Whole-genome resequencing of Hu sheep identifies candidate genes associated with agronomic traits.

The phenotypic diversity resulting from artificial or natural selection of sheep has made a significant contribution to human civilization. Hu sheep are a local sheep breed unique to China with high reproductive rates and rapid growth. Genome selection signatures have been widely used to investigate the genetic mechanisms underlying phenotypic variation in livestock. Here, we conduct whole-genome sequencing of 207 Hu sheep and compare them with the wild ancestors of domestic sheep (Asiatic mouflon) to investigate the genetic characteristics and selection signatures of Hu sheep. Based on six signatures of selection approaches, we detect genomic regions containing genes related to reproduction (BMPR1B, BMP2, PGFS, CYP19, CAMK4, GGT5, and GNAQ), vision (ALDH1A2, SAG, and PDE6B), nervous system (NAV1), and immune response (GPR35, SH2B2, PIK3R3, and HRAS). Association analysis with a population of 1299 Hu sheep reveal those missense mutations in the GPR35 (GPR35 g.952651 A>G; GPR35 g.952496 C>T) and NAV1 (NAV1 g.84216190 C>T; NAV1 g.84227412 G>A) genes are significantly associated (P < 0.05) with immune and growth traits in Hu sheep, respectively. This research offers unique insights into the selection characteristics of Hu sheep and facilitates further genetic improvement and molecular investigations.

Relationship between sheep feces scores and gastrointestinal microorganisms and their effects on growth traits and blood indicators.

Fecal scores are crucial for assessing the digestive and gastrointestinal status of animals. The Bristol fecal scoring system is a commonly used method for the subjective evaluation of host feces, there is limited research on fecal scoring standards for fattening Hu sheep. In this study, Hu sheep were collected for rumen, rectum, and colon contents for 16S rDNA sequencing. 514 Hu sheep feces were scored based on the Bristol fecal scoring system, and production performance at each stage was measured. Finally, we developed the scoring standard of the manure of Hu sheep in the fattening period (a total of five grades). The result shows that moisture content significantly increased with higher grades (p < 0.05). We analyzed the relationship between fecal scores and production traits, blood indices, muscle nutrients, and digestive tract microorganisms. The growth traits (body weight, body height, body length, average daily gain (ADG), and average daily feed intake (ADFI) during 80-180 days), body composition traits of the F3 group, and the carcass traits were found to be significantly higher (p < 0.05) than those of the F1 and F2 groups. There was no significant difference in gastrointestinal microflora diversity among all groups (p > 0.05). Significant differences were observed in Aspartate aminotransferase, Glucose, Total bilirubin, and Red Blood Cell Count between groups (p < 0.05). The mutton moisture content in group F4 was significantly higher than in the other groups, and the protein content was also the lowest (p < 0.05). The results of the correlation analysis demonstrated that Actinobacteria, Peptostreptococcaceae, Acidaminococcales, Gammaproteobacteria, and Proteobacteria were the significant bacteria affecting fecal scores. In addition, Muribaculaceae and Oscillospiraceae were identified as the noteworthy flora affecting growth performance and immunity. This study highlights the differences in production traits and blood indicators between fecal assessment groups and the complex relationship between intestinal microbiota and fecal characteristics in Hu sheep, suggesting potential impacts on animal performance and health, which suggest strategies for improved management.

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.

Effect of Codonopsis pilosula polysaccharide on the quality of sheep semen preservation at 4°C.

Objective: This study aimed to investigate the effect of Codonopsis pilosula polysaccharide (CPP) on the motility, mitochondrial integrity, acrosome integrity rate, and antioxidant ability of sheep sperm after preservation at 4 °C. Methods: Semen from healthy adult rams were collected and divided into four groups with separate addition of 0, 200, 400, and 1000 mg/L CPP. Sperm motility was analyzed using the Computer-Assisted Semen Analysis software after preservation at 4 °C for 24, 72, 120, and 168 h. Sperm acrosome integrity rate was analyzed by Giemsa staining at 24, 72, and 120 h, and mitochondrial membrane integrity was analyzed by Mito-Tracker Red CMXRos. The total antioxidant capacity (T-AOC) and malondialdehyde (MDA) content of spermatozoa were measured after 120 h of preservation. Results: The sperm viability and forward-moving sperm under 200 mg/L CPP were significantly higher than that in the control group at 72 h (61.28% ± 3.89% vs. 52.83% ± 0.70%,51.53% ±4.06% vs.42.84% ± 1.14%), and 168 h (47.21% ±0.85% vs.41.43% ± 0.37%, 38.68% ±0.87% vs. 31.68% ± 0.89%). The percentage of fast-moving sperm (15.03% ± 1.10% vs.11.39% ± 1.03%) and slow-moving sperm (23.63% ± 0.76% vs.20.29% ± 1.11%) in the 200 mg/L group was significantly higher than control group at 168 h. The mitochondrial membrane integrity of the sperm in the group with 200 mg/L CPP was significantly higher than those in the control group after storage at 4 °C for 120 h (74.76% ± 2.54% vs. 65.67% ± 4.51%, p < 0.05). The acrosome integrity rate in the group with 200 mg/L (87.66%±1.26%) and 400 mg/L （84.00%±2.95%）was significantly higher than those in the control group (80.65%±0.16%) after storage for 24 h (p < 0.05). CPP also increased T-AOC and decreased the MDA concentration after preservation at 4 °C (p< 0.05). Conclusion: Adding CPP could improve the T-AOC of sperm, inhibit lipid peroxidation, and facilitate semen preservation.

Localization and expression of phospholipase A2 and polyunsaturated fatty acid profile in the testis tissues of Hu sheep.

The fatty acid content and the localization and expression of phospholipase A2 (PLA2) in the testis of Hu sheep were investigated. A total of 18 six-month-old Hu sheep were divided into small group (S, with left testis weight < 50 g), medium group (M, with left testis weight among 90-110 g), and large group (L, with left testis weight >160 g), which had six individuals each. The expression of PLA2 in testicular tissues of different sizes was analyzed by immunohistochemistry, RT-qPCR, and Western blot. The fatty acid profile was detected by gas chromatography. Immunohistochemical labeling determined that PLA2 protein was expressed in the Leydig and Sertoli cells of testis, and the immunohistochemical average optional density in the S group was significantly greater than the L group (P < 0.05). RT-qPCR and Western blot analysis showed that PLA2 in the S group was greater than that in the L group (P < 0.05). Docosahexaenoic acid, ω-3 polyunsaturated fatty acid (PUFA), and total PUFA content in the testis of the L group were significantly less than those of the S and M groups (P < 0.01). This study showed that PLA2 content in the S group was greater than that in the L group.

Identification of AGO2 and PLEC genes polymorphisms in Hu sheep and their relationship with body size traits.

The body size traits are major traits in livestock, which intuitively displays the development of the animal's bones and muscles. This study used PCR amplification, Sanger sequencing, KASPar genotyping, and quantitative real-time reverse transcription PCR (qRT-PCR) to analyze the Single-nucleotide polymorphism and expression characteristics of Argonaute RISC catalytic component 2 (AGO2) and Plectin (PLEC) genes in Hu sheep. Two intron mutations were found in Hu sheep, which were AGO2 g.51700 A > C and PLEC g.23157 C > T, respectively. Through association analysis of two mutation sites and body size traits, it was found that AGO2 g.51700 A > C mainly affects the chest and cannon circumference of Hu sheep of while PLEC g.23157 C mainly affects body height and body length. The combined genotypes of AGO2 and PLEC genes with body size traits showed SNPs at the AGO2 g.51700 A > C and PLEC g.23157 C > T loci significantly improved the body size traits of Hu sheep. In addition, the AGO2 gene has the highest expression levels in the heart, rumen, and tail fat, and the PLEC gene is highly expressed in the heart. These two loci can provide new research ideas for improving the body size traits of Hu sheep.

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.

iTRAQ-based proteomic analysis provides novel insight into the postnatal testicular development of Hu sheep.

Testicular development is an intricate and coordinated process in which thousands of proteins are involved in the regulation of somatic cells development and spermatogenesis. However, knowledge about the proteomic changes during postnatal testicular development in Hu sheep is still elusive. The study was conducted to characterize the protein profiles at four key stages during postnatal testicular development, including infant (0-month-old, M0), puberty (3-month-old, M3), sexual maturity (6-month-old, M6) and body maturity (12-month-old, M12), and between the large- and small-testis groups at 6 months in Hu sheep. Consequently, 5252 proteins were identified using isobaric tags for relative and absolute quantification (iTRAQ) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, and 465, 1261, 231 and 1080 differentially abundant proteins (DAPs) were found between M0_vs_M3, M3_vs_M6L, M6L_vs_M12, and M6L_vs_M6S, respectively. GO and KEGG analysis revealed that the majority of DAPs were involved in cellular process, metabolic process and immune system-related pathways. Furthermore, a protein-protein interaction network was constructed using 86 fertility-related DAPs, and five proteins with the highest degree were represented as hub proteins, including CTNNB1, ADAM2, ACR, HSPA2 and GRB2. This study provided new insights into the regulation mechanisms of postnatal testicular development and identified several potential biomarkers for selecting the high-fertility rams. SIGNIFICANCE OF THE STUDY: Testicular development is an intricate developmental process in which thousands of proteins are involved in regulating the somatic cells development and spermatogenesis. However, knowledge about the proteome changes during postnatal testicular development in Hu sheep is still elusive. This study provides comprehensive insights into the dynamic changes in the sheep testis proteome during postnatal testicular development. Additionally, testis size is positively correlated with semen quality and ejaculation volume, also for the merits of easy measurement, high heritability and selection efficiency, is an important indicator to select candidate rams with high fertility. The functional analyses of the acquired candidate proteins may help us gain a better understanding of the molecular regulatory mechanisms of testicular development.

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.

Effect of corn straw or corncobs in total mixed ration during peri-puberty on testis development in Hu lambs.

Corn straw and corncobs contain large amounts of crude fibers and are widely used in mutton sheep husbandry in northwest China. The aim of this study was to determine whether feeding with corn straw or corncobs affects lamb testis development. A total of 50 healthy Hu lamb at two-month-old (average body weight of 22.3 ± 0.1 kg) were randomly and equally divided into two groups, and the lambs were equally allocated to five pens in each group. The corn straw group (CS) received a diet containing 20% corn straw, whereas the corncobs group (CC) received a diet containing 20% corncobs. After a 77-day feeding trial, the lambs, except the heaviest and lightest in each pen, were humanely slaughtered and investigated. Results revealed no differences in body weight (40.38 ± 0.45 kg vs. 39.08 ± 0.52 kg) between the CS and CC groups. Feeding diet containing corn straw significantly (P < 0.05) increased testis weight (243.24 ± 18.78 g vs. 167.00 ± 15.20 g), testis index (0.60 ± 0.05 vs. 0.43 ± 0.04), testis volume (247.08 ± 19.99 mL vs. 162.31 ± 14.15 mL), diameter of seminiferous tubule (213.90 ± 4.91 µm vs. 173.11 ± 5.93 µm), and the number of sperm in the epididymis (49.91 ± 13.53 × 108/g vs. 19.34 ± 6.79 × 108/g) compared with those in the CC group. The RNA sequencing results showed 286 differentially expressed genes, and 116 upregulated and 170 downregulated genes were found in the CS group compared with the CC group. The genes affecting immune functions and fertility were screened out. Corn straw decreased the mtDNA relative copy number in the testis (P < 0.05). These results suggest that compared with corncobs, feeding corn straw in the early reproductive development stage of lambs increased the testis weight, diameter of seminiferous tubule and the number of cauda sperm.

Differences of the anti-oxidative capability, GPX3, and Cu/ZnSOD expression in Hu sheep testis with different size at six-month-old.

This study aimed to investigate the differences in the anti-oxidant capabilities and related gene expressions of six-month-old Hu sheep with different testis sizes. A total of 201 Hu ram lambs were fed up to 6 months in the same environment. Based on their testis weight and sperm count, 18 individuals were selected and divided into large (n = 9) and small (n = 9) groups, with an average testis weight of 158.67 g ± 5.21 g and 44.58 g ± 4.14 g, respectively. The total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), and malondialdehyde (MDA) concentration in testis tissue were tested. The localization of antioxidant-related genes, GPX3 and Cu/ZnSOD in testis were detected by immunohistochemistry. The GPX3, Cu/ZnSOD expression, and relative mitochondrial DNA (mtDNA) copy number were detected by quantitative real-time PCR. Compared with the small group, the T-AOC (2.69 ± 0.47 vs. 1.16 ± 0.22 U/mgprot) and T-SOD (22.35 ± 2.59 vs. 9.92 ± 1.62 U/mgprot) in the large group were significantly higher, whereas the MDA (0.72 ± 0.13 vs. 1.34 ± 0.17 nM/mgprot) and relative mtDNA copy number in the large group was significantly lower (p < .05). Immunohistochemistry results indicated that the GPX3 and Cu/ZnSOD were expressed in Leydig cells and seminiferous tubule. The expressions of GPX3 and Cu/ZnSOD mRNA in the large group were significantly higher than those in the small group (p < .05). In conclusion, Cu/ZnSOD and GPX3 widely expressed in the Leydig cells and seminiferous tubule, high expression of Cu/ZnSOD and GPX3 in a large group has a higher potential in addressing oxidative stress and contribute to spermatogenesis.

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.

Transcriptomic analysis reveals that non-forage or forage fiber source promotes rumen development through different metabolic processes in lambs.

Dietary fiber supplementation can stimulate rumen development in lambs during the pre-weaning period. However, it is unclear whether different sources of fiber have varying effects on rumen development. This study aimed to investigate the molecular mechanism of rumen morphological and functional development based on non-forage or forage as a starter dietary fiber source. Twenty-four male Hu lambs with similar body weights (BW, 3.67 ± 0.08 kg) were selected and divided into two groups that received diets supplemented with either alfalfa hay (AH) or soybean hull (SH). At the age of 70 days, six lambs were slaughtered from each treatment group for rumen fermentation and morphological analyses. Three samples of the rumen tissue from the ventral sac were collected for transcriptomic analysis. The results identified 633 differentially expressed genes (DEGs), of which 210 were upregulated and 423 were downregulated in the SH group compared with those in the AH group. The upregulated DEGs were most enriched in the immune function and proteolysis pathways, whereas the downregulated DEGs were mainly involved in cell proliferation, apoptosis, and differentiation pathways. These findings indicated that non-forage as a starter dietary fiber source improved immune function and enhanced nitrogen utilization, whereas forage facilitated rumen morphological development.

Composition of Fatty Acids and Localization of SREBP1 and ELOVL2 Genes in Cauda Epididymides of Hu Sheep with Different Fertility.

The epididymis is an organ that transports, matures and stores sperm, and has functions such as secretion and absorption. Polyunsaturated fatty acid (PUFA) compositions in sperm membrane were changed during the process of epididymis maturation and influence the male fertility. This study aimed to investigate differences in crude fat and fatty acid content in cauda epididymis between high and low fertility of Hu sheep. One hundred and seventy-nine Hu ram lambs were fed from 56 days to 6 months under the same environment. After the feeding trial, all rams were slaughtered, and the body weight, testicular weight, epididymal weight and sperm density were measured. Pearson correlation analysis showed significantly moderate positive correlation between epididymal weight and sperm density and testicular weight. Eighteen rams were selected and divided into the high fertility group (H, n = 9) and low fertility group (L, n = 9) according to the epididymal weight, sperm density and histomorphology. The crude fat content, fatty acid profile and genes related to fatty acid metabolism were detected. The crude fat content, total fatty acid, total n-3 PUFA and docosahexaenoic acid (C22:6n-3, DHA) content of cauda epididymis in high fertility group was significantly higher than those in low fertility group (p < 0.05). However, the ratio of n-6/n-3 PUFA was significantly lower than that in group L (p < 0.05). Immunohistochemistry results showed that SREBP1 and ELOVL2 were expressed in pseudostratified columnar ciliated epithelium and smooth muscle cells. The mRNA expression of SREBP1 (p = 0.09) and ELOVL2 (p < 0.05) in the high fertility group were increased. In conclusion, the high expression of SREBP1 and ELOVL2 may contribute to high n-3 PUFA content in cauda epididymis of high-fertility Hu sheep.

Early feeding strategies in lambs affect rumen development and growth performance, with advantages persisting for two weeks after the transition to fattening diets.

This study aimed to explore the effects of early feeding strategies on the growth and rumen development of lambs from pre-weaning to the transition to fattening diets. Ninety-six newborn, male lambs with similar body weights were randomly assigned to three treatments: fed starter at 42 days old + weaned at 56 days old (Ctrl, n = 36), fed starter at 7 days old + weaned at 56 days old (ES, n = 36), and fed starter at 7 days old + weaned at 28 days old (ES + EW, n = 24). The fattening diets of all lambs were gradually replaced from 60 to 70 days of age. Six randomly selected lambs from each treatment were slaughtered at 14, 28, 42, 56, 70, and 84 days of age. The results showed that the richness and diversity of rumen microbiota of lambs in the Ctrl group were distinct from those of lambs in the other groups at 42 days of age. Moreover, transcriptome analysis revealed 407, 219, and 1,211 unique differentially expressed genes (DEGs) in the rumen tissue of ES vs. Ctrl, ES vs. ES + EW, and ES + EW vs. Ctrl groups, respectively, at 42 days of age. Different early feeding strategies resulted in differences in ruminal anatomy, morphology, and fermentation in lambs from 42 to 84 days of age (P < 0.05). Lambs in the ES + EW group had a higher average starter diet intake than those in the other groups (P < 0.05) from 28 to 56 days of age, which affected their growth performance. After 42 days of age, the body and carcass weights of lambs in the ES and ES + EW groups were higher than those in the Ctrl group (P < 0.05). These findings demonstrate that feeding lambs with a starter diet at 7 days of age and weaning them at 28 days of age can promote rumen development and improve growth performance, and this advantage persists for up to 2 weeks after transition to the fattening diet.

Antioxidant properties and transcriptome of cauda epididymis with different levels of fertility in Hu lambs.

Oxidative stress damages the structure of sperm and affects its fertility. Further maturation and storage of spermatozoa occur in the epididymis. Thus, a balanced environment between free-radical production and the antioxidant-enzyme system is important. This study aimed to investigate differences in antioxidative activity in epididymis between high and low fertility levels of Hu sheep. A total of 179 Hu ram lambs aged 56 days were fed up to 6 months in the same environment. After feeding trial, all rams were slaughtered. According to the epididymis weight, cauda sperm concentration, and histo-morphological observation, 18 individuals were selected and divided into low fertility (n = 9) and high fertility (n = 9) groups. The RNA sequencing results showed 1404 differentially expressed genes (DEGs), with 735 genes upregulated and 669 genes downregulated in the high fertility group compared with the low fertility group. Sixteen DEGs related to lipid metabolism and oxidative stress, including GPX3, GPX5, LRP1, PLCB1, CRISP2, BSPH1, and ACSBG1, were screened by qPCR, and results were correlated well with the sequencing data. Antioxidative indices such as total antioxidant capacity (21.29 ± 5.97 vs. 30.23 ± 4.56 µM/gprot) and glutathione peroxidase (39.75 ± 7.61 vs. 68.84 ± 12.51 U/mgprot) increased in the high fertility group. However, the relative expression of mtDNA and malondialdehyde concentration (0.75 ± 0.15 vs. 0.54 ± 0.10 nmol/mgprot) and the reactive oxygen species (ROS) content significantly decreased (P < 0.05). Therefore, epididymis from high-fertility group had strong antioxidative activity and protected spermatozoa from ROS.

Polymorphisms in SHISA3 and RFC3 genes and their association with feed conversion ratio in Hu sheep.

In animal husbandry, feed efficiency is a crucial economic trait. In this study, the general linear model was used to perform association analysis for various genotypes and feed conversion ratio (FCR)-related traits. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect the expression of SHISA3 and RFC3 mRNA levels in 10 tissues from 6 sheep. The results showed that SNPs in the NC_040257.1:c.625 T > C and NC_040261.1:g.9905 T > C were analyzed whether they were associated to feed efficiency parameters in Hu sheep (body weight, feed intake, average daily growth, and feed conversion ratio). NC_040257.1:c.625 T > C was shown to be significantly associated with body weight at 80, 100, and 120 days as well as feed conversion ratio (P < 0.05), whereas NC_040261.1:g.9905 T > C was found to be significantly associated with average daily weight gain from 80-140 days (ADG80-140) and FCR (P < 0.05). In Hu sheep, the CC genotypes of SHISA3 and RFC3 were the most common genotypes related to feed efficiency traits. Furthermore, the feed conversion ratio of the combined genotypes TT SHISA3-CC RFC3, TT SHISA3-CT RFC3, TT SHISA3-TT RFC3, CT SHISA3-CC RFC3 and CT SHISA3-CT RFC3 was significantly better than the FCR of CC SHISA3-TT RFC3. RT-qPCR results showed that the expression levels of SHISA3 were lower in the lung than in spleen, kidney, muscle and lymph (P < 0.05), and RFC3 was the lung had a highly significant higher expression level than the heart, liver, spleen, and muscle (P < 0.01). In conclusion, SHISA3 and RFC3 polymorphisms can be used as genetic markers for improving feed conversion efficiency in Hu sheep.

Dietary supplementation of grape seed tannin extract stimulated testis development, changed fatty acid profiles and increased testis antioxidant capacity in pre-puberty hu lambs.

Grape seed tannin extract (GPE) from wine grape pomace has many effective anti-oxidative effects and is used as a promising natural feed additive in the animal feed industry. This study investigated the effect of GPE as a source of tannin on the antioxidant capacity and testis development in Hu lambs. Twenty-seven 3-month-old ram lambs were randomly assigned to three groups. For each treatment group, nine lambs were allocated to nine pens (one lamb per pen). The lambs in the control group were fed a control diet without GPE for 61 days from D21 to D80. Group I (TAN1) was fed with 0.36% GPE diet, and Group II (TAN2) was fed with 0.72% GPE diet. After an 81-day feeding trial, all lambs except the heaviest and lightest in each group were humanely slaughtered and investigated. Results showed that feeding GPE did not affect the body weight, average daily gain, dry matter intake, scrotal circumference, and testis index. Meanwhile, feeding with 0.36% GPE diet increased testis weight, testis volume, and epididymis weight (P ≤ 0.05) compared with those of the control, but no difference was found between TAN1 and TAN2 groups. Copper-zinc superoxide dismutase (Cu-ZnSOD), steroid acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), follicle-stimulating hormone receptor (FSHR), elongation of very long chain fatty acid protein 2 (ELOVL2), fatty acid desaturase (FADS2), and proliferating cell nuclear antigen (PCNA) mRNA in TAN1 and TAN2 groups were significantly up-regulated (P < 0.05). GPE also markedly increased the antioxidant status of testis. Compared with the control group, the treatment groups showed significantly increased superoxide dismutase (SOD) activity (314.23 ± 18.64 U/mg prot in control, 505.22 ± 63.47 U/mg prot in TAN1 and 587.88 ± 55.94 U/mg prot in TAN2, P < 0.05) and total antioxidant capacity (T-AOC) (98.23 ± 18.99 U/g prot in control, 202.15 ± 34.19 U/g prot in TAN1 and 189.57 ± 18.95 U/g prot in TAN2, P < 0.05). Consuming 0.72% GPE also changed the fatty acid profiles in testis with increased C15:1, C22:6n3, and total n-3 fatty acids (P < 0.05) but decreased C22:5n3 (P < 0.05). Therefore, feeding lambs with GPE stimulated testis seminiferous tubule development and increased the number of Sertoli cells (10.56 ± 0.44 in control, 14.10 ± 0.57 in TAN1 and 13.60 ± 0.42, P < 0.05), and seminiferous tubule diameter (109.30 ± 4.56 µm in control, 164.49 ± 5.37 µm in TAN1 and 146.56 ± 4.53 µm in TAN2, P < 0.05). These results suggested that feeding GPE in the early reproductive development stage of lambs upregulated the expression of antioxidative, steroidogenesis, and polyunsaturated fatty acid metabolism-related genes, changed the fatty acid profiles, increased the antioxidant capacity in lamb's testis, and contributed to testis development and spermatogenesis.

Supplementation with dietary linseed oil during peri-puberty stimulates steroidogenesis and testis development in rams.

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs), such as α-linolenic acid (ALA), eicosapentaenoic acid, and docosahexaenoic acid, are involved in male reproductive function. In this study, we investigated the effects of linseed oil (LO) as a source of ALA on the steroidogenesis and changes of testicular histology in rams. Sixteen 3-month old rams during peri-puberty were randomly assigned into two groups. Eight rams were assigned as the control group, and the other received LO (4% dry matter of total diet) as the LO treatment group. After an 81-day feeding trial, the rams were slaughtered and investigated. Results revealed that compared with control group, diet containing LO did not affect body weight (36.87 ± 0.53 kg vs. 37.65 ± 0.64 kg, respectively; P = 0.361), average daily gain (227.47 ± 5.82 g vs. 237.95 ± 9.22 g, respectively; P = 0.353) and epididymis weight (40.77 ± 4.41 g vs. 45.53 ± 4.01 g, respectively; P = 0.398), however, it up-regulated PUFAs metabolism and steroidogenesis-related genes mRNA expression (P < 0.05), and increased plasma estradiol concentration (14.88 ± 0.67 pg/mL vs. 19.50 ± 1.27 pg/mL, respectively; P < 0.05). Therefore, LO stimulated seminiferous tubule development and increased the number of Sertoli cells (19.17 ± 2.14 vs. 27.2 ± 2.39, respectively; P < 0.01), germ-cell layers, as well as testis weight (148.65 ± 22.66 g vs. 249.96 ± 30.63 g, respectively; P < 0.05). All these results suggested that LO can improve testis development during peri-puberty by regulating steroidogenesis in rams' testes.