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.

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.

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 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.

Contamination and Health Risk Assessment of Polycyclic Aromatic Hydrocarbons in Seasoning Flour Products in Hunan, China.

Polycyclic aromatic hydrocarbons (PAHs) are persistent organic pollutants with carcinogenic, teratogenic, and mutagenic effects. Dietary intake is one of the significant exposure pathways of PAHs. In this study, gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) was used to detect 16 priority PAHs listed by the United States Environmental Protection Agency (USEPA) in seasoning flour products distributed in Hunan Province. The consumption of seasoning flour products by the Hunan population was investigated by questionnaire. The results showed that the detection rate of PAHs in seasoning flour products in Hunan Province was 92.41%. Among them, benzo[a]anthracene (BaA), phenanthrene (PHE), fluoranthene (FLA), and chrysene (CHR) were dominant. The total PAHs and benzo[a]pyrene (BaP) contents of soggy seasoning flour product samples were higher than those of crisp samples and chewy samples. The total amount of PAHs in rod-shaped and flaky samples were higher than that in filamentous and granular samples. The margin of exposure (MOE) values of various seasoning flour products and all age groups (children, adolescents, and adults) was much more significant than 10,000. Moreover, the incremental lifetime of cancer risk (ILCR) values of all age groups were below 1 × 10-5. The above results indicate that PAHs in seasoning flour products have a relatively low health risk for the Hunan population. Still, it is recommended that susceptible populations (children, adolescents, etc.) should control their intake of flour products.

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.

Relationship between rumen microbial differences and traits among Hu sheep, Tan sheep, and Dorper sheep.

Rumen microbes play an important role in the growth and development of ruminants. Differences in variety will affect the rumen community structure. The three excellent sheep breeds were selected for this study (Hu sheep, Tan sheep, and Dorper sheep) have different uses and origins. The sheep were raised on the same diet to 180 d of age in a consistent environment. 16S rDNA V3 to V4 region sequencing was used to assess the rumen microbes of 180 individuals (60 per breed). There were differences in microbial diversity among different sheep breeds (P < 0.05). Principal coordinate analysis showed that the three varieties were separated, but also partially overlapped. Linear discriminant analysis effect size identified a total of 19 biomarkers in three breeds. Of these biomarkers, five in Hu sheep were significantly negatively correlated with average feed conversion rate (P < 0.05). Six biomarkers were identified in the rumen of Dorper sheep, among which Ruminococcus was significantly positively correlated with body weight at 80 d (P < 0.05). In Tan sheep, Rikenellaceae_RC9_gut_group was significantly positively correlated with meat fat, and significantly positively correlated with volatile fatty acids (VFAs), such as butyric acid and isobutyric acid (P < 0.05). The Rikenellaceae_RC9_gut_group may regulate Tan mutton fat deposition by affecting the concentration of VFAs. Functional prediction revealed enrichment differences of functional pathways among different sheep breeds were small. All were enriched in functions, such as fermentation and chemoheterotrophy. The results show that there are differences in the rumen microorganisms of the different sheep breeds, and that the microorganisms influence the host.

The rumen is the most important digestive organ of ruminants, and the rumen microflora plays an important role in the process of digestion. This study compared the differences in rumen microbes of different breeds of sheep, identified key biomarkers of each breed, and analyzed their correlation with important economic traits of sheep. The results showed that the biomarkers of various breeds were significantly correlated with key traits. Therefore, we believe that there is a link between sheep rumen microbes and the differential traits of different breeds of sheep. This provides a new idea for sheep trait improvement.

Identification of TRAPPC9 and BAIAP2 Gene Polymorphisms and Their Association With Fat Deposition-Related Traits in Hu Sheep.

Fat deposition is an important economic trait that is closely related to feed efficiency and carcass performance in livestock. In this study, the fat deposition-related traits of 1,293 Hu sheep were measured and descriptive statistical analysis was conducted. The results showed that the coefficient of variation of all fat deposition-related traits was higher than 24%. In addition, single nucleotide polymorphisms and the expression characteristics of TRAPPC9 (encoding trafficking protein particle complex subunit 9) and BAIAP2 (encoding brain-specific Angiogenesis inhibitor 1-associated protein 2) genes in Hu sheep were detected using PCR amplification, Sanger sequencing, KASPar genotyping, and quantitative real-time reverse transcription PCR (qRT-PCR). The associations between SNPs and fat deposition-related traits were also analyzed. Two intronic mutations, TRAPPC9 g.57654 A > G and BAIAP2 g.46061 C > T, were identified in Hu sheep. The result of association analysis showed that TRAPPC9 g.57654 A > G and BAIAP2 g.46061 C > T were both significantly associated with the weight of tail fat, tail fat relative weight (body weight), and tail fat relative weight (carcass) (P < 0.05). Comprehensive effects analysis showed that there were significant differences between the combined genotypes and tail fat and perirenal fat deposition. Moreover, qRT-PCR analysis showed that TRAPPC9 and BAIAP2 are widely expressed, and their expression levels were significantly higher in the small-tail group compared with those in the big-tail group (P < 0.01). These results provided important candidate molecular markers that could be used in strategies to reduce tail fat deposition in Hu sheep.

Molecular characterization and expression of RPS23 and HPSE and their association with hematologic parameters in sheep.

Ribosomal protein S23 (RPS23) and Heparanase (HPSE) were located on chromosome 5 and chromosome 6, respectively, which play vital roles in protein synthesis and immunity. The objective of this study was to clone RPS23 and HPSE and to detect the expression levels of RPS23 and HPSE and the polymorphisms of RPS23 and HPSE associated with the hematologic parameters by using qRT-PCR, DNA sequencing and KASPar assay. The quantitative real-time PCR (RT-qPCR) showed that the two genes were expressed widely in the ten tissues of sheep. The expression levels of RPS23 and HPSE were the highest in lung and liver, respectively. The expression levels of RPS23 and HPSE in lung and liver increased from 0 to 3 months, decreased from 3 to 6 months, respectively. Furthermore, two mutations g.720 A > G and g.1077 G > A were detected in the RPS23 and HPSE, respectively, which were confirmed to be significantly associated with hematologic parameters. These results supported RPS23 g.720 A > G and HPSE g.1077 G > A as genetic markers of sheep.

Whole Genome Sequencing Analysis to Identify Candidate Genes Associated With the rib eye Muscle Area in Hu Sheep.

In sheep meat production, the rib eye area is an important index to evaluate carcass traits. However, conventional breeding programs have led to slow genetic progression in rib eye muscle area. Operationalizing molecular marker assisted breeding is an optimized breeding method that might improve this situation. Therefore, the present study used whole genome sequencing data to excavate candidate genes associated with the rib eye muscle. Male Hu lambs (n = 776) with pedigrees and 274 lambs with no pedigree were included. The genetic parameters of the rib eye area were estimated using a mixed linear mixed model. The rib eye area showed medium heritability (0.32 ± 0.13). Whole-genome sequencing of 40 large rib eye sheep [17.97 ± 1.14, (cm2)] and 40 small rib eye sheep [7.89 ± 0.79, (cm2)] was performed. Case-control genome-wide association studies and the fixation index identified candidate rib eye-associated genes. Seven single nucleotide polymorphisms (SNPs) in six genes (ALS2, ST6GAL2, LOC105611989, PLXNA4, DPP6, and COL12A1) were identified as candidates. The study population was expanded to 1050 lambs to perform KASPar genotyping on five SNPs, which demonstrated that SNPs in LOC105611989, DPP6, and COL12A1 correlated significantly with the rib eye area, which could be used as genetic markers for molecular breeding of the rib eye area. The results provided genetic parameters estimated on the rib eye area and information for breeding based on carcass traits in Hu sheep.

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.

[Determination of thiourea dioxide in lotus seed paste fillings by solid phase extraction-liquid chromatography-tandem mass spectrometry].

A method of solid phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) was developed to determine thiourea dioxide which was illegally added into lotus seed paste fillings. An amount of 0.05% (v/v) acetic acid was used to extract thiourea dioxide from fillings, and the BOND ELUT PLEXA column (60 mg/3 mL) was used as the SPE column to clean-up the extraction. Then, an Agilent HILIC column (100 mm x 2.1 mm, 3.5 microm) was applied to separate target compounds by using the mobile phases of 0.01 mol/L ammonium acetate (pH 3.5) and acetonitrile. Qualitative and quantitative analyses were operated by the multiple reaction monitoring (MRM) mode. The calibration curve showed a good linearity for the target compound in the detection range of 10 - 1 000 microg/L. The limit of detection (LOD) and limit of quantitation (LOQ) of this method were 8.0 microg/kg and 30.0 microg/kg, respectively. The recoveries were in the ranges of 75.3% - 80.7% with the RSDs of no more than 4.83%. This proposed method was rapid, highly specific and suitable for the confirmation and quantitative determination of thiourea dioxide in lotus seed paste fillings.

Anti human immunodeficiency virus type 1 (HIV-1) agents 4. Discovery of 5,5'-(p-phenylenebisazo)-8-hydroxyquinoline sulfonates as new HIV-1 inhibitors in vitro.

To search for compounds with superior anti-human immunodeficiency virus type 1 (HIV-1) activity, ten 5,5'-(p-phenylenebisazo)-8-hydroxyquinoline sulfonates (4a-j) were synthesized and preliminarily evaluated as HIV-1 inhibitors in vitro for the first time. Some compounds demonstrated anti-HIV-1 activity, especially 5,5'-(p-phenylenebisazo)-8-hydroxyquinoline p-ethylbenzenesulfonate (4g) and 5,5'-(p-phenylenebisazo)-8-hydroxyquinoline p-chlorobenzenesulfonate (4i) showed the more potent anti-HIV-1 activity with 50% effective concentration (EC(50)) values of 2.59 and 4.01 microg/ml, and therapeutic index (TI) values of 31.77 and 24.51, respectively.

Synthesis of diaryl-azo derivatives as potential antifungal agents.

As compared with a commercially available agricultural fungicide hymexazol, some phenyl-azo phenol derivatives (e.g., 4a, 4b, 4f, 4n, 4q, 4u, and 4v) exhibited the more promising and pronounced antifungal activities in vitro against seven phytopathogenic fungi. It seemed that 4-((un)substituted phenylazo)-phenol and 4-((un)substituted phenylazo)-3-methylphenol might be considered as new lead structures for further design of agricultural fungicides.