Supplemented Organic and Inorganic Selenium Affects Milk Performance and Selenium Concentration in Milk and Tissues in the Guanzhong Dairy Goat.

Trace amounts of selenium (Se) are essential for several organisms, and deficiencies therein have adverse effects on growth, development, and reproduction; this is particularly significant in animals raised for milk and livestock production. To study the effect of Se on Guanzhong dairy goats, their diets were supplemented with different sources (inorganic or organic) and Se concentrations (0.2 or 0.4 mg Se/kg). A non-Se-fortified basal diet served as a negative control, and a sixth treatment group received both inorganic and organic Se sources (0.2 mg Se/kg diet each). Dietary Se supplementation increased milk production, with organic Se being more effective than inorganic Se. Selenium supplementation also increased Se concentration and glutathione peroxidase activity in whole blood, with organic Se more effective than inorganic Se at the same Se concentration. With increasing Se in diets, the Se content in milk increased markedly, reaching a plateau value at day 30 in all groups, and organic Se (0.4 mg/kg diet) had the best effect. In addition, dietary Se sources and concentrations markedly affected Se concentrations in different tissues and organs. Thus, organic Se supplementation of a basal diet at 0.4 mg/kg is practically applicable for Se-enriched milk and meat production in Guanzhong dairy goats.

MicroRNA-10b suppresses goat granulosa cell proliferation by targeting brain-derived neurotropic factor.

Brain-derived neurotropic factor (BDNF) and its high-affinity receptor, tyrosine kinase receptor B, have been assumed to be involved in female reproduction and have recently shown to play an essential role in follicle activation and oocyte maturation. In this study, we analyzed the expression of miR-10b and BDNF in the ovary and discovered that the expression of miR-10b was higher in monotocous goat ovaries than in polytocous goat ovaries, whereas the expression pattern of BDNF in ovary was opposite. Moreover, human chorionic gonadotropin induced rapid and transient expression of BDNF messenger RNA and protein. In contrast, human chorionic gonadotropin upregulated miR-10b expression in a time-dependent manner. The BDNF gene was identified as a direct target of miR-10b using a dual-luciferase reporter assay. Transfection of granulosa cells with miR-10b decreased BDNF messenger RNA and protein levels. MiR-10b overexpression inhibited cell proliferation, whereas BDNF promoted cell proliferation. However, a combined treatment with miR-10b and BDNF promoted cell proliferation, indicating that the reintroduction of BDNF reversed the suppressive effect of miR-10b. These results demonstrate that miR-10b downregulates BDNF expression in granulosa cells by directly targeting the 3' untranslated regions and plays an important role in inhibiting granulosa cell proliferation by targeting BDNF.

Two mutations in the 5'-flanking region of the KITLG gene are associated with litter size of dairy goats.

In this study, Xinong Saanen (SN) and Guanzhong (GZ) dairy goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the 5'-flanking region of the KITLG gene by DNA sequencing and primer-introduced restriction analysis-polymerase chain reaction. Two novel SNPs (g.13090G>T and g.13664C>A) were identified (GenBank Accession no. KM658964). Furthermore, g.13090G>T and g.13664C>A loci were closely linked in SN and GZ breeds (r(2) > 0.33). Association analysis results showed that g.13090G>T and g.13664C>A SNPs significantly affected litter size (P < 0.05). The litter size of individuals with the combined genotype GG/CC from both dairy goat breeds was greater than that of individuals with TT/AA in average parity (P < 0.05). Known biochemical and physiological functions, along with our results, indicated that GG/CC could be used in marker-assisted selection to choose individuals with greater litter size from both breeds. These results extend the spectrum of genetic variation in the caprine KITLG gene and may contribute to genetic resources and breeding of goats.

Molecular characterization and hormonal regulation of tissue inhibitor of metalloproteinase 1 in goat ovarian granulosa cells.

Tissue inhibitor of metalloproteinase 1 (TIMP1) belongs to a group of endogenous inhibitors that control the activity of matrix metalloproteinases and other metalloproteinases. TIMP1 is ubiquitously expressed and implicated in many physiological and pathologic processes. In this study, the full-length complementary DNA of goat (Capra hircus) Timp1 was cloned from adult goat ovary for the first time to better understand the regulatory role of TIMP1. The putative TIMP1 protein shared a high amino acid sequence identity with other species. Real-time polymerase chain reaction results showed that Timp1 was widely expressed in adult goat tissues, and messenger RNA expression was higher in the ovary than in other tissues; meanwhile, increasing expression of Timp1 was also discovered during the process of follicle growth and corpus luteum. We then investigated Timp1 expression patterns in different types of ovarian follicular cells from goats. In small or large antral follicles, Timp1 expression was higher (P < 0.05) in theca cells than in granulosa cells, cumulus cells, and oocytes. Increasing expression of Timp1 in theca and granulosa cells was observed as the variation of the follicle size. Immunohistochemical analyses further revealed the presence of the TIMP1 proteins in follicles at all antral stages of development. The most intense staining for TIMP1 was observed in the theca cells and granulosa cells of large antral follicles and corpus luteum. Timp1 was highly (P < 0.05) induced in granulosa cells in vitro after treatment with the luteinizing hormone agonist, human chorionic gonadotropin. Treatments with forskolin, phorbol 12-myristate 13-acetate, or phorbol 12-myristate 13-acetate + forskolin could also stimulate Timp1 messenger RNA expression. The effects of human chorionic gonadotropin were reduced (P < 0.05) by the inhibitors of protein kinase A, protein kinase C, MAPK kinase, or p38 kinase, indicating that Timp1 expression could be adjusted by luteinizing hormone-initiated activation of these signaling mediators. Our results suggested that TIMP1 may be involved in regulating ovarian follicle development and ovulation.

Polymorphism and DNA methylation in the promoter modulate KISS1 gene expression and are associated with litter size in goats.

Polymorphisms in the promoter region are likely to impact KISS1 gene transcription and reproductive traits. In this study, Guanzhong (GZ, n=350) and Boer (BE, n=196) goats were used to detect polymorphism in the promoter of the goat KISS1 gene by DNA sequencing. In the GZ goats, the g.1384G>A mutation was identified in the promoter of the goat KISS1 gene. Guanzhong goats were in Hardy-Weinberg disequilibrium at g.1384G>A locus (P<0.05). The 1384A allele was predicted to eliminate methylation, AHR-arnt heterodimers and AHR-related factors (AHRR) and myoblast determining factors (MYOD) transcription factor-binding sites. Statistical results indicated that the g.1384G>A SNP was associated with litter size in the GZ goats (P<0.05). Luciferase assay analysis suggested that the 1384A allele increased luciferase activity when compared to the 1384G allele. The RT-qPCR assay also demonstrated that the 1384A allele had greater amounts of KISS1 mRNA than the 1384G allele in homozygous individuals. Functional analysis suggested that this g.1384G>A SNP may be an important genetic regulator of KISS1 gene expression with effects on downstream processes that are modulated by KISS1 gene because of the changes of methylation and transcription factor-binding sites. Therefore, the current study provides evidence in goats for genetic markers that might be used in breeding programs.

Association analysis between variants in KITLG gene and litter size in goats.

Xinong Saanen (SN) and Guanzhong (GZ) goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the coding regions with their intron-exon boundaries and the proximal flanking regions of KITLG gene by DNA sequencing and genotyped by PCR-restriction fragment (PCR-RFLP). Four novel SNPs (g.12654G>A, g.12772G>A, g.12829T>C and g.23683C>T) were identified (GenBank accession No. KM609289). It was shown that Xinong Saanen and Guanzhong goat breeds were in Hardy-Weinberg disequilibrium at g.12654G>A, g.12772G>A and g.12829T>C loci (P<0.05). The g.12654G>A, g.12772G>A and g.12829T>C loci were closely linked in both goat breeds (r(2)>0.33). Results of an association analysis indicated that SNPs g.12654G>A, g.12772G>A and g.12829T>C had significant effects on litter size (P<0.05). The combined genotypes of four SNP loci also affected litter size with the C7(GG/GG/CC/CC) genotype in the SN goat breed and C1(AA/GG/CC/CC) and C7(GG/GG/CC/CC) genotypes in the GZ goat breed having the highest litter size. The biochemical and physiological functions, together with the results obtained in our investigation, suggest that C7(GG/GG/CC/CC) could be used in marker-assisted selection to select the individuals with higher litter size in both goat breeds. The results extend the spectrum of genetic variation of the caprine KITLG gene, which might contribute to goat genetic resources and breeding.

Two missense mutations in exon 9 of caprine PRLR gene were associated with litter size.

Guanzhong (n = 321) and Boer (n = 191) goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the coding regions of the prolactin receptor (PRLR) gene by DNA sequencing and PCR-RFLP. Two SNPs (c.1457G>A and c.1645G>A) were identified that caused amino acid variations p.Ser485Asn and p.Val548Met respectively. Statistical results indicated that the c.1457G>A and c.1645G>A SNPs were significantly associated with litter size in Boer and Guanzhong goat breeds. Further analysis revealed that combined genotype C4 (GGGG) and haplotype G-G were better than the others for litter size in both goat breeds. These results might contribute to goat genetic resources and breeding.

Polymorphisms of PRLR and FOLR1 genes and association with milk production traits in goats.

We investigated the polymorphisms of PRLR and FOLR1 genes in Xinong Saanen, Guanzhong, and Boer goat breeds by DNA sequencing and PCR-RFLP. Two novel SNPs were identified: KC109741: g.62130C>T in the 3ꞌ-UTR of goat gene PRLR, and KC136296: g.7884A>C in exon 3 of goat gene FOLR1. In the three goat breeds, the polymorphism information content was 0.20-0.27 at the g.62130C>T locus. At the g.7884A>C locus, it was 0.36 in Boer goats. The three goat breeds were in Hardy-Weinberg disequilibrium at the g.62130C>T locus. The g.62130C>T SNP was found to be significantly associated with milk production traits in Xinong Saanen and Guanzhong breeds. These results are consistent with the regulatory function of PRLR in mammary gland development, milk secretion, and expression of milk protein genes; they extend the spectrum of genetic variation of the goat PRLR gene, which could be useful for breeding programs.

Combined effects of four SNPs within goat PRLR gene on milk production traits.

Xinong Saanen (SN, n=323) and Guanzhong (GZ, n=197) goat breeds were used to detect single nucleotide polymorphisms (SNPs) in the coding regions with their intron-exon boundaries of prolactin receptor (PRLR) gene by DNA sequencing, primer-introduced restriction analysis-polymerase chain reaction (PIRA-PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Four novel SNPs (g.40452T>C, g.40471G>A, g.61677G>A and g.61865G>A) were identified. The g.61677G>A and g.61865G>A SNPs caused amino acid variations p.Ser485Asn and p.Val548Met, respectively. Both g.40452T>C and g.40471G>A loci were closely linked in SN and GZ goat breeds (r(2)>0.33). In addition, there was also a close linkage between g.61677G>A and g.61865G>A loci in both goat breeds. Statistical results indicated that the g.40452T>C, g.61677G>A and g.61865G>A SNPs were significantly associated with milk production traits in SN and GZ breeds. Further analysis revealed that combinative genotype C1 (TTAAGGGG) was better than the others for milk yield in SN and GZ goat breeds. These results are consistent with the regulatory function of PRLR in mammary gland development, milk secretion, and expression of milk protein genes, and extend the spectrum of genetic variation of the caprine PRLR gene, which might contribute to goat genetic resources and breeding.

Molecular cloning and characterization of KISS1 promoter and effect of KISS1 gene mutations on litter size in the goat.

Kisspeptins, the product of the KISS1 gene, play an essential role in the regulation of reproductive functions, acting primarily at the hypothalamic level of the gonadotropic axis. We detected polymorphisms of the goat KISS1 gene in 723 individuals from three goat breeds (Xinong Saanen, Guanzhong, and Boer) by DNA pooling, PCR-RFLP, and DNA sequencing methods. We cloned the promoter sequence of this gene and found it to share high similarity with that of the bovine KISS1 promoter. Six TATA boxes were found in the goat KISS1 promoter region. Two novel SNPs (g.2124T>A and g.2270C>T) were identified in the intron 1 of the KISS1 gene of all three goat breeds. The three goat breeds were in Hardy-Weinberg disequilibrium at g.2124T>A and g.2270C>T loci. The g.2124T>A and g.2270C>T loci were closely linked in the three goat breeds (r2 > 0.33). The g.2124T>A and g.2270C>T SNPs were significantly associated with litter size, and the C1 female goats had a larger litter size than did those with the other genotypes. These results extend the spectrum of genetic variation of the goat KISS1 gene, which contributes to our knowledge of goat genetic resources for breeding programs.

Polymorphism identification in goat GNRH1 and GDF9 genes and their association analysis with litter size.

This study investigated the polymorphisms of GNRH1 and GDF9 genes in 641 goats of three breeds: Xinong Saanen, Guanzhong and Boer. Two allelic variants were identified in the GNRH1 gene (JN645280:g.3548A>G and JN645281:g.3699G>A) and one allelic variant was found in the GDF9 gene (JN655693:g.4093G>A). Furthermore, g.4093G>A was a missense mutation (p.Val397Ile of GDF9). Results of an association analysis indicated that SNPs g.3548A>G and g.4093G>A had significant effects on litter size (P < 0.05). The combination genotypes of SNPs g.3548A>G, g.3699G>A and g.4093G>A also affected litter size with the C5 genotype having the highest litter size in the first, third, fourth and average parity. Hence, the biochemical and physiological functions, together with the results obtained in our investigation, suggest that the GNRH1 and GDF9 genes could serve as genetic markers for litter size in goat breeding.

Polymorphism identification in the goat KITLG gene and association analysis with litter size.

This study reported the analysis of KIT ligand (KITLG) gene polymorphisms in 681 goats of three breeds: Xinong Saanen (SN), Guanzhong (GZ), and Boer (BG). In addition, the study identified three allelic variants: g.769T>C and g.817G>T in SN and GZ breeds, and g.9760G>C in the three goat breeds. The g.769T>C and g.817G>T loci were closely linked (r(2) > 0.33). All the single nucleotide polymorphism loci were in Hardy-Weinberg disequilibrium (P < 0.05). Significant associations were found for litter size with all three loci. Therefore, these results suggest that the KITLG gene is a strong candidate gene affecting litter size in goats.

Analysis of differentially expressed genes in ovaries of polytocous versus monotocous dairy goats using suppressive subtractive hybridization.

In this study, suppressive subtractive hybridization (SSH) was performed to screen differentially expressed genes in ovarian tissues between polytocous and monotocous goats. From the SSH cDNA library, we obtained 29 differentially expressed sequence tags (ESTs) that have high similarity with known genes in the public database, which were involved in signal transduction, transcriptional regulation, cellular molecular dynamics, cytoskeleton, metabolism and oxidation-reduction. In addition, one novel EST that has no similarity with known genes in the public database was obtained. Eight ESTs, TIMP1, NUCB1, OAZ1, CXXC5, CAPNS2, ATP5A1, Z and RPL5, were further examined for the reproducibility of the SSH data by the real-time quantitative PCR. The results confirmed an increased expression of respective mRNA in ovarian tissues of polytocous goats compared with those of monotocous goats. The study has identified several genes (known or unknown) that may have effect on follicular development, ovulation and egg activation in goats.

Novel polymorphisms of the growth hormone gene and their effect on growth traits in Chinese goats.

The polymorphisms of the growth hormone (GH) gene were analyzed in 686 individuals from four goat populations, Three haplotypes (A, B and C) and three observed genotypes (AA, AB and AC) were detected at the P2 locus, and three haplotypes (E, F and G) and three observed genotypes (EE, EF and EG) were also detected at the P4 locus. In addition, five single nucleotide polymorphisms (SNPs)-A112G, C142T (Gly>Ser), C214T (P2 locus), C266A (Pro>His) and C214T (P4 locus, Arg>Trp), were identified by GH gene sequencing and PCR-SSCP analysis. The SNPs loci were in Hardy-Weinberg disequilibrium in three goat populations (P<0.05). Association of polymorphisms with growth traits was done in BG, F1 and F1 populations, which were shown to be associated with growth traits in three goat populations. The SNPs in the goat GH gene had significant effects on growth traits (P<0.05). suggesting that the GH gene is a strong candidate gene that affects growth traits in goat.