Polymorphism in IGFALS gene and its association with scrotal circumference in Hu lambs.

Scrotal circumference is an important reproductive index of breeding rams, which has a high genetic correlation with ejaculation volume and semen quality. In this study, the scrotal circumference of 1353 male Hu sheep at different stages of development was measured and descriptive statistical analysis was performed. The results showed that the coefficient of variation of scrotal circumference at each stage was greater than 10%, and its heritability were moderately to high, ranging from 0.318 to 0.719. We used PCR amplification and Sanger sequencing to scan the polymorphisms of the IGFALS gene, and performed association analysis with the circumference of the scrotum at different stages. We identified a synonymous mutation g.918 G > C in exon 1 of the IGFALS gene, and this mutation was significantly associated with scrotal circumference at 100, 120, 140, 160 and 180 days (p < 0.05). Therefore, IGFALS gene polymorphism can be used as a molecular marker affecting scrotal circumference of Hu sheep, which can provide a reference for future molecular marker-assisted selection of scrotal circumference in sheep.

Analysis of liver miRNA in Hu sheep with different residual feed intake.

Feed efficiency (FE), an important economic trait in sheep production, is indirectly assessed by residual feed intake (RFI). However, RFI in sheep is varied, and the molecular processes that regulate RFI are unclear. It is thus vital to investigate the molecular mechanism of RFI to developing a feed-efficient sheep. The miRNA-sequencing (RNA-Seq) was utilized to investigate miRNAs in liver tissue of 6 out of 137 sheep with extreme RFI phenotypic values. In these animals, as a typical metric of FE, RFI was used to distinguish differentially expressed miRNAs (DE_miRNAs) between animals with high (n = 3) and low (n = 3) phenotypic values. A total of 247 miRNAs were discovered in sheep, with four differentially expressed miRNAs (DE_miRNAs) detected. Among these DE_miRNAs, three were found to be upregulated and one was downregulated in animals with low residual feed intake (Low_RFI) compared to those with high residual feed intake (High_RFI). The target genes of DE_miRNAs were primarily associated with metabolic processes and biosynthetic process regulation. Furthermore, they were also considerably enriched in the FE related to glycolysis, protein synthesis and degradation, and amino acid biosynthesis pathways. Six genes were identified by co-expression analysis of DE_miRNAs target with DE_mRNAs. These results provide a theoretical basis for us to understand the sheep liver miRNAs in RFI molecular regulation.

Relationship between hindgut microbes and feed conversion ratio in Hu sheep and microbial longitudinal development.

Feed efficiency is an important indicator in the sheep production process, which plays an important role in improving economic benefits and strengthening energy conservation and emission reduction. Compared with the rumen, the fermentation of the hindgut microorganisms can also provide part of the energy for the host, and the composition of the hindgut microorganisms will affect the feed efficiency. Therefore, we hope to find new ways to regulate sheep feed efficiency by studying the sheep gut microbes. In this study, male Hu sheep with the same birth date were raised under the same conditions until 180 d old. The sheep were divided into high and low groups according to the feed conversion ratio (FCR) at 80 to 180 d old, and the differences in rectal microorganisms between the two groups were compared. The permutational multivariate analysis (PERMANOVA) test showed that there were differences in microorganisms between the two groups (P < 0.05). Combined with linear fitting analysis, a total of six biomarkers were identified, including Ruminobacter, Eubacterium_xylanophilum_group, Romboutsia, etc. Functional enrichment analysis showed that microorganisms may affect FCR through volatile fatty acids synthesis and inflammatory response. At the same time, we conducted a longitudinal analysis of the hindgut microbes, sampling nine-time points throughout the sheep birth to market stages. The microbiota is clearly divided into two parts: before weaning and after weaning, and after weaning microbes are less affected by before weaning microbial composition.

The level of feed efficiency determines the input of sheep production costs and the income of economic benefits. Improving sheep feed efficiency can effectively save energy and reduce emissions. Gut microbes play an important role in the process of feed fermentation. In this study, biomarkers associated with feed efficiency were identified by exploring the relationship between microbes and feed conversion ratio. At the same time, the longitudinal development of microorganisms was explored. It provides a basis for the regulation of intestinal microbes in sheep.

Heritability and recursive influence of host genetics on the rumen microbiota drive body weight variance in male Hu sheep lambs.

BACKGROUND: Heritable rumen microbiota is an important modulator of ruminant growth performance. However, no information exists to date on host genetics-rumen microbiota interactions and their association with phenotype in sheep. To solve this, we curated and analyzed whole-genome resequencing genotypes, 16S rumen-microbiota data, and longitudinal body weight (BW) phenotypes from 1150 sheep. RESULTS: A variance component model indicated significant heritability of rumen microbial community diversity. Genome-wide association studies (GWAS) using microbial features as traits identified 411 loci-taxon significant associations (P < 10-8). We found a heritability of 39% for 180-day-old BW, while also the rumen microbiota likely played a significant role, explaining that 20% of the phenotypic variation. Microbiota-wide association studies (MWAS) and GWAS identified four marker genera (Bonferroni corrected P < 0.05) and five novel genetic variants (P < 10-8) that were significantly associated with BW. Integrative analysis identified the mediating role of marker genera in genotype influencing phenotype and unravelled that the same genetic markers have direct and indirect effects on sheep weight. CONCLUSIONS: This study reveals a reciprocal interplay among host genetic variations, the rumen microbiota and the body weight traits of sheep. The information obtained provide insights into the diverse microbiota characteristics of rumen and may help in designing precision microbiota management strategies for controlling and manipulating sheep rumen microbiota to increase productivity. Video Abstract.

Polymorphism and expression level of the FADS3 gene and associated with the growth traits in Hu sheep.

Growth traits are the economically important traits of sheep, and screening for genes related to growth and development is helpful for the genetic improvement of ovine growth traits. The fatty acid desaturase 3 (FADS3) is one of the important genes affecting the synthesis and accumulation of polyunsaturated fatty acids in animals. In this study, the expression levels of the FADS3 gene and polymorphism of the FADS3 gene associated with growth traits in Hu sheep were detected using quantitative real-time PCR (qRT-PCR), Sanger sequencing, and KAspar assay. The result showed that the expression levels of the FADS3 gene were widely expressed in all tissues, and the expression level of FADS3 in the lung was significantly higher than in other tissues (p < .05). Then, the polymorphism locus g. 2918 A > C was detected in intron 2 of the FADS3 gene, and associated analysis showed that the mutation in the FADS3 gene was associated significantly with growth traits (including body weight, body height, body length, and chest circumference, p < .05). Therefore, individuals with AA genotype showed significantly better growth traits than those with CC genotype, and FADS3 gene could be a candidate gene for improving growth traits in Hu sheep.

Expression profiles of the CD274 and PLEKHH2 gene and association of its polymorphism with hematologic parameters in sheep.

CD274 and PLEKHH2 genes have been identified as immune- and multiple diseases-related genes, and have recently garnered significant interest. However, their role in regulating immune functions in sheep remains largely unexplored. In this study, we aimed to investigate the effects of polymorphisms in CD274 and PLEKHH2 on hematologic parameters in 915 sheep. Our results showed that the CD274 and PLEKHH2 genes were most highly expressed in the spleen and tail fat, respectively, as determined by qRT-PCR. We also identified a G to A mutation (g 0.11858 G > A) in the exon 4 region of CD274, and a C to G mutation (g 0.38384 C > G) in the intron 8 region of PLEKH2. Association analysis revealed that CD274 g 0.11858 G > A was significantly associated with RBC, HCT, MCHC, and MCV (P < 0.05), while PLEKHH2 g 0.38384 C > G was significantly associated with HCT, MPV, MCHC, and MCV (P < 0.05). These results suggest that CD274 and PLEKHH2 genes may play a role in regulating blood physiological indicators and could be potential functional candidates for influencing immune traits in sheep breeding programs.

Detection of single nucleotide polymorphism in HTR4 and its relationship with growth traits in sheep.

In this study, a single nucleotide polymorphism of HTR4 (hydroxytryptamine receptor 4) was detected using DNA sequencing and KASPar (Kompetitive Allele-Specific PCR) technique with the aim of analyzing its effect on growth traits in 1102 sheep. A synonymous mutation g.101220 C > T located on the fifth intron of the ovis HTR4 gene was detected, and association analysis showed that this mutation was significantly associated with growth traits in sheep (p ＜.05), with TT genotypes having significantly lower body weight, height, length and chest circumference than TC and CC genotypes. It showed that the polymorphism of this locus was significantly associated with growth traits in sheep. In addition, qRT-PCR results showed that HTR4 was expressed in different tissues of sheep. It is highly expressed in the liver, spleen and duodenum. As important metabolic, immune and digestive absorption organs in animals, the above tissues can regulate the excitability of intestinal smooth muscle by participating in the body metabolism and nutrient metabolism of sheep, so that sheep can show better growth characteristics. In conclusion, the polymorphic locus identified in HTR4 gene can be used as candidate molecular marker in sheep breeding.

Expression of the Ovine Gene and the Relationship Between Its Polymorphism and Feed Efficiency Traits.

In the mutton industry, feed efficiency traits have the greatest influence on the economic benefits of sheep raised in housing conditions. In this study, quantitative real-time PCR (qRT-PCR), Sanger sequencing, and KASPar methods were used to detect the expression levels of the B cell scaffold protein with ankyrin repeats 1 (BANK1) gene and the relationship between its polymorphism and feed efficiency traits in Hu sheep. The qRT-PCR results showed that the BANK1 gene was extensively expressed in 10 tissues and it was expressed at remarkably higher levels in lymph than in other tissues (p < 0.05). Then, the polymorphism locus, g.93888 A > T, was detected in intron 4 of the BANK1 gene and proved to be remarkably associated with feed efficiency traits (p < 0.05). Hence, the BANK1 gene can be used as a candidate gene for improving the feed efficiency of Hu sheep and this locus could be used as a potential molecular marker for breeding high-feed efficiency sheep in future breeding efforts.

Expression and polymorphisms of CD8B gene and its associations with body weight and size traits in sheep.

The growth traits are economically important traits in sheep. Improving growth rates will increase the profitability of producers. The aim of this study was to identify alleles of CD8B (encoding T-cell surface glycoprotein CD8 beta chain) that are aberrantly expressed in different tissues and to assess the effects and associations of its different genotypes on weight and size traits in sheep. Using quantitative real-time reverse transcription PCR arrays, expression profiling of CD8B was performed in various organs and tissues. CD8B was ubiquitously expressed, with very high expression in the lung, spleen, lymph, duodenum, and liver. One intronic mutation (chr3:62,718,030 (Oar_rambouillet_v1.0, same below) G > A) was identified using pooled DNA sequencing. Subsequently, the variants (AA, AG, and GG) were genotyped using the KASPar® PCR single nucleotide polymorphism (SNP) genotyping system. The results of association analysis with body weight and body size traits in 1304 sheep showed that increases in multiple phenotypic traits correlated with the AA genotype (body weight, p < 0.05; body length, p < 0.05). Thus, SNP chr3:62,718,030 G > A is a promising molecular marker for marker-assisted selection in sheep breeding.

Polymorphism of sheep PRKAA2 gene and its association with growth traits.

Small ruminants farming plays an important role in the livelihood of a large part of the population. Herein we aimed to analyze the effects of single nucleotide polymorphisms in PRKAA2 gene on the growth-related traits of Hu sheep and Dorper sheep. The body weight and body type of 1254 sheep were measured at 80, 100, 120, 140, 160 and 180d, and 37620 phenotypic data were collected. RT-qPCR analysis was performed to test PRKAA2 gene expressed in different tissues of sheep, with the highest expression level in spleen, followed by kidney. In the present study, the PRKAA2 gene sequencing revealed one polymorphism located on Chr1 (Oar_rambouillet_v1.0), termed as chr1:32832382 G > A, and were significantly associated with growth traits of sheep (p < 0.05). The body weight, body length, chest circumference, and cannon circumference of individuals with AA genotype were significantly higher than those with the GG and GA genotypes (p < 0.05). Our findings reveal that PRKAA2 gene could be used as a marker-assisted selection to improve the growth-related traits of sheep.

Polymorphism and expression of the HMGA1 gene and association with tail fat deposition in Hu sheep.

Hu sheep is an excellent short fat-tailed breed in China. Fat deposition in Hu sheep tail affects carcass quality and consumes a lot of energy, leading to an increase in feed cost. The objective of this study was to analyze the effects of HMGA1 polymorphism on tail fat weight in Hu sheep. Partial coding and non-coding sequences of HMGA1 were amplified with PCR and single nucleotide polymorphisms (SNP) of HMGA1 in 1163 Hu sheep were detected using DNA sequencing and KASPar technology. RT-qPCR analysis was performed to test HMGA1 expression in different tissues. The results showed that the expression of HMGA1 was higher in the duodenum, liver, spleen, kidney, and lung than in the heart, muscle, rumen, tail fat, and lymph. A mutation, g.5312 C > T, was detected in HMGA1; g.5312 C > T was significantly associated with tail fat weight, relative weight of tail fat (body weight), and relative weight of tail fat (carcass) (p < 0.05). The tail fat weight of the TT genotype was remarkably higher than that of the CC and TC genotypes. Therefore, HMGA1 can be used as a genetic marker for marker-assisted selection of tail fat weight in Hu sheep.

Polymorphisms in the ovine GP5 gene associated with blood physiological indices.

This study aimed to analyze the effects of polymorphisms in GP5 on blood physiological indices of 1065 sheep. The coefficients of variation of the red blood cell count (RBC), hemoglobin concentration (HGB), mean platelet volume (MPV), mean erythrocyte hemoglobin content (MCH), and red blood cell distribution-coefficient of variation (RDW-CV) were greater than 10%, and there was a very significant correlation between the main indices such as RBC, white blood cell, and platelet count (PLT) and most other indices (p < 0.01). qRT-PCR showed that GP5 was expressed in the heart, liver, spleen, lung, kidney, rumen, duodenum, muscle, tail fat, and lymph tissue, with significantly higher expression in the lymph. Subsequently, we detected single nucleotide polymorphisms (SNPs) in GP5 from group, which identified synonymous mutation g.657 T > C in the first exon of GP5. Association analysis showed significant correlations between the SNP and the physiological traits (p < 0.05), in which the RBC, neutrophilic granulocyte (NEUT) and RDW-CV values in sheep with the TC genotype and TT genotype were markedly lower than those in the CC genotype (p < 0.05). Thus, GP5 polymorphisms could be candidate biomarkers to screen blood physiological indices.

Identification of polymorphic loci in OSMR and GHR genes and analysis of their association with growth traits in sheep.

The aim of this study was to analyze the effect of OSMR and GHR genes polymorphisms on growth traits in sheep. The single nucleotide polymorphisms of sheep OSMR and GHR genes were identified by DNA sequencing technology. A total of two intronic mutations g.2443 T > C and g.170196 A > G were identified in OSMR and GHR, respectively. Correlation analysis was carried out between the obtained genotypes and the growth traits of sheep. The results showed that at the OSMR g.2443 T > C locus, the body weight, chest circumference and cannon circumference of the TT genotype sheep were significantly higher than those of the CC genotype sheep (p < .05). At the GHR g.170196 A > G locus, the body weight, body length, chest circumference and cannon circumference of the AA genotype sheep were significantly higher than those of the AG genotype and GG genotype sheep (p < .05). Moreover, the body weight of sheep of combination TTOSMR/AAGHR genotype was significantly higher than that of other combination genotypes (p < .05). Therefore, we believe that the polymorphic sites identified in the OSMR and GHR genes can be used as candidate molecular markers for breeding good traits in sheep.

Identification of SNPs and expression patterns of ALB, AHSG and GC genes and their association with growth traits in Hu sheep.

Growth traits are economically important traits in sheep breeding. This study was conducted to evaluate the polymorphisms of ALB, AHSG and GC genes and their association with growth traits in Hu sheep. We measured and recorded the body weight (BW), body height (BH), body length (BL) and feed conversion ratio (FCR) of 1418 male Hu sheep raised in the same environment from 80 to 180 days of age. The total of four SNPs in the ALB, AHSG and GC genes were identified by direct sequencing technology. The results of association analysis showed that two loci (g.8699 A>T and g.13458 T>C) of ALB gene significantly affect average daily gain (ADG; P < 0.05). The genotypes of SNP g.2454 T>C in AHSG gene were significantly associated with ADG and FCR (P < 0.05). There were significant associations between GC g.19484 A>C and BW, BH and BL (P < 0.05). The results of qRT-PCR showed that ALB, AHSG, and GC genes were extremely significantly higher in H_BW sheep compared with those in the L_BW sheep (P < 0.01). These results revealed that ALB-1 g.8699 A>T, ALB-2 g.13458 T>C, AHSG g.2454 T>C and GC g.19484 A>C loci are potential molecular markers for Hu sheep breeding.

Estimation of genetic correlations of two key feed efficiency traits with production traits in male Hu sheep.

Increased body growth and feed efficiency are important for breeding due to expensive feed costs. This study estimated the genetic parameters for two feed efficiency traits [the feed conversion ratio (FCR) and the residual feed intake (RFI)] and other important economic traits in male Hu sheep using 1642 male Hu lambs. The heritability of the RFI was estimated at 0.31 (±0.10)-0.54 (±0.1) and the heritability of the FCR was 0.05 (±0.07)-0.77 (±0.12). There was a negative genetic correlation between rib-eye muscle area traits and RFI. A positive genetic correlation was found between fat deposition traits and feed efficiency traits. The digestive tract weight decreased with the direction of RFI: however, the FCR showed the opposite: A low FCR was accompanied by a high digestive tract weight. The genetic correlation between feed efficiency (RFI and FCR) and rumen weight was negative. Heritability estimates for feed efficiency generate fluctuations with different experimental intervals. The weight of other traits should be reasonably allocated to avoid losses in production and feed efficiency in the selection of feed efficiency traits. The present study added to our understanding of the genetic parameters of sheep under the condition of house feeding.

Novel polymorphism at KLF15 gene and its association with growth traits in Hu sheep.

Growth traits are important economic characteristics of livestock and poultry. In the present study, the expression features of KLF15 and the relationship between KLF15 gene polymorphisms and growth traits in Hu sheep were investigated by using real-time quantitative PCR technology (qPCR), Sanger sequencing, and Kaspar genotyping technology. The qPCR results showed that the KLF15 gene is expressed widely in the tested tissues of Hu sheep, and the expression level of the KLF15 gene in the heart and the muscle was significantly higher than in other tissues (p < 0.05). Missense mutation c.62565119 A > G was found in KLF15, and an association analysis showed that it was correlated with the growth traits (body weight, body height, and body length) of Hu sheep (p < 0.05). The body weight, body height, and body length of the sheep carrying the AA genotype were remarkably higher than those of the GG and AG genotypes (p < 0.05). These results showed that novel polymorphisms at the KLF15 gene can be used as a genetic marker of growth traits of Hu sheep.

Polymorphism in ovine ADCY8 gene and its association with residual feed intake in Hu sheep.

Feed efficiency makes up a large part of sheep production, which also has a crucial impact on the economic benefits of producers. This study explores the polymorphism of Adenylyl cyclase 8 gene associated with feed efficiency and detects the expression characteristics of ADCY8 in ten tissues of Hu sheep. The polymorphism of ADCY8 was recognized by using PCR amplification and Sanger sequencing, KASPar technology was used for genotyping subsequently, and the relationship between SNP and RFI is also studied. The results indicated that an intronic mutation g.24799148 C > T (rs 423395741) was identified in ADCY8, and association analysis showed that the SNP g.24799148 C > T (rs 423395741) was significantly associated with RFI at 100-120, 100-140, 100-160, and 100-180 days (p < 0.05). The quantitative real-time PCR (qRT-PCR) result showed that ADCY8 was expressed in ten tissues, and the expression of ADCY8 gene in rumen tissue was significantly higher than in the other tested tissues. Therefore, these results indicated that the ADCY8 mutation locus may be used as a candidate molecular marker for evaluating the feed efficiency of Hu sheep.

Association between rumen microbiota and marbling grade in Hu sheep.

The marbling fat regulates the flavor of mutton and measures the fat density in the loin eye and is the most important parameter of carcass grading. The objective of this study was to explore the relationship of rumen microbiota and mutton marbling grade. One hundred and eighty-seven feedlot-finished Hu male lambs (Age: 180 day; Final BW: 46.32 ± 6.03 kg) were slaughtered, and ruminal contents and marbling grade were collected. Ruminal microbial DNA extraction and 16S rRNA gene sequencing was performed to investigate microbial composition and to predict microbial metabolic pathways. The animal cohort was then grouped based on marbling grades [low marbling (LM), marbling grade ≤ 1; Medium marbling (MM), 1 < marbling grade ≤ 3; High Marbling (HM), 3 < marbling grade ≤ 5] and intramuscular fat-associated microorganisms were pinpointed using LEfSe and random forest classification model. Intramuscular fat content had significantly differences among the three groups (P < 0.05), and was significantly correlated with VFAs profiling. HM sheep showed a higher abundance of one bacterial taxon (Kandleria), and two taxa were overrepresented in the MM sheep (Pseudobutyrivibrio and Monoglobus), respectively. In addition, the main intramuscular fat deposition pathway was found to involve peroxisome proliferator-activated receptor (PPAR) fatty acid synthesis. By studying the effect of the ruminal microbiome on the marbling of sheep, the present study provides insights into the production of high-quality mutton.

Sheep fecal transplantation affects growth performance in mouse models by altering gut microbiota.

Animal growth traits are important and complex traits that determine the productivity of animal husbandry. There are many factors that affect growth traits, among which diet digestion is the key factor. In the process of animal digestion and absorption, the role of gastrointestinal microbes is essential. In this study, we transplanted two groups of sheep intestinal microorganisms with different body weights into the intestines of mice of the same age to observe the effect of fecal bacteria transplantation on the growth characteristics of the mouse model. The results showed that receiving fecal microbiota transplantation (FMT) had an effect on the growth traits of recipient mice (P < 0.05). Interestingly, only mice receiving high-weight donor microorganisms showed differences. Use 16S rDNA sequencing technology to analyze the stool microorganisms of sheep and mice. The microbial analysis of mouse feces showed that receiving FMT could improve the diversity and richness of microorganisms (P < 0.05), and the microbial composition of mouse feces receiving low-weight donor microorganisms was similar to that of the control group, which was consistent with the change trend of growth traits. The feces of high-weight sheep may have higher colonization ability. The same five biomarkers were identified in the donor and recipient, all belonging to Firmicutes, and were positively correlated with the body weight of mice at each stage. These results suggest that FMT affects the growth traits of receptors by remodeling their gut microflora.

The rate of development of growth traits determines the productivity of sheep. Shortening the fattening cycle of sheep can effectively improve economic efficiency. Gut microbes have important effects on host growth traits. In this study, the method of fecal microbiota transplantation (FMT) was used to transplant the intestinal microbes of sheep into the mouse model, and the changes of the growth traits and intestinal microbes of the mice were observed. The results suggest that FMT can effectively affect growth traits by altering recipient gut microbes. This provides a new idea for the regulation of sheep growth traits.

Ovine RAP1GAP and rBAT gene polymorphisms and their association with tail fat deposition in Hu sheep.

Excessive fat deposition in the tail of sheep will affect its feed efficiency, which will increase the feeding cost. The purpose of this study was to identify the single nucleotide polymorphisms (SNPs) of RAP1GAP and rBAT genes by PCR amplification and Sanger sequencing, the SNPs were genotyped by KASP genotyping assays to evaluate their association with tail fat deposition traits. The results showed that two intronic mutations of g.13561 G > A and g.1460 T > C were found in RAP1GAP and rBAT, respectively. There were three genotypes of GG, AG, AA and CC, CT and TT at these two loci, respectively. Association analysis showed that g.13561 G > A of RAP1GAP was associated with tail width, tail fat weight and relative tail fat weight (P < 0.05). The g.1460 T > C of rBAT was associated with tail width and tail fat weight (P < 0.05). Different combinations of genotypes also differed significantly with tail fat deposition traits. In the tail fat tissue, the expression levels of RAP1GAP gene was significantly higher in small-tailed sheep than in big-tailed sheep, and the expression levels of rBAT gene was significantly higher in big-tailed sheep than in small-tailed sheep. In the liver, the expression levels of RAP1GAP and rBAT gene was significantly higher at 6 months than at 0 and 3 months. In conclusion, RAP1GAP and rBAT polymorphisms can be used as a candidate molecular marker to reduce tail fat deposition in sheep.