0.01), and the expression trend of transcription factor ASCL2 was consistent with ALAS1. Additionally, the ALAS1 g.48791372C>A mutation created a new binding site for the transcription factor ASCL2. The luciferase activity assay indicated that the mutation increased the promoter activity of ALAS1. Overexpression of the transcription factor ASCL2 induced increased expression of ALAS1 in goat granulosa cells (p < 0.05). The opposite trend was shown for the inhibition of ASCL2 expression. The results of real-time quantitative PCR, EdU and Cell Counting Kit-8 assays indicated that the transcription factor ASCL2 increased the proliferation of goat granulosa cells by mediating the expression of ALAS1. In conclusion, the transcription factor ASCL2 positively regulated the transcriptional activity and expression levels of ALAS1, altering granulosa cell proliferation and the kidding number in goats.

Comparative analysis of the genetic diversity of the neutral microsatellite loci and second exon of the goat MHC-DQB1 gene.

This study compared and analyzed the genetic diversity and population structure of exon 2 of the DQB1 gene and 13 autosomal neutral microsatellite markers from 14 Chinese goat breeds to explore the potential evolutionary mechanism of the major histocompatibility complex (MHC). A total of 287 haplotypes were constructed from MHC-DQB1 exon 2 from 14 populations, and 82 nucleotide polymorphic sites (SNPs, 31.78%) and 172 heterozygous individuals (79.12%) were identified. The FST values of the microsatellites and MHC-DQB ranged between 0.01831-0.26907 and 0.00892-0.38871, respectively. Furthermore, 14 goat populations showed rich genetic diversity in the microsatellite loci and MHC-DQB1 exon 2. However, the population structure and phylogenetic relationship represented by the two markers were different. Positive selection and Tajima's D test results showed the occurrence of a diversified selection mechanism, which was primarily based on a positive and balancing selection in goat DQB. This study also found that the DQB sequences of bovines exhibited trans-species polymorphism (TSP) among species and families. In brief, this study indicated that positive and balancing selection played a major role in maintaining the genetic diversity of DQB, and TSP of MHC in bovines was common, which enhanced the understanding of the MHC evolution.

Identification of WNT4 alternative splicing patterns and effects on proliferation of granulosa cells in goat.

Increasing ovulation numbers is one of the most important ways to promote reproduction in mammals, and follicular granulosa cells (GCs) provide the necessary nutrients and microenvironment for oocytes to ovulate. WNT4 has been shown to be a key factor in regulating the proliferation of GCs in mammalian ovarian tissues. Our previous transcriptome sequencing (RNA-seq) results have identified two alternatively spliced products of WNT4;however, little is known about the splicing mechanism and its effect on GC proliferation. In this study, two alternatively spliced products of WNT4, designated WNT4-α and WNT4-ß, were identified by cloning and analyzed for their function by bioinformatics. The RT-qPCR and Western blot results showed that the expression of WNT4-α was significantly higher than that of WNT4-ß in the ovary tissues and GCs of Yunshang black goats. We therefore hypothesized that WNT4-α was the main isoform affecting the proliferation of goat GCs. Subsequently, goat GC proliferation assays showed that overexpression of WNT4-α significantly promoted GC proliferation, and the opposite was true after WNT4-α inhibition. The expression of marker genes of the Wnt signaling pathway was also examined and WNT4-α was found to affect the proliferation and hormone secretion of goat GCs by regulating the Wnt signaling pathway. In addition, a series of splicing factors were involved in in the alternative splicing; in this study, SRSF6 was found to be involved as a splicing factor in the generation of WNT4 alternative splicing. In summary, WNT4 alternative splicing was mediated by the splicing factor SRSF6, and WNT4-α alternative splicing played an important role in follicle development and had a significant effect on the proliferation of goat GCs. The results of this study provide a theoretical foundation for further understanding the molecular regulatory mechanisms of the WNT4 in follicle development in goats.

Oviduct Transcriptomic Reveals the Regulation of mRNAs and lncRNAs Related to Goat Prolificacy in the Luteal Phase.

The oviduct is associated with embryo development and transportation and regulates the pregnancy success of mammals. Previous studies have indicated a molecular mechanism of lncRNAs in gene regulation and reproduction. However, little is known about the function of lncRNAs in the oviduct in modulating goat kidding numbers. Therefore, we combined RNA sequencing (RNA-seq) to map the expression profiles of the oviduct at the luteal phase from high- and low-fecundity goats. The results showed that 2023 differentially expressed mRNAs (DEGs) and 377 differentially expressed lncRNAs (DELs) transcripts were screened, and 2109 regulated lncRNA-mRNA pairs were identified. Subsequently, the genes related to reproduction (IGF1, FGFRL1, and CREB1) and those associated with embryonic development and maturation (DHX34, LHX6) were identified. KEGG analysis of the DEGs revealed that the GnRH- and prolactin-signaling pathways, progesterone-mediated oocyte maturation, and oocyte meiosis were related to reproduction. GSEA and KEGG analyses of the target genes of DELs demonstrated that several biological processes and pathways might interact with oviduct functions and the prolificacy of goats. Furthermore, the co-expression network analysis showed that XLOC_029185, XLOC_040647, and XLOC_090025 were the cis-regulatory elements of the DEGs MUC1, PPP1R9A, and ALDOB, respectively; these factors might be associated with the success of pregnancy and glucolipid metabolism. In addition, the GATA4, LAMA2, SLC39A5, and S100G were trans-regulated by lncRNAs, predominantly mediating oviductal transport to the embryo and energy metabolism. Our findings could pave the way for a better understanding of the roles of mRNAs and lncRNAs in fecundity-related oviduct function in goats.

Characterization of circular RNA profiles of oviduct reveal the potential mechanism in prolificacy trait of goat in the estrus cycle.

The mammalian oviduct is functionally highly diverse during the estrus cycle. It provides a suitable milieu for oocyte maturation, sperm capacitation, fertilization, early embryo development and transportation. While there have been many studies of molecular mechanisms on the kidding number of goats, a systematic analysis by which the underlying circular RNAs (circRNAs) changes in the oviduct related to prolificacy traits is lacking. Herein, we present a comprehensive circRNA atlas of the oviduct among high- and low-fecundity goats in the follicular phase (FH vs. FL), luteal phase (LH vs. LL), and estrus cycle (FH vs. LH; FL vs. LL) to unravel their potential regulatory mechanisms in improving kidding number. We generated RNA sequencing data, and identified 4,078 circRNAs from twenty sampled Yunshang black goats. Many of these circRNAs are exon-derived and differentially expressed between each comparison group. Subsequently, eight differentially expressed (DE) circRNAs were validated by RT‒qPCR, which was consistent with the RNA-seq data. GO and KEGG enrichment analyses suggested that numerous host genes of DE circRNAs were involved in the hormone secretion, gamete production, fertilization, and embryo development processes. The competing endogenous RNA (ceRNA) interaction network analysis revealed that 2,673 circRNA-miRNA-mRNA axes (including 15 DE circRNAs, 14 miRNAs, and 1,699 mRNAs) were formed, and several target genes derived from the ceRNA network were associated with oviduct functions and reproduction, including SMAD1, BMPR1B, IGF1, REV1, and BMP2K. Furthermore, miR-15a-5p, miR-181b-5p, miR-23b-5p, miR-204-3p, and miR-145-5p might play important roles in reproduction. Finally, a novel circRNA, circIQCG, was identified as potentially involved in embryo development. Overall, our study provides a resource of circRNAs to understand the oviductal function and its connection to prolificacy trait of goats in the differentiation estrus cycle.

Moraxella nasovis sp. nov., isolated from a sheep with respiratory disease.

A novel Gram-stain-negative, aerobic, coccus-shaped bacteria, designated ZY201115T, was isolated from the nasal cavity of a sheep with respiratory disease in Yunnan Province, south-west China, and its taxonomic affiliation was studied by applying a polyphasic approach. The strain grew at 18-41 °C (optimum, 37 °C), at pH 6.0-9.0 (optimum, pH 8.0) and in 0.5-3.0% (w/v) NaCl (optimum, 1.0 % NaCl). Phylogenetic analysis based on 16S rRNA gene sequences showed that the strain is affiliated to the genus Moraxella with highest similarity to Moraxella bovis ATCC 10900T (96.6 %). Phylogenomic analysis based on 811 single-copy genes also indicated that the strain represents a novel species in the genus Moraxella and formed a deep and separated clade with Moraxella caviae NCTC 10293T. The highest genomic orthologous average nucleotide identity and digital DNA-DNA hybridization values between the strain and the type strains in the genus Moraxella were 73.7% (M. caviae NCTC 10293T) and 25.3% (Moraxella osloensis CCUG 350T), respectively. The G+C content of the complete genome sequence was 42.1 mol%. The predominant fatty acids (>5 %) were C18:1 ω9c, C17:1 ω8c, C12:03OH and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The major polar lipids were phosphatidylglycerol, cardiolipin, monolysocardiolipin, phosphatidylethanolamine and hemibismonoacylglycerophosphate. The major respiratory quinone was CoQ-8. On the basis of the results of phylogenetic, phenotypic and chemotaxonomic characterizations, strain ZY201115T clearly represents a novel species of the genus Moraxella, for which the name Moraxella nasovis sp. nov. is proposed. The type strain is ZY201115T (=CCTCC AB 2021473T=CCUG 75922T).

Effect of dietary peNDF levels on digestibility and rumen fermentation, and microbial community in growing goats.

Physically effective neutral detergent fiber (peNDF) is a concept that accounts for the particle length of NDF in diets, sustaining the normal chewing behavior and rumen fermentation of ruminants. Specifically, peNDF>1.18 is the commonest one that is calculated from NDF and the percentage of feed dry matter left on the 1.18, 8.00, and 19.00 mm sieves. This study aimed to investigate the effects of different levels of peNDF>1.18 on the rumen microbiome and its correlation with nutrient digestibility and rumen fermentation in goats. A total of 30 Lezhi black goats were randomized and blocked to five dietary treatments (n = 6). All the diets were identical in composition but varied in hay lengths, leading to the different peNDF>1.18 content of the diets: 32.97, 29.93, 28.14, 26.48, and 24.75%. The results revealed that the nutrient digestibility increased when dietary peNDF>1.18 levels decreased from 32.97% to 28.14%, with the highest digestibility at 28.14% peNDF>1.18 treatment, after which nutrient digestibility decreased with the decreasing of dietary peNDF levels. Ruminal NH3-N concentrations in the 29.93% and 28.14% groups were higher than that in the 24.75% group (p < 0.05). Ruminal microbial protein concentration was the highest in the 32.97% group (p < 0.05). Daily CH4 production in the 32.97% and 24.75% peNDF>1.18 treatments was lower than that in the 26.48% group (p < 0.05) and no differences were observed among other groups. The relative abundance of rumen fungi at the phylum and genus levels and archaea at the species were affected by dietary peNDF>1.18 content. In conclusion, decreasing dietary peNDF>1.18 levels within a certain range can improve nutrient digestibility and change the rumen microbial community structure of goats. Dietary peNDF>1.18 level should be 28.14% (roughage length around 1 cm) among the five levels for 4 months Lezhi black goats with the purpose of optimal nutrient digestibility.

Effect of Upregulation of Transcription Factor TFDP1 Binding Promoter Activity Due to RBP4 g.36491960G>C Mutation on the Proliferation of Goat Granulosa Cells

Retinol-binding protein 4 (RBP4), a member of the lipocalin family, is a specific carrier of retinol (vitamin A) in the blood. Numerous studies have shown that RBP4 plays an important role in mammalian embryonic development and that mutations in RBP4 can be used for the marker-assisted selection of animal reproductive traits. However, there are few studies on the regulation of reproduction and high-prolificacy traits by RBP4 in goats. In this study, the 5' flanking sequence of RBP4 was amplified, and a G>C polymorphism in the promoter region -211 bp (g.36491960) was detected. An association analysis revealed that the respective first, second and third kidding number and mean kidding number of nanny goats with CC and GC genotypes (2.167 ± 0.085, 2.341 ± 0.104, 2.529 ± 0.107 and 2.189 ± 0.070 for CC and 2.052 ± 0.047, 2.206 ± 0.057, 2.341 ± 0.056 and 2.160 ± 0.039 for GC) were significantly higher (p < 0.05) than those with the GG genotype (1.893 ± 0.051, 2.027 ± 0.064, 2.107 ± 0.061 and 1.74 ± 0.05). The luciferase assay showed that luciferase activity was increased in C allele individuals compared with that in G allele individuals. A competitive electrophoretic mobility shift assay (EMSA) showed that individuals with the CC genotype had a stronger promoter region binding capacity than those with the GG genotype. In addition, transcription factor prediction software showed that the RBP4 g.36491960G>C mutation added a novel binding site for transcription factor DP-1 (TFDP1). RT−qPCR results showed that the expression of TFDP1 was significantly higher in the high-prolificacy group than in the low-prolificacy group, and the expression of RBP4 was higher in both the CC and GC genotypes than that in the GG genotype. TFDP1 overexpression significantly increased the expression of RBP4 mRNA (p < 0.05) and the expression of the cell proliferation factors cyclin-D1, cyclin-D2 and CDK4 (p < 0.05). The opposite trend was observed after interference with TFDP1. Both the EdU and CCK-8 results showed that TFDP1 expression could regulate the proliferation of goat ovarian granulosa cells. In summary, our results showed that RBP4 g.36491960G>C was significantly associated with fecundity traits in goats. The g.36491960G>C mutation enhanced the transcriptional activity of RBP4 and increased the expression of RBP4, thus improving the fertility of Yunshang black goats.

Transcriptome Analysis Reveals Key miRNA-mRNA Pathways in Ovarian Tissues of Yunshang Black Goats With Different Kidding Numbers.

The granulosa cell (GC) is the basic functional unit of follicles, and it is important for promoting follicle growth and sex hormones, as well as growth factor secretion in the process of reproduction. A variety of factors influence granulocyte proliferation, yet there are still many gaps to be filled in target and non-coding RNA regulation. In our study, the differentially expressed (DE) mRNAs and miRNAs were detected by using RNA-seq, and we constructed a mRNA-miRNA network related to goat prolificacy. Then, the goat primary GCs were isolated from the follicle for the function validation of candidate genes and their regulator miRNAs. A total of 2,968 DE mRNAs and 99 DE miRNAs were identified in the high- and low-prolificacy goat by RNA-seq, of which there were 1,553 upregulated and 1,415 downregulated mRNAs, and 80 upregulated and 19 downregulated miRNAs, respectively. JAK3 was identified as highly expressed in the low-prolificacy goats (3 times higher than high-prolificacy goats), and the integrated analysis showed that chi-miR-493-3p was a potential regulator of JAK3. The analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that JAK3 was involved in the PI3K-Akt signaling pathway, the Jak-STAT signaling pathway, and signaling pathways regulating pluripotency of stem cells. In particular, the PI3K-Akt signaling pathway was a typical pathway for cell proliferation, differentiation, apoptosis, and migration. We found that the chi-miR-493-3p targets JAK3 directly via RT-qPCR, dual fluorescence assays, and Western blot. Furthermore, the expression of JAK3 was significantly decreased by the chi-miR-493-3p mimic and increased by the chi-miR-493-3p inhibitor. The CCK-8 assay showed that overexpression of JAK3 promoted cell proliferation, while inhibiting JAK3 had the opposite effect. The expression of cell proliferation markers CDK4 and cyclin D2 also showed the same results. Moreover, the enzyme-linked immunosorbent assay showed that steroid hormones E2 and PROG were increased by overexpressing JAK3 and decreased by inhibiting JAK3. Therefore, our results identified a chi-miR-439-3p-JAK3 regulatory pathway, which provided a new insight into the GC proliferation and prolificacy of goat.

Integrated Analysis of mRNAs and Long Non-Coding RNAs Expression of Oviduct That Provides Novel Insights into the Prolificacy Mechanism of Goat (Capra hircus).

Artificial directional selection has replaced natural selection and resulted in trait differences across breeds in domestic animal breeding. However, the molecular mechanism by which the oviduct regulates litter size remains largely elusive in goats during the follicular phase. Accumulating data have linked lncRNAs to reproductive activities; however, little is known about the modulation mechanism in the oviduct. Herein, RNA-seq was used to measure mRNA and lncRNA expression levels in low- and high-fecundity goats. We observed distinctive differences in mRNA and lncRNA in terms of different kidding numbers and detected the differential expression of 1640 mRNA transcripts and 271 lncRNA transcripts. Enrichment analysis of differentially expressed mRNAs (DEGs) suggested that multiple pathways, such as the AMPK, PI3K-Akt, calcium signaling pathway, oocyte meiosis, ABC transporter, and ECM-receptor interaction pathways, directly or indirectly affected goat reproduction. Additionally, coexpression of differentially expressed lncRNAs (DEL)-genes analysis showed that XLOC_021615, XLOC_119780, and XLOC_076450 were trans-acting as the DEGs ATAD2, DEPDC5, and TRPM6, respectively, and could regulate embryo development. Moreover, XLOC_020079, XLOC_107361, XLOC_169844, XLOC_252348 were the trans-regulated elements of the DEGs ARHGEF2 and RAPGEF6, and the target DEGs CPEB3 of XLOC_089239, XLOC_090063, XLOC_107409, XLOC_153574, XLOC_211271, XLOC_251687 were associated with prolificacy. Collectively, our study has offered a thorough dissection of the oviduct lncRNA and mRNA landscapes in goats. These results could serve as potential targets of the oviduct affecting fertility in goats.

A Novel SNP in the Promoter Region of IGF1 Associated With Yunshang Black Goat Kidding Number via Promoting Transcription Activity by SP1.

IGF1, a member of the insulin-like growth factor (IGF) superfamily, is also known as the growth-promoting factor (somatomedin C). IGF1 is involved in vertebrate growth and development, immunity, cell metabolism, reproduction, and breeding. However, there are relatively few studies on the relationship between IGF1 and goat reproduction. In this study, a new transcription factor SP1 bound to the IGF1 g. 64943050T>C promoted granulosa cell (GC) proliferation. A mutation g.64943050T>C located in the promoter region of IGF1 was identified. Association analysis revealed that the kidding number in the first and second litters and the average number of first three litters of the CC genotype (2.206 ± 0.044, 2.254 ± 0.056, and 2.251 ± 0.031) were significantly higher than those in the TC genotype (1.832 ± 0.049, 1.982 ± 0.06, and 1.921 ± 0.034) and TT genotype (1.860 ± 0.090, 1.968 ± 0.117, and 1.924 ± 0.062) (p < 0.05). The kidding number in the third litter of the CC genotype (2.355 ± 0.057) was significantly higher than that in the TT genotype (2.000 ± 0.107) (p < 0.05). Then, the function of this mutation was validated by the dual-luciferase reporter assay and EMSA. The results showed that the luciferase activity of IGF1-mutant-C was significantly higher than that of IGF1-Wild-T (p < 0.05). The EMSA also showed that the binding ability of IGF1-mutant-C was higher than that of IGF1-Wild-T (p < 0.05). Subsequently, the transcription factor SP1 was predicted to bind to the mutation of IGF1 (g.64943050T>C). Overexpression of SP1 promotes the expression of IGF1 in the primary granulosa cells (GCs). The results of the CCK-8 assay and the expression of GC proliferation factors (CDK4, cyclin D1, and cyclin D2) demonstrated that SP1 promoted GC proliferation by regulating IGF1 expression. Our results suggested that the IGF1 g.64943050T>C was significantly associated with the kidding number of Yunshang black goats, and SP1 as a transcription factor of IGF1 binding to the mutation T>C regulated the expression of IGF1. Furthermore, SP1 promoted goat GC proliferation by regulating the expression of IGF1, which provides a new insight for the goat fertility trait.

Differentially Expressed Circular RNA Profile Signatures Identified in Prolificacy Trait of Yunshang Black Goat Ovary at Estrus Cycle.

CircRNAs acting as miRNA sponges play important roles in the growth process of animal individuals. The prolificacy trait of goats is involved in many pathways, however, the variation of circRNA expression profiles in the different phases of the estrus cycle at high and low fecundity groups is still unknown. Here, we analyzed the circRNA profiles of ovarian tissues among high and low fecundity groups in the follicular phase (HF vs LF), high and low fecundity groups in the luteal phase (HL vs LL), and high and low fecundity in the whole estrus cycle (HF vs HL and LF vs LL) using RNA-seq. A total of 283 (114 upregulated and 169 downregulated), 559 (299 upregulated and 260 downregulated), 449 (254 upregulated and 195 downregulated), and 314 (210 upregulated and 104 downregulated) differentially expressed (DE) circRNAs were screened in HF vs LF, HF vs HL, HL vs LL, and LF vs LL groups, respectively. Enrichment analysis suggested that the targeting of DE circRNAs was mainly enriched in oocyte meiosis, the GnRH signaling pathway, and estrogen signaling pathway. After integrating our previous study of miRNA-seq, there were 56 miRNAs that could target to 192 DE circRNAs, including the miR-133 family (including miR-133a-3p and miR-133b), miR-129-3p, and miR-21, which also had important influence on the prolificacy trait of goats. Then, 18 circRNAs with coding potential were obtained by four software predictions, and 9 circRNAs were validated by RT-qPCR. Together, circRNAs play a key role in the prolificacy trait and the transformation of the follicular phase to the luteal phase in the estrus cycle of goats.

Genetic and Pathogenic Characterisation of a Virulent Akabane Virus Isolated from Goats in Yunnan, China.

Introduction: Akabane virus (AKAV) has been detected in a variety of host species in China, but there are only limited records of its occurrence in goats. However, more attention needs to be paid to understanding the diversity of viruses in this species. The aim of the study was to explore the genotype characteristics and variation trend of AKAV and their relationship with virulence in Yunnan, China. Material and Methods: Blood samples were collected from goats during routine surveillance of goat diseases in Yunnan province in 2019. The AKAV CX-01 strain was isolated using BHK-21 cells. To understand pathogenicity, the virus was intraperitoneally (IP) and intracerebrally (IC) inoculated into suckling mice and tissue samples were subsequently analysed histopathologically and immunohistochemically. Results: Akabane virus CX-01 strain induced encephalitis and impairment of the central nervous system with fatal consequences. Phylogenetic analysis based on the ORF sequences of the small segments indicated that the AKAV isolate used was most closely related to the GD18134/2018 Chinese midge and bovine NM BS/1strains, while phylogenetic analysis based on the medium segments showed a close relationship between CX-01 and the Chinese GLXCH01 strain. Conclusion: The CX-01 isolate was related to AKAV genogroup Ia and probably originated from a recombination of different strains.

Astaxanthin Supplementation Improves the Subsequent Developmental Competence of Vitrified Porcine Zygotes.

Cryopreservation of embryos has been confirmed to cause oxidative stress as a factor responsible for impaired developmental competence. Currently, astaxanthin (Ax) raises considerable interest as a strong exogenous antioxidant and for its potential in reproductive biology. The present study aimed to investigate the beneficial effects of Ax supplementation during in vitro culture of vitrified porcine zygotes and the possible underlying mechanisms. First, the parthenogenetic zygotes were submitted to vitrification and then cultured in the medium added with various concentrations of Ax (0, 0.5, 1.5, and 2.5 µM). Supplementation of 1.5 µM Ax achieved the highest blastocyst yield and was considered as the optimal concentration. This concentration also improved the blastocyst formation rate of vitrified cloned zygotes. Moreover, the vitrified parthenogenetic zygotes cultured with Ax exhibited significantly increased mRNA expression of CDX2, SOD2, and GPX4 in their blastocysts. We further analyzed oxidative stress, mitochondrial and lysosomal function in the 4-cell embryos and blastocysts derived from parthenogenetic zygotes. For the 4-cell embryos, vitrification disturbed the levels of reactive oxygen species (ROS) and glutathione (GSH), and the activities of mitochondria, lysosome and cathepsin B, and Ax supplementation could fully or partially rescue these values. The blastocysts obtained from vitrified zygotes showed significantly reduced ATP content and elevated cathepsin B activity, which also was recovered by Ax supplementation. There were no significant differences in other parameters mentioned above for the resultant blastocysts. Furthermore, the addition of Ax significantly enhanced mitochondrial activity and reduced lysosomal activity in resultant blastocysts. In conclusion, these findings revealed that Ax supplementation during the culture period improved subsequent embryonic development and quality of porcine zygotes after vitrification and might be used to ameliorate the recovery culture condition for vitrified embryos.

Changes in Rumen Bacterial Community Induced by the Dietary Physically Effective Neutral Detergent Fiber Levels in Goat Diets.

Physically effective neutral detergent fiber (peNDF) is a concept that accounts for the particle length of NDF in a feed, sustaining the normal chewing behavior and rumen fermentation of ruminants. This study aimed to elucidate the effects of dietary peNDF on growth performance and bacterial communities in the rumen of goats through a high-throughput sequencing technique. A total of 30 male Lezhi black goats were randomly assigned to five groups, corresponding to five diets with identical compositions and nutrient levels but with varying forage lengths (the peNDF1.18 contents of the diets were 33.0, 29.9, 28.1, 26.5, and 24.8%, respectively). The whole trial lasted for 44 days. As results show, feed intake and average daily gain were highest when peNDF1.18 content was 26.5%, in which the papilla length of the dorsal sac in rumen was the highest. Chao1 and ACE indexes were similar among the treatments, while Shannon and Simpson indexes of the peNDF1.18 = 28.1% group were the highest (p < 0.05). As the level of dietary peNDF1.18 decreased, the dominant phylum transitioned from Bacteroidetes to Firmicutes. The top three dominant genera of rumen bacteria were Prevotella 1, Ruminococcaceae NK4A214 group, and Christensenellaceae R-7 group. They all showed a quadratic correlation with dietary peNDF1.18 level (p < 0.05). The relative abundance of Ruminococcaceae UCG-011 was positively correlated, while that of Prevotella 1 was negatively correlated, with amino acid metabolism and energy metabolism (p < 0.01). In conclusion, dietary peNDF level influenced goat growth performance, rumen development, and rumen bacterial community structures, and a peNDF1.18 level between 26.5 and 28.1% was considered optimal for goat diet.

Comparison of coated and uncoated trace elements on growth performance, apparent digestibility, intestinal development and microbial diversity in growing sheep.

The suitable supplement pattern affects the digestion and absorption of trace minerals by ruminants. This study aimed to compare the effects of coated and uncoated trace elements on growth performance, apparent digestibility, intestinal development and microbial diversity in growing sheep. Thirty 4-month-old male Yunnan semi-fine wool sheep were randomly assigned to three treatments (n = 10) and fed with following diets: basal diet without adding exogenous trace elements (CON), basal diet plus 400 mg/kg coated trace elements (CTE, the rumen passage rate was 65.87%) and basal diet plus an equal amount of trace elements in uncoated form (UTE). Compared with the CON group, the average daily weight gain and apparent digestibility of crude protein were higher (P < 0.05) in the CTE and UTE groups, while there was no difference between the CTE and UTE groups. The serum levels of selenium, iodine and cobalt were higher (P < 0.05) in the CTE and UTE groups than those in the CON group, the serum levels of selenium and cobalt were higher (P < 0.05) in the CTE group than those in the UTE group. Compared with the CON and UTE groups, the villus height and the ratio of villus height to crypt depth in duodenum and ileum were higher (P < 0.05) in the CTE groups. The addition of trace minerals in diet upregulated most of the relative gene expression of Ocludin, Claudin-1, Claudin-2, ZO-1, and ZO-2 in the duodenum and jejunum and metal ion transporters (FPN1 and ZNT4) in small intestine. The relative abundance of the genera Christensenellaceae R-7 group, Ruminococcus 1, Lachnospiraceae NK3A20 group, and Ruminococcaceae in ileum, and Ruminococcaceae UCG-014 and Lactobacillus in colon was higher in the CTE group that in the CON group. These results indicated that dietary trace mineral addition improved the growth performance and intestinal development, and altered the structure of intestinal bacteria in growing sheep. Compared to uncoated form, offering trace mineral elements to sheep in coated form had a higher absorption efficiency, however, had little effect on improving growth performance of growing sheep.

chi-miR-324-3p Regulates Goat Granulosa Cell Proliferation by Targeting DENND1A.

Granulosa cell (GC) proliferation provides essential conditions for ovulation in animals. A previous study showed that DENND1A plays a significant role in polycystic ovary syndrome. However, the modulation of DENND1A in GCs remains unclear. Our previous integrated analysis of miRNA-mRNA revealed that the 3'-untranslated region of DENND1A could be a target of chi-miR-324-3p. In this study, we used quantitative reverse transcription polymerase chain reaction (RT-qPCR) to investigate DENND1A expression in ovarian tissues of high- and low-yielding goats. Furthermore, dual-fluorescent reporter vector experiments, Cell Counting Kit-8 (CCK-8) assay, and RT-qPCR were used to elucidate the regulatory pathway of chi-miR-324-3p-DENND1A in GCs. The results revealed an opposite tendency between the expressions of chi-miR-324-3p and DENND1A in the ovaries of high- and low-yielding goats. The CCK-8 assay indicated that chi-miR-324-3p overexpression significantly suppressed GC proliferation, whereas chi-miR-324-3p inhibition promoted GC proliferation. In addition, the expressions of GC proliferation markers LHR, Cylin D2, and CDK4 showed the same tendency. The dual-fluorescent reporter assay revealed that chi-miR-324-3p directly targeted DENND1A, and the RT-qPCR results revealed that DENND1A expression was inhibited by chi-miR-324-3p. In summary, chi-miR-324-3p inhibited the proliferation of GCs by targeting DENND1A.

Faecalibacter bovis sp. nov., isolated from cow faeces.

A Gram-stain-negative, non-spore-forming, yellow-pigmented, aerobic, pleomorphic rod-shaped bacterium, designated ZY171143T, was isolated from faeces of a cow with diarrhoea in Wenshan, Yunnan Province, south-west China and its taxonomic position was studied. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain ZY171143T belonged to the family Weeksellaceae and was most closely related to the only species of the genus Faecalibacter, Faecalibacter macacae CCTCC AB 2016016T with a sequence similarity of 97.8 %. The genomic OrthoANI and digital DNA-DNA hybridization values between the strain and F. macacae CCTCC AB 2016016T were 86.2 and 30.5 %, respectively. The genomic G+C content was 31.1 mol%. The predominant fatty acids (>5 %) were C15 : 0 iso, C17 : 0 iso 3OH, C16 : 0, C16 : 1 ω5c and summed feature 3 (C16 : 1 ω7c and/or 16 : 1 ω6c). The major polar lipids were phosphatidylethanolamine, triacylglycerol and sulfonolipid. The sole respiratory quinone was MK-6. These chemotaxonomic characterizations also revealed that strain ZY171143T was a member of the genus Faecalibacter. Based on the phenotypic, chemotaxonomic and genotypic data, strain ZY171143T represents a novel species within the genus Faecalibacter, for which the name Faecalibacter bovis sp. nov. is proposed. The type strain is ZY171143T (=CGMCC 1.13663T=KCTC 62642T).

Integrated analyses of miRNA-mRNA expression profiles of ovaries reveal the crucial interaction networks that regulate the prolificacy of goats in the follicular phase.

BACKGROUND: Litter size is an important index of mammalian prolificacy and is determined by the ovulation rate. The ovary is a crucial organ for mammalian reproduction and is associated with follicular development, maturation and ovulation. However, prolificacy is influenced by multiple factors, and its molecular regulation in the follicular phase remains unclear. METHODS: Ten female goats with no significant differences in age and weight were randomly selected and divided into either the high-yielding group (n = 5, HF) or the low-yielding group (n = 5, LF). Ovarian tissues were collected from goats in the follicular phase and used to construct mRNA and miRNA sequencing libraries to analyze transcriptomic variation between high- and low-yield Yunshang black goats. Furthermore, integrated analysis of the differentially expressed (DE) miRNA-mRNA pairs was performed based on their correlation. The STRING database was used to construct a PPI network of the DEGs. RT-qPCR was used to validate the results of the predicted miRNA-mRNA pairs. Luciferase analysis and CCK-8 assay were used to detect the function of the miRNA-mRNA pairs and the proliferation of goat granulosa cells (GCs). RESULTS: A total of 43,779 known transcripts, 23,067 novel transcripts, 424 known miRNAs and 656 novel miRNAs were identified by RNA-seq in the ovaries from both groups. Through correlation analysis of the miRNA and mRNA expression profiles, 263 negatively correlated miRNA-mRNA pairs were identified in the LF vs. HF comparison. Annotation analysis of the DE miRNA-mRNA pairs identified targets related to biological processes such as "estrogen receptor binding (GO:0030331)", "oogenesis (GO:0048477)", "ovulation cycle process (GO:0022602)" and "ovarian follicle development (GO:0001541)". Subsequently, five KEGG pathways (oocyte meiosis, progesterone-mediated oocyte maturation, GnRH signaling pathway, Notch signaling pathway and TGF-ß signaling pathway) were identified in the interaction network related to follicular development, and a PPI network was also constructed. In the network, we found that CDK12, FAM91A1, PGS1, SERTM1, SPAG5, SYNE1, TMEM14A, WNT4, and CAMK2G were the key nodes, all of which were targets of the DE miRNAs. The PPI analysis showed that there was a clear interaction among the CAMK2G, SERTM1, TMEM14A, CDK12, SYNE1 and WNT4 genes. In addition, dual luciferase reporter and CCK-8 assays confirmed that miR-1271-3p suppressed the proliferation of GCs by inhibiting the expression of TXLNA. CONCLUSIONS: These results increase the understanding of the molecular mechanisms underlying goat prolificacy. These results also provide a basis for studying interactions between genes and miRNAs, as well as the functions of the pathways in ovarian tissues involved in goat prolificacy in the follicular phase.

Hypothalamic Transcriptome Analysis Reveals the Crucial MicroRNAs and mRNAs Affecting Litter Size in Goats.

The hypothalamus was the coordination center of the endocrine system, which played an important role in goat reproduction. However, the molecular mechanism of hypothalamus regulating litter size in goats was still poorly understood. This study aims to investigate the key functional genes associated with prolificacy by hypothalamus transcriptome analysis of goats. In this research, an integrated analysis of microRNAs (miRNAs)-mRNA was conducted using the hypothalamic tissue of Yunshang black goats in the follicular stage. A total of 72,220 transcripts were detected in RNA-seq. Besides, 1,836 differentially expressed genes (DEGs) were identified between high fecundity goats at the follicular phase (FP-HY) and low fecundity goats at the follicular phase (FP-LY). DEGs were significantly enriched in 71 Gene Ontology (GO) terms and 8 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. The transcriptome data suggested that DEGs such as BMPR1B, FGFR1, IGF1 and CREB1 are directly or indirectly involved in many processes like hypothalamic gonadal hormone secretion. The miRNA-seq identified 1,837 miRNAs, of which 28 differentially expressed miRNAs (DEMs). These DEMs may affect the nerve cells survival of goat hypothalamic regulating the function of target genes and further affect the hormone secretion activities related to reproduction. They were enriched in prolactin signaling pathway, Jak-STAT signaling pathway and GnRH signaling pathway, as well as various metabolic pathways. Integrated analysis of DEMs and DEGs showed that 87 DEGs were potential target genes of 28 DEMs. After constructing a miRNA-mRNA pathway network, we identified several mRNA-miRNAs pairs by functional enrichment analysis, which was involved in hypothalamic nerve apoptosis. For example, NTRK3 was co-regulated by Novel-1187 and Novel-566, as well as another target PPP1R13L regulated by Novel-566. These results indicated that these key genes and miRNAs may play an important role in the development of goat hypothalamus and represent candidate targets for further research. This study provides a basis for further explanation of the basic molecular mechanism of hypothalamus, but also provides a new idea for a comprehensive understanding of prolificacy characteristics in Yunshang black goats.