Enhancing milk quality and modulating rectal microbiota of dairy goats in starch‑rich diet:the role of bile acid supplementation

Background Diets rich in starch have been shown to increase a risk of reducing milk fat content in dairy goats.While bile acids(BAs)have been used as a lipid emulsifier in monogastric and aquatic animals,their effect on ruminants is not well understood.This study aimed to investigate the impact of BAs supplementation on various aspects of dairy goat physiology,including milk composition,rumen fermentation,gut microbiota,and BA metabolism.Results We randomly divided eighteen healthy primiparity lactating dairy goats(days in milk=100±6 d)into two groups and supplemented them with 0 or 4 g/d of BAs undergoing 5 weeks of feeding on a starch-rich diet.The results showed that BAs supplementation positively influenced milk yield and improved the quality of fatty acids in goat milk.BAs supplementation led to a reduction in saturated fatty acids(C16:0)and an increase in monounsaturated fatty acids(cis-9 C18:1),resulting in a healthier milk fatty acid profile.We observed a significant increase in plasma total bile acid concentration while the proportion of rumen short-chain fatty acids was not affected.Furthermore,BAs supplementation induced significant changes in the composition of the gut microbiota,favoring the enrichment of specific bacterial groups and altering the balance of microbial populations.Correlation analysis revealed associations between specific bacterial groups(Bacillus and Christensenellaceae R-7 group)and BA types,suggesting a role for the gut microbiota in BA metabolism.Functional prediction analysis revealed notable changes in pathways associated with lipid metabolism,suggesting that BAs supplementation has the potential to modulate lipid-related processes.Conclusion These findings highlight the potential benefits of BAs supplementation in enhancing milk production,improving milk quality,and influencing metabolic pathways in dairy goats.Further research is warranted to elucidate the underlying mechanisms and explore the broader implications of these findings.

Whole-genome methylation analysis reveals epigenetic variation between wild-type and nontransgenic cloned,ASMT transgenic cloned dairy goats generated by the somatic cell nuclear transfer

Background:SCNT(somatic cell nuclear transfer)is of great significance to biological research and also to the livestock breeding.However,the survival rate of the SCNT cloned animals is relatively low compared to other transgenic methods.This indicates the potential epigenetic variations between them.DNA methylation is a key marker of mammalian epigenetics and its alterations will lead to phenotypic differences.In this study,ASMT(acetylserotonin-Omethyltransferase)ovarian overexpression transgenic goat was produced by using SCNT.To investigate whether there are epigenetic differences between cloned and WT(wild type)goats,WGBS(whole-genome bisulfite sequencing)was used to measure the whole-genome methylation of these animals.Results:It is observed that the different m Cp G sites are mainly present in the intergenic and intronic regions between cloned and WT animals,and their CG-type methylation sites are strongly correlated.DMR(differentially methylated region)lengths are located around 1000 bp,mainly distributed in the exonic,intergenic and intronic functional domains.A total of 56 and 36 DMGs(differentially methylated genes)were identified by GO and KEGG databases,respectively.Functional annotation showed that DMGs were enriched in biological-process,cellularcomponent,molecular-function and other signaling pathways.A total of 10 identical genes related to growth and development were identified in GO and KEGG databases.Conclusion:The differences in methylation genes among the tested animals have been identified.A total of 10 DMGs associated with growth and development were identified between cloned and WT animals.The results indicate that the differential patterns of DNA methylation between the cloned and WT goats are probably caused by the SCNT.These novel observations will help us to further identify the unveiled mechanisms of somatic cell cloning technology,particularly in goats.

TLR7/8 signalling affects X-sperm motility via the GSK3α/β-hexokinase pathway for the efficient production of sexed dairy goat embryos

Background:Goat milk is very similar to human milk in terms of its abundant nutrients and ease of digestion.To derive greater economic benefit,farmers require more female offspring(does);however,the buck-to-doe offspring sex ratio is approximately 50%.At present,artificial insemination after the separation of X/Y sperm using flow cytometry is the primary means of controlling the sex of livestock offspring.However,flow cytometry has not been successfully utilised for the separation of X/Y sperm aimed at sexing control in dairy goats.Results:In this study,a novel,simple goat sperm sexing technology that activates the toll-like receptor 7/8(TLR7/8),thereby inhibiting X-sperm motility,was investigated.Our results showed that the TLR7/8 coding goat Xchromosome was expressed in approximately 50%of round spermatids in the testis and sperm,as measured from cross-sections of the epididymis and ejaculate,respectively.Importantly,TLR7/8 was located at the tail of the Xsperm.Upon TLR7/8 activation,phosphorylated forms of glycogen synthase kinaseα/β(GSK3α/β)and nuclear factor kappa-B(NF-κB)were detected in the X-sperm,causing reduced mitochondrial activity,ATP levels,and sperm motility.High-motility Y-sperm segregated to the upper layer and the low-motility X-sperm,to the lower layer.Following in vitro fertilisation using the TLR7/8-activated sperm from the lower layer,80.52±6.75%of the embryos were XX females.The TLR7/8-activated sperm were subsequently used for in vivo embryo production via the superovulatory response;nine embryos were collected from the uterus of two does that conceived.Eight of these were XX embryos,and one was an XY embryo.Conclusions:Our study reveals a novel TLR7/8 signalling mechanism that affects X-sperm motility via the GSK3α/β-hexokinase pathway;this technique could be used to facilitate the efficient production of sexed dairy goat embryos.

circRNA landscape of non-pregnant endometrium during the estrus cycle in dairy goats

Endometrial development is a complicated process involving numerous regulatory factors.Circular RNAs(circRNAs)have been known as a member of the naturally occurring non-coding RNA family,and are reportedly crucial for a variety of physiological processes.This study investigated the circRNA landscape of non-pregnant endometrium of dairy goats during estrus.Non-pregnant endometrial samples of goats at estrus day 5(Ed5)and estrus day 15(Ed15)were used to methodically analyze the circRNA landscape using strand-specific Ribo-Zero RNA-Seq.A total of 2331 differentially expressed(P<0.05)circRNAs(DEciRs)between Ed5 and Ed15 were discovered in the goat endometrium.It was found that Nipped-B-like(NIPBL)and calcium responsive transcription factor(CARF)may participate in the development of the endometrium by decreasing(P<0.05)the levels of their circRNA-transcript forms.Furthermore,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analyses of DEciR host genes(hgDEciRs)revealed that tight junctions and GTPases may be involved in endometrial development during the estrus cycle.A total of 2331 DEciRs were discovered in the endometrium at Ed5 and Ed15.Based on GO and KEGG enrichment analyses,it could be inferred that tight junctions and GTPases are likely to play an important role in the development of goat endometrium during the estrus cycle.This circRNA study greatly enhances our knowledge of global trends in the development of non-pregnant endometrium during the estrus cycle in goats;these results help us to better understand the molecular regulation of endometrial development in dairy goats.

Genome-wide association studies of lactation yields of milk, fat, protein and somatic cell score in New Zealand dairy goats

Background: Identifying associations between genetic markers and traits of economic importance will provide practical benefits for the dairy goat industry, enabling genomic prediction of the breeding value of individuals, and facilitating discovery of the underlying genes and mutations. Genome-wide association studies were implemented to detect genetic regions that are significantly associated with effects on lactation yields of milk(MY), fat(FY),protein(PY) and somatic cell score(SCS) in New Zealand dairy goats.Methods: A total of 4,840 goats were genotyped with the Caprine 50 K SNP chip(Illumina Inc., San Diego, CA).After quality filtering, 3,732 animals and 41,989 SNPs were analysed assuming an additive linear model. Four GWAS models were performed, a single-SNP additive linear model and three multi-SNP Bayes C models. For the single-SNP GWAS, SNPs were fitted individually as fixed covariates, while the Bayes C models fit all SNPs simultaneously as random effects. A cluster of significant SNPs were used to define a haplotype block whose alleles were fitted as covariates in a Bayesian model. The corresponding diplotypes of the haplotype block were then fit as class variables in another Bayesian model.Results: Across all four traits, a total of 43 genome-wide significant SNPs were detected from the SNP GWAS. At a genome-wide significance level, the single-SNP analysis identified a cluster of variants on chromosome 19 associated with MY, FY, PY, and another cluster on chromosome 29 associated with SCS. Significant SNPs mapped in introns of candidate genes(45%), in intergenic regions(36%), were 0–5 kb upstream or downstream of the closest gene(14%) or were synonymous substitutions(5%). The most significant genomic window was located on chromosome 19 explaining up to 9.6% of the phenotypic variation for MY, 8.1% for FY, 9.1% for PY and 1% for SCS.Conclusions: The quantitative trait loci for yield traits on chromosome 19 confirms reported findings in other dairy goat populations. There is benefit to be gained from using these results for genomic selection to improve milk production in New Zealand dairy goats.

Comparing successful gene knock-in efficiencies of CRISPR/Cas9 with ZFNs and TALENs gene editing systems in bovine and dairy goat fetal fibroblasts

This study aimed to compare the efficiencies of clustered regulatory interspaced short palindromic repeat（CRISPR）/Cas9-mediated gene knock-ins with zinc finger nucleases（ZFNs） and transcription activator-like effector nucleases（TALENs） in bovine and dairy goat fetal fibroblasts. To test the knock-in efficiency, a set of ZFNs and CRISPR/Cas9 plasmids were designed to edit the bovine myostatin（MSTN） gene at exon 2, while a set of TALENs and CRISPR/Cas9 plasmids were designed for editing the dairy goat β-casein gene at exon 2. Donor plasmids utilizing the ZFNs, TALENs, and CRISPR/Cas9 cutting sites were constructed in theGFP-PGK-Neo R plasmid background, including a 5′ and 3′ homologous arm flanking the genes humanized Fat-1（h Fat-1） or enhanced green fluorescent protein（eGFP）. Subsequently, the ZFNs, TALENs, or CRISPR/Cas9 and thehFat-1 or eGFP plasmids were co-transfected by electroporation into bovine and dairy goat fetal fibroblasts. After G418（Geneticin） selection, single cells were obtained by mouth pipetting, flow cytometry or a cell shove. The gene knock-in events were screened by PCR across the homologous arms. The results showed that in bovine fetal fibrobalsts, the efficiencies of ZFNs-mediated eGFP andhFat-1 gene knock-ins were 13.68 and 0%, respectively. The efficiencies of CRISPR/Cas9-mediated eGFP andhFat-1 gene knock-ins were 77.02 and 79.01%, respectively. The eGFP gene knock-in efficiency using CRISPR/Cas9 was about 5.6 times higher than when using the ZFNs gene editing system. Additionally, thehFat-1 gene knock-in was only obtained when using the CRISPR/Cas9 system. The difference of knockin efficiencies between the ZFNs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. In the dairy goat fetal fibroblasts, the efficiencies of TALENs-mediated eGFP andhFat-1 gene knock-ins were 32.35 and 26.47%, respectively. Theefficiencies of eGFP and hFat-1 gene knock-ins using CRISPR/Cas9 were 70.37 and 74.29%, respectively. The knock-in efficiencies difference between the TALENs and CRISPR/Cas9 systems were extremely significant（P〈0.01）. This study demonstrated that CRISPR/Cas9 was more efficient at gene knock-ins in domesticated animal cells than ZFNs and TALENs. The CRISPR/Cas9 technology offers a new era of precise gene editing in domesticated animal cell lines.

Exploring the Novel Genetic Variant of PITX1 Gene and Its Effect on Milk Performance in Dairy Goats

Paired-like homeodomain transcription factor 1 （PITX1） plays an important role in pituitary development by indirectly regulating the expression of the GH and PRL genes, and therefore PITX1 gene is regarded as a potential candidate gene for building the relationship between the gene polymorphism and milk traits. The aim of this study was to explore the novel genetic variant in PITX1 gene and its effect on milk performance in dairy goats. Herein, a novel genetic variation （NW_00314033： g.201GA or IVS1＋41GA） located at nt41 position of the first intron of the goat PITX1 gene was reported at the P1 locus, which can be genotyped by the Msp I PCR-RFLP. In the Msp I PCR-RFLP analyis, the GG variant was a major genotype, and the A variant was a minor allele in Guanzhong dairy goats which was at Hardy-Weinberg disequilibrium （chi-square χ2=140, P0.01）. The establishment of associations between different genotypes and milk performance was performed in the analyzed population. A total of three significant associations of the polymorphism with average milk fat content （%） （P=0.045）, morning milk fat content （%） （P=0.049）, and afternoon milk fat content （%） （P=0.050）, were found, respectively. A significant relationship between the polymorphism and average total solid content （P=0.029） was also detected. This novel single nucleotide polymorphism （SNP） extended the spectrum of genetic variation of the goat PITX1 gene, and its significant association with milk performance would benefit from the application of DNA markers related to improving milk performance through marker-assisted selection （MAS） in dairy goats.

Long-term and combined effects of N-[2-(nitrooxy)ethyl]-3-pyridinecarboxamide and fumaric acid on methane production,rumen fermentation, and lactation performance in dairy goats

Background:In recent years,nitrooxy compounds have been identified as promising inhibitors of methanogenesis in ruminants.However,when animals receive a nitrooxy compound,a high portion of the spared hydrogen is eructated as gas,which partly offsets the energy savings of CH4mitigation.The objective of the present study was to evaluate the long-term and combined effects of supplementation with N-[2-(nitrooxy)ethyl]-3-pyridinecarboxamide(NPD),a methanogenesis inhibitor,and fumaric acid(FUM),a hydrogen sink,on enteric CH4production,rumen fermentation,bacterial populations,apparent nutrient digestibility,and lactation performance of dairy goats.Results:Twenty-four primiparous dairy goats were used in a randomized complete block design with a 2×2factorial arrangement of treatments:supplementation without or with FUM(32 g/d)or NPD(0.5 g/d).All samples were collected every 3 weeks during a 12-week feeding experiment.Both FUM and NPD supplementation persistently inhibited CH4yield(L/kg DMI,by 18.8%and 18.1%,respectively)without negative influence on DMI or apparent nutrient digestibility.When supplemented in combination,no additive CH4suppression was observed.FUM showed greater responses in increasing the molar proportion of propionate when supplemented with NPD than supplemented alone(by 10.2%vs.4.4%).The rumen microbiota structure in the animals receiving FUM was different from that of the other animals,particularly changed the structure of phylum Firmicutes.Daily milk production and serum total antioxidant capacity were improved by NPD,but the contents of milk fat and protein were decreased,probably due to the bioactivity of absorbed NPD on body metabolism.Conclusions:Supplementing NPD and FUM in combination is a promising way to persistently inhibit CH4emissions with a higher rumen propionate proportion.However,the side effects of this nitrooxy compound on animals and its residues in animal products need further evaluation before it can be used as an animal feed additive.

Effects of Different Dietary Cation-Anion Difference on Fiber Degradation in Rumen of Laoshan Dairy Goats

The experiment was conducted to determine effects of different dietary cation-anion difference（DCAD） in diets on ruminal fluid pH and fiber degradation in rumen of Laoshan dairy goats. A 4×4 latin square design was adopted. DCAD in different groups was 0, 50, 100, 200 mEq·kg^-1 of DM, respectively. The results of ruminal pH indicated that different DCAD could significantly influence the ruminal pH （P〈0.05） and ruminal fluid pH increased as DCAD increased from 0 to 200 mEq·kg^-1 of DM at different sampling time-points. There was no effect of DCAD on carboxymethyl cellulase in ruminal fluid at 4 h and 8 h postfeeding （P〉0.05）. Degradation ofNDF, ADF, CF peaked at a DCAD of 100 mEq·kg^-1 of DM. It could be concluded that DCAD of 100 mEq·kg^-1 of DM was advantage to non-pregnancy, non-lactication Laoshan dairy goat.

Potential Genes for Regulation of Milk Protein Synthesis in Dairy Goat Mammary Gland

The lactating mammary gland is a prodigious protein-producing factory, but the milk protein synthesis mechanisms are not well understood. The major objective of this paper was to elucidate which genes and pathways were involved in the regulation of milk protein synthesis in the dairy goat mammary gland. Total 36 primiparous Guanzhong dairy goats were allotted in 12 groups according to their mammary development stages： days 90 and 150 of virgin, days 30, 90, and 150 of pregnancy, days 1, 10, 35, and 60 of lactation and days 3, 7, and 21 of involution （three animals per group）. Mammary tissue RNA was isolated for quantitative real- time RT-PCR of four casein genes alpha-s 1 casein （CSN 1S 1 ）, alpha-s2 casein （CSN 1 S2）, beta-casein （CSN2） and casein kappa （CSN3）, four whey protein genes lactoglobulin （LGB）, laetalbumin （LALBA）, laetofarrin （LTF）, and Whey acidic protein （WAP） and the genes which were potentially to regulate dairy goat milk protein synthesis at the level of transcription or translation [prolactin receptor （PRLR）, AKT1, signal transducers and activators of transcription 5 （STAT5）, E74-Like Factor 5 （ELF5）, eukaryotic translation initiation factor 4E binding protein 1 （EIF4E-BP1）, S6kinase （S6K） and caveolin 1]. The results showed that all genes were up-regulated in lactation period. The expressions of PRLR, AKT1, STAT5, ELF5, and S6K were similar to mRNA expressions of milk proteins. Our results indicated that milk protein synthesis in dairy goat mammary gland was possibly regulated by these genes.

Active dry yeast supplementation benefits ruminal fermentation,bacterial community,blood immunoglobulins,and growth performance in young dairy goats,but not for intermittent supplementation

This study evaluated the effects of active dry yeast(ADY)supplementation and supplementation stra-tegies on ruminal fermentation,bacterial community,blood metabolites,and growth performance in young dairy goats.Sixty young female Guanzhong dairy goats of similar age(4.00±0.50 months)and BW(19.65±0.41 kg)were randomly divided into 3 groups(n=20):(1)basal diet group(CON);(2)basal diet continuously supplemented with 3.0 g/goat per day commercial ADY(a proprietary strain of Saccharomyces cerevisiae with 5.0×10^(9)cfu/g)group(CSY);(3)basal diet with intermittently supple-mented ADY group(ISY;5 d supplementation with ADY at 4.5 g/goat per day following 5 d of no sup-plementation).The experiment lasted 67 d with the first 7 d as an adaptive period.Rumen fluid and blood samples were collected bi-weekly.Data were analyzed using the MIXED procedure combined with the SLICE option in SAS.Specific orthogonal contrasts of ADY vs.CON and CSY vs.ISY were also analyzed.During the experimental period,ADY supplementation resulted in greater DMI(P=0.03),ruminal ac-etate proportion(P<0.01)and acetylesterase activity(P=0.01),and blood contents of glucose(P=0.01)and IgM(P=0.02)and tended to have greater ADG(P=0.05)and paunch girth(P=0.06)than the CON,despite the propionate proportion(P=0.03)and contents of total protein(P=0.04)and IgA(P=0.03)being lower.The lower ruminal NH_(3)-N(P<0.01)and blood urea nitrogen(P=0.07)contents indicated greater nitrogen utilization with ADY supplementation.ADY supplementation showed persistent effects after it was stopped because the BW at 12 months of age(P=0.03)and birth weight of lambs(P=0.02)were greater than the CON.However,the ISY did not show those benefits and had significantly lower relative abundances of fiber-degrading related bacteria than the CSY.In conclusion,ADY supplementa-tion,especially continuously supplemented,may enhance ADG and ADG:DMI ratio by improving DMI,ruminal cellulolytic bacteria abundance and enzyme activity,nitrogen utilization,and immune status.These findings provide a theoretical basis for the rational application of ADY and have important practical implications for the design of nutritional strategies in growing dairy goats.

High rumen degradable starch diet induced blood bile acids profile changes and hepatic inflammatory response in dairy goats

The objective of this study was to reveal the effect of rumen degradable starch(RDS)on bile acid metabolism and liver transcription in dairy goats using metabolomics and transcriptomics.Eighteen Guanzhong dairy goats of a similar weight and production level(body weight=45.8±1.54 kg,milk yield=1.75±0.08 kg,and second parity)were randomly assigned to 3 treatment groups where they were fed a low RDS(LRDS,RDS=20.52%DM)diet,medium RDS(MRDS,RDS=22.15%DM)diet,or high RDS(HRDS,RDS=24.88%DM)diet,respectively.The goats were fed with the experimental diets for 5weeks.On the last day of the experiment,all goats were anesthetized,and peripheral blood and liver tissue samples were collected.The peripheral blood samples were used in metabolomic analysis and white blood cell(WBC)count,whereas the liver tissue samples were used in transcriptomic analysis.Based on the metabolomics results,the relative abundances of primary bile acids in the peripheral blood were significantly reduced in the group that was fed the HRDS diet(P<0.05).The WBC count was significantly increased in the HRDS group compared with that in the LRDS and MRDS groups(P<0.01),indicating that there was inflammation in the HRDS group.Transcriptomic analysis showed that 4 genes related to bile acid secretion(genes:MDR1,RXRa,AE2,SULT2A1)were significantly downregulated in the HRDS group.In addition,genes related to the immune response were upregulated in the HRDS group,suggesting the HRDS diet induced a hepatic inflammatory response mediated by lipopolysaccharides(LPS)(gene:LBP),activated the Toll-like receptor 4 binding(genes:S100A8,S100A9)and the NF-kappa B signaling pathway(genes:LOC106503980,LOC108638497,CD40,LOC102180880,LOC102170970,LOC102175177,LBP,LOC102168903,LOC102185461,LY96 and CXCL8),triggered inflammation and complement responses(genes:C1QB,C1QC,and CFD).The HRDS diet induced a hepatic inflammatory response may be mediated by activating the Toll-like receptor 4 binding and NF-kappa B signaling pathway after free LPS entered the liver.The changes of bile acids profile in blood and the downregulation of 4 key genes(MDR1,RXRa,AE2,SULT2A1)involved in bile secretion in liver are probably related to liver inflammation.

Ingredients from integral valorization of Isabel grape to formulate goat yogurt with stimulatory effects on probiotics and beneficial impacts on human colonic microbiota in vitro

Isabel grape(IG)products have high contents of phenolic compounds and fiber recognized for their positive impacts on microorganisms associated with health benefits to host.This study evaluated the effects of goat yogurts formulated with ingredients from IG integral valorization on the growth and metabolism of different probiotic strains,as well as on the population of selected bacterial groups and metabolic activity of human colonic microbiota in vitro.Goat yogurts with IG ingredients(IGI)stimulated the growth of tested Lactobacillus and Bifidobacterium probiotic strains during a 48-h cultivation,as well as decreased the pH values and enhanced the organic acid production.Goat yogurts with IGI increased the population of Lactobacillus spp.and Bifidobacterium spp.during a 24-h in vitro colonic fermentation.A stable Firmicutes:Bacteroidetes ratio close to 1 was found in media with goat yogurt formulations during the colonic fermentation,being similar to the effect caused by fructooligosaccharides.Goat yogurt formulations with IGI caused increased production of short-chain fatty acids and sugar consumption during colonic fermentation.Goat yogurts with IGI should be a valuable strategy for development of novel added-value foods with beneficial effects on gut microbiota and human health.

High rumen degradable starch decreased goat milk fat via trans-10,cis-12 conjugated linoleic acid-mediated downregulation of lipogenesis genes,particularly,INSIG1

Background:Starch is an important substance that supplies energy to ruminants.To provide sufficient energy for high-yielding dairy ruminants,they are typically fed starch-enriched diets.However,starch-enriched diets have been proven to increase the risk of milk fat depression(MFD)in dairy cows.The starch present in ruminant diets could be divided into rumen-degradable starch(RDS)and rumen escaped starch(RES)according to their different degradation sites(rumen or intestine).Goats and cows have different sensitivities to MFD.Data regarding the potential roles of RDS in milk fat synthesis in the mammary tissue of dairy goats and in regulating the occurrence of MFD are limited.Results:Eighteen Guanzhong dairy goats(day in milk=185±12 d)with similar parity,weight,and milk yield were selected and randomly assigned to one of three groups(n=6),which were fed an LRDS diet(Low RDS=20.52%),MRDS diet(Medium RDS=22.15%),or HRDS diet(High RDS=24.88%)for 5 weeks.Compared with that of the LRDS group,the milk fat contents in the MRDS and HRDS groups significantly decreased.The yields of short-,mediumand long-chain fatty acids decreased in the HRDS group.Furthermore,increased RDS significantly decreased ruminal B.fibrisolvens and Pseudobutyrivibrio abundances and increased the trans-10,cis-12 conjugated linoleic acid(CLA)and trans-10 C18:1 contents in the rumen fluid.A multiomics study revealed that the HRDS diet affected mammary lipid metabolism down-regulation of ACSS2,MVD,AGPS,SCD5,FADS2,CERCAM,SC5D,HSD17B7,HSD17B12,ATM,TP53RK,GDF1 and LOC102177400.Remarkably,the significant decrease of INSIG1,whose expression was depressed by trans-10,cis-12 CLA,could reduce the activity of SREBP and,consequently,downregulate the downstream gene expression of SREBF1.Conclusions:HRDS-induced goat MFD resulted from the downregulation of genes involved in lipogenesis,particularly,INSIG1.Specifically,even though the total starch content and the concentrate-to-fiber ratio were the same as those of the high-RDS diet,the low and medium RDS diets did not cause MFD in lactating goats.

Transplantation of Goat Bone Marrow Mesenchymal Stem Cells (gMSCs) Help Restore Spermatogenesis in Endogenous Germ Cells-Depleted Mouse Models

Mesenchymal stem cells （MSCs） derived from bone marrow are a well-characterized population of adult stem cells that can be maintained and propagated in culture for a long time with the capacity to form a variety of cell types. This study investigated the characteristics of dairy goat bone marrow MSCs （gMSCs） and their differentiation potential toward germ cells in vitro, and to test their potential in vivo, these ceils were transplanted into seminiferous tubes of endogenous germ cells-depleted mouse models. The results showed that characteristic gMSC lines were established and a small population of gMSCs transdifferentiated into male germ cell-like cells which expressed Stra8 after induction with retinoic acid （RA）, as analysed by reverse transcription-polymerase chain reaction （RT-PCR） and immunofluorescence. Further, we transplanted the gMSCs into endogenous germ cells-depleted mouse models. A variety of analysis demonstrated that gMSCs might differentiate into male germ cells and helped spermatogenesis in endogenous germ cells depleted mouse models at 30 d after transplantation. The gMSCs could be used as a potential source of cells for reproductive studies and a neoadjuvant therapy for the spermatogenesis anomaly. Moreover, these cells may offer a new strategy for male infertility and an alternative approach for production of transgenic animals.

1,25-Dihydroxyvitamin D_3 modulates calcium transport in goat mammary epithelial cells in a dose- and energydependent manner

Background： Calcium is a vital mineral and an indispensable component of milk for ruminants. The regulation of transcellular calcium transport by 1,25-dihydroxyvitamin D3 （1,25-（OH）2D3, the active form of vitamin D） has been confirmed in humans and rodents, and regulators, including vitamin D receptor （VDR）, calcium binding protein Dgk （calbindin-Dgk）, plasma membrane Ca2＋-ATPase ] b （PMCAlb）, PMAC2b and Oral1, are involved in this process. However, it is still unclear whether 1,25-（OH）2D3 could stimulate calcium transport in the ruminant mammary gland. The present trials were conducted to study the effect of 1,25-（OH）2D3 supplementation and energy availability on the expression of genes and proteins related to calcium secretion in goat mammary epithelial cells. Methods： An in vitro culture method for goat secreting mammary epithelial cells was successfully established. The cells were treated with different doses of 1,25-（OH）2D3 （0, 0.1, 1.0, 10.0 and 100.0 nmol/L） for calcium transport research, followed by a 3-bromopyruvate （3-BrPA, an inhibitor of glucose metabolism） treatment to determine its dependence on glucose availability. Cell proliferation ratios, glucose consumption and enzyme activities were measured with commercial kits, and real-time quantitative polymerase chain reaction （RT-qPCR）, and western blots were used to determine the expression of genes and proteins associated with mammary calcium transport in dairy goats, respectively. Results： 1,25-（OH）2D3 promoted cell proliferation and the expression of genes involved in calcium transport in a dose-dependent manner when the concentration did not exceed 10.0 nmol/L. In addition, 100.0 nmol/L 1,25-（OH） 2D3 inhibited cell proliferation and the expression of associated genes compared with the 10.0 nmol/L treatment. The inhibition of hexokinase 2 （HK2）, a rate-limiting enzyme in glucose metabolism, decreased the expression of PMCA1 b and PMCA2b at the mRNA and protein levels as well as the transcription of Oral1, indicating that glucose avaitability was required for goat mammary calcium transport. The optimal concentration of 1,25-（OH）2D3 that facilitated calcium transport in this study was 10.0 nmol/L. Conclusions： Supplementation with 1,25-（OH）2D3 influenced cell proliferation and regulated the expression of calcium transport modulators in a dose- and energy-dependent manner, thereby highlighting the role of 1,25-（OH）2D3 as an efficacious regulatory agent that produces calcium-enriched milk in ruminants when a suitable energy status was guaranteed.

Effects of perinatal stress on the metabolites and lipids in plasma of dairy goats

Dairy goats experience metabolic stress during the peripartal period,and their ability to navigate this stage of lactation is related to the occurrence and development of metabolic diseases.Unlike dairy cows,there is a lack of comprehensive analysis of changes in the plasma profiles of peripartal dairy goats,particularly using high-throughput techniques.A subset of 9 clinically-healthy dairy goats were used from a cohort of 96 primiparous Guanzhong dairy goats(BCS,2.75±0.15).Blood samples were collected at seven time points around parturition(d 21,14,7 before parturition,the day of kidding,and d 7,14,21 postpartum),were analyzed using untargeted metabolomics and targeted lipidomics.The orthogonal partial least squares discriminant analysis model revealed a total of 31 differential metabolites including p-cresol sulfate,pyruvic acid,cholic acid,and oxoglutaric acid.The pathway enrichment analysis identified phenylalanine metabolism,aminoacyl-tRNA biosynthesis,and citrate cycle as the top three significantly-altered pathways.The Limma package identified a total of 123 differentially expressed lipids.Phosphatidylserine(PS),free fatty acids(FFA),and acylcarnitines(ACs)were significantly increased on the day of kidding,while diacylglycerols(DAG)and triacylglycerols(TAG)decreased.Ceramides(Cer)and lyso-phosphatidylinositols(LPI)were significantly increased during postpartum period,while PS,FFA,and ACs decreased postpartum and gradually returned to antepartum levels.Individual species of FFA and phosphatidylcholines(PC)were segregated based on the differences in the saturation and length of the carbon chain.Overall,this work generated the largest repository of the plasma lipidome and metabolome in dairy goats across the peripartal period,which contributed to our understanding of the multifaceted adaptations of transition dairy goats.

Genome characterization of the Caprine arthritis-encephalitis virus in China:A retrospective genomic analysis of the earliest Chinese isolates

Caprine arthritis-encephalitis virus(CAEV) is an under-studied virus infecting caprines and ovines worldwide. Over the last four decades, CAEV has spread in China, obtaining genomic data on CAEV strains circulating in China is of importance for developing diagnostic methods and eradicating associated diseases. However, there is limited information on the genome, including characterizations, and the probable origin. This work aimed to characterize Chinese CAEV genomes and population structures. Five CAEV strains isolated from infected dairy goats between 1989and 1994 in Gansu, Guizhou, Shaanxi, Shandong and Sichuan provinces were cloned and sequenced. The Chinese CAEV had a 58–93% genome similarities to strains outside of China, and they belonged to subgenotype B1. The highest similarity levels(98.3–99.3%) were with two other Chinese strains, and they shared a 91.8–92.3% similarity with the strain Clements(GenBank accession no. NC_001463.1) from outside of China. The Chinese CAEV strains isolated from different provinces over five years were still highly homologous and contained unique ancestral population components,indicating that these Chinese strains had a common origin that differed from other known strains. Our results provide genomic data on circulating Chinese CAEV strains and will be useful for future epidemiological investigations and CAEV eradication programs.

Cloning RPL11 cDNA 3' End by SMART RACE Technique

RT-PCR and RACE techniques were used to clone 3＇ end real sequence of RPL11 gene from a goat. The sequences included some non-structural protein （3D） genes, and 3D gene immediately downstream 3＇ non-coding region （UTR） and poly （A） tail. The results showed that the length of specific amplified fragment was 566 bp （not including polyA tail）. RPL11 gene encoded 188 amino acids, amino acid sequence was conducted for homology analysis by NCBI BLAST tool. The highest homology was Mus （RPL11, mRNA homology 99%）, followed by Bos （RPL11, mRNA homology 96%）, and the next was Hs （RPL11, mRNA homology of 93%）.