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Background: In quantitative real-time polymerase chain reaction (qRT-PCR) experiments, accurate and reliable target gene expression results are dependent on optimal amplification of house-keeping genes (HKGs). RNA-seq technology offers a novel approach to detect new HKGs with improved stability. Goat (Capra hircus) is an economically important livestock species and plays an indispensable role in the world animal fiber and meat industry. Unfortunately, uniform and reliable HKGs for skin research have not been identified in goat. Therefore, this study seeks to identify a set of stable HKGs for the skin tissue of C. hircus using high-throughput sequencing technology. Results: Based on the transcriptome dataset of 39 goat skin tissue samples, 8 genes (SRP68, NCBP3, RRAGA, EIF4H, CTBP2, PTPRA, CNBP, and EEF2) with relatively stable expression levels were identified and selected as new candidate HKGs. Commonly used HKGs including SDHA and YWHAZ from a previous study, and 2 conventional genes (ACTB and GAPDH) were also examined. Four different experimental variables: (1) different development stages, (2) hair follicle cycle stages, (3) breeds, and (4) sampling sites were used for determination and validation. Four algorithms (geNorm, NormFinder, BestKeeper, and ΔCt method) and a comprehensive algorithm (ComprFinder, developed in-house) were used to assess the stability of each HKG. It was shown that NCBP3 + SDHA + PTPRA were more stably expressed than previously used genes in all conditions analysis, and that this combination was effective at normalizing target gene expression. Moreover, a new algorithm for comprehensive analysis, ComprFinder, was developed and released. Conclusion: This study presents the first list of candidate HKGs for C. hircus skin tissues based on an RNA-seq dataset. We propose that the NCBP3 + SDHA + PTPRA combination could be regarded as a triplet set of HKGs in skin molecular biology experiments in C. hircus and other closely related species. In addition, we also encourage researchers who perform candidate HKG evaluations and who require comprehensive analysis to adopt our new algorithm, ComprFinder. [ABSTRACT FROM AUTHOR]

Improving the hair follicle density (HFD) has long been a breeding goal, as it can directly affect the fiber yield of animals, such as cashmere goats, sheep, rabbits and alpacas. Due to sample size and time limitations, the HFD cannot be easily obtained from a large histological area. If the area is too small, the experimental sampling error cannot be ignored. The number of view-fields (fieldN), the area of view-field (fieldS), and the total statistical area (total SA, fieldN×fieldS) needed in the HFD examination have not been determined. Thus, we aimed to develop a strategy to address this unclear topic. Goatskin was obtained as the sample, and paraffin sectioning and hematoxylin-eosin staining were done. Images of each skin sample were captured, stitched, and clipped. All hair follicles (HFs) were marked manually as color dots, and the background noise was removed. We acquired 52 clean images (50 mm2 for each) and considered 10 types of fieldN, fieldN∈[1,10] and 14 types of fieldS, from 32.0 mm2 to 0.10 mm2. A Matlab-based algorithm/program, MinSA, was developed to calculate the mean relative error (MRE) under different fieldN and fieldS. In fieldN = 1, the field area was 5.04 mm2 where MRE reached 5%, indicating that if the field area were less than 5.04 mm2 (one field) for the HFD examination, the MRE would be higher than 5%. As the fieldN increased from 2 to 10, the fieldS at 5% MRE gradually reduced, but the total SA increased from 4.45 to 5.49 mm2. This result demonstrates that using multiple view-field was not a good choice. In goats, 1–3 fields are recommended, and the total statistical area should be over 5 mm2 of stained skin section. Furthermore, this algorithm could apply to other economic fur-bearing animals. • Hair follicle density (HFD) of goats was measured at the histological level. • MinSA tool was developed to measure the accuracy of HFD examination. • Fewer large view-fields are more appropriate than multiple smaller view-fields. • In HFD examination of goat, over 5 mm2 of the stained section should be inspected. • MinSA algorithm can be applied to other fur-bearing animals. [ABSTRACT FROM AUTHOR]

Despite the significant advantages of using indigenous Chinese goat breeds in cashmere production, yield improvement is an ongoing challenge to cashmere goat breeding. This study aimed to investigate the occupied proportion of hair follicle group (HFG) and examine its influence on total hair follicle density (THFD). A total of 459 skin samples from the Inner Mongolia cashmere goat were processed for paraffin embedment, transverse sectioning, and H&E staining. An image of 9.26 mm2 was captured for each sample. All hair follicles were marked as color dots and the areas occupied by HFGs were filled by color blocks. Color blocks and the corresponding color dots were then analysed to calculate parameters. Our results showed that those samples with the higher THFD had the following characteristics: the size of the HFG (Size-HFG) was smaller, the HFGs (Num-HFG) was denser, the area proportion of the HFGs (Ratio-HFG) was larger, and the hair follicle density within HFG (Int-THFD) was higher. For each sample, its THFD was directly determined by the Int-THFD and Ratio-HFG. By improving one or both of Int-THFD and Ratio-HFG, a higher THFD can be achieved, thereby increasing the cashmere yield. This work implied that the Inner Mongolia cashmere goat still has upward potential for THFD improvement. • Histological image analysis was done on 459 skin samples of Inner Mongolia cashmere goat. • The total hair follicle density (THFD) of Inner Mongolia cashmere goats varied greatly. • The occupied proportion of hair follicle groups (Ratio-HFG) in goat skin was investigated. • The higher the Ratio-HFG, the higher the THFD. • The Inner Mongolia cashmere goat has upward potential for THFD improvement. [ABSTRACT FROM AUTHOR]

• The histological examinations of hair follicle density were performed on 14 body sites from 11 Inner Mongolia cashmere goats. • The pattern of total hair follicle density (THFD) was unevenly distributed. • The THFD was high in front and rear, and low in the bottom of the body of Inner Mongolia cashmere goat. • Sites belonging to the body side can be the representative sampling area. In this study, hair follicle density (HFD) was compared among different body sites in Inner Mongolia cashmere goat (Capra hirus). Skin samples from 14 different body sites of 11 Inner Mongolia cashmere goats were collected. A total of 154 skin samples were processed for paraffin embedment, sectioning and H&E staining. Images of 9.26 mm2 were captured by a microscope camera, and primary hair follicle density (PHFD), secondary hair follicle density (SHFD), the ratio of second to primary hair follicle density (S/P) and total hair follicle density (THFD) were calculated. The results showed that there was no significant difference in HFD between females and males. The 14 body sites were clustered into three categories according to the HFD. The THFD of high-density, medium-density and low-density groups was 50.0 ± 8.5, 41.6 ± 7.1 and 26.6 ± 6.2 follicles/mm2 of stained skin section, respectively, and the significant differences (P < 0.01) were found among them. High and low HFD existed in front and rear, and bottom of the body, respectively. These sites from the body of Inner Mongolia cashmere goats had medium value, but had very high value to represent all sites. [ABSTRACT FROM AUTHOR]

Coat color is one of the major quality traits of animals, and miR-129-5p acts as an important regulator for melanin biosynthesis in mammals. In this study, real-time PCR and western blotting were used to examine the expression of miR-129-5p and its targets genes in the skin of different coat color goats. The results showed that the expression of miR-129-5p in the skin samples of Inner Mongolia cashmere goats (IMCG) was higher than that of Dazu black goat (DBG). Also, the target genes (tyrosinase (TYR), frizzled 6 (FZD6) and glycogen synthase kinase 3β (GSK3β)) of miR-129-5p was highly expressed in the skin samples of DBG. The expression of miR-129-5p firstly increased and then decreased with age in F1 hybrid generation of DBG and IMCG. In addition, the expression of TYR decreased with age, while the expression of MITF increased with age but then decreased. The expression of FZD6 and GSK3β in the skin samples of F1 of different ages were irregular. Our results indicated that miR-129-5p mainly affects the formation of coat color of goats by decreasing the expression of TYR. This study suggests that miR-129-5p can act as a suppressor in the formation of coat color to lay the foundation for studying the effect of miR-129-5p on melanin synthesis. [ABSTRACT FROM AUTHOR]

Simple Summary: To explore the genomic variations associated with goats' body conformation traits, we performed a genome-wide association study (GWAS) on Tashi goats. We assessed eight body conformation traits in 155 Tashi goats and performed whole-genome sequencing on 100 of them. We obtained 1676.4 Gb of raw data and identified 11,257,923 qualified single nucleotide polymorphisms (SNPs). GWAS revealed 109, 20, 52, 14, 62, 51, 70, and 7 SNPs significantly associated with body height, body length, chest depth, chest width, chest girth, rump width, rump height, and cannon bone circumference, respectively. We annotated 183 genes based on the significant SNPs' physical locations. Notably, several SNPs have been identified multiple times in two regions, chr.10:25988403-26102739 and chr.11:88216493-89250659, where candidate genes such as FNTB, CHURC1, and RNF144 could be crucial for goat body conformation traits. Our findings offer significant insights into body conformation and support the use of molecular breeding in meat goats. Identifying genetic markers of economically valuable traits has practical benefits for the meat goat industry. To better understand the genomic variations influencing body conformation traits, a genome-wide association study was performed on Tashi goats, an indigenous Chinese goat breed. A total of 155 Tashi goats were phenotyped for eight body conformation traits: body height, body length, chest depth, chest width, chest girth, rump width, rump height, and cannon bone circumference. Then, 100 Tashi goats were randomly selected for whole-genome sequencing and genotyped. We obtained 1676.4 Gb of raw data with an average sequencing depth of 6.2X. Clean reads were aligned to the ARS1.2 reference genome, and 11,257,923 single nucleotide polymorphisms (SNPs) were identified. The structure analysis showed that these Tashi goats were almost not genetically related. The 109, 20, 52, 14, 62, 51, 70, and 7 SNPs were significantly associated with body height, body length, chest depth, chest width, chest girth, rump width, rump height, and cannon bone circumference. Within the ±500 kb region of significant SNPs, 183 genes were annotated. The most significantly enriched KEGG pathway was "olfactory transduction", and the most significantly enriched gene ontology (GO) terms were "cellular process", "cellular anatomical entity", and "molecular transducer activity". Interestingly, we found several SNPs on chromosomes 10 and 11 that have been identified multiple times for all eight body conformation traits located in two fragments (114 kb and 1.03 Mb). In chr.10:25988403-26102739, the six SNPs were tightly linked, the TACTAG genotype was the highest at 91.8%, and the FNTB (Farnesyltransferase, CAAX Box Beta) and CHURC1 (Churchill Domain Containing 1) genes were located. In chr.11:88216493-89250659, ten SNPs were identified with several dependent linkage disequilibrium (LD) blocks, and seven related genes were annotated, but no significant SNP was located in them. Our results provide valuable biological information for improving growth performance with practical applications for genomic selection in goats. [ABSTRACT FROM AUTHOR]

Reproductive traits are essential economic traits in goats. This study aimed to analyze the relationship between single nucleotide polymorphisms (SNPs) within the genes of GLRB, GRIA2, and GASK1B, and reproductive traits (kidding traits and placental traits) in goats. We used the resequencing data of 150 Dazu Black Goats to perform correlation analysis with the average litter size. We screened thirteen SNPs loci in introns and then used the Sanger method to genotype the remaining 150 Dazu Black Goats. The results showed that a total of six SNPs were screened. Three SNPs related to litter size and live litter size (g.28985790T > G, g.28986352A > G, and g.28987976A > G); one SNP related to total cotyledon area (g.29203243G > A); two SNPs related to placental efficiency (g.30189055G > A and g.30193974C > T); one SNP associated with cotyledon support efficiency (g.30193974C > T). The qPCR results showed that GLRB, GRIA2, and GASK1B were all highly expressed in the udder, kidney, uterus, and ovary. It indicated that these three candidate genes might affect the reproductive traits, which could be used as candidate markers for reproductive traits in Dazu Black Goats. Moreover, association studies on a large scale are still needed to figure out what effect these SNPs have on reproductive traits. [ABSTRACT FROM AUTHOR]

Reproductive traits are the basic economic traits of goats and important indicators in goat breeding. In this study, Dazu black goats (DBGs; n = 150), an important Chinese local goat breed with excellent reproductive performance, were used to screen for important variation loci and genes of reproductive traits. Through genome-wide association studies (GWAS), 18 SNPs were found to be associated with kidding traits (average litter size, average litter size in the first three parity, and average litter size in the first six parity), and 10 SNPs were associated with udder traits (udder depth, teat diameter, teat length, and supernumerary teat). After gene annotation of the associated SNPs and in combination with relevant references, the candidate genes, namely ATP1A1, LRRC4C, SPCS2, XRRA1, CELF4, NTM, TMEM45B, ATE1, and FGFR2, were associated with udder traits, while the ENSCHIG00000017110, SLC9A8, GLRB, GRIA2, GASK1B, and ENSCHIG00000026285 genes were associated with litter size. These SNPs and candidate genes can provide useful biological information for improvement of the reproductive traits of goats. [ABSTRACT FROM AUTHOR]

Simple Summary: Body conformation traits are economically important in the goat meat industry. Good growth performance in goats, including an accelerated growth rate, can improve carcass weight and meat yield. The identification of genetic variants associated with these traits provides a basis for the genetic improvement of growth performance. In this study, we measured six body conformation traits, including body height, body length, cannon circumference, chest depth, chest width, and heart girth. By a genome-wide association study of a Chinese meat goat breed, 53 significant single nucleotide polymorphisms and 42 candidate genes associated with these traits were detected. These findings improve our understanding of the genetic basis of body conformation traits in goats. Identifying associations between genetic markers and economic traits has practical benefits for the meat goat industry. To better understand the genomic regions and biological pathways contributing to body conformation traits of meat goats, a genome-wide association study was performed using Dazu black goats (DBGs), a Chinese indigenous goat breed. In particular, 150 DBGs were genotyped by whole-genome sequencing, and six body conformation traits, including body height (BH), body length (BL), cannon circumference (CC), chest depth (CD), chest width (CW), and heart girth (HG), were examined. In total, 53 potential SNPs were associated with these body conformation traits. A bioinformatics analysis was performed to evaluate the genes located close to the significant SNPs. Finally, 42 candidate genes (e.g., PSTPIP2, C7orf57, CCL19, FGF9, SGCG, FIGN, and SIPA1L) were identified as components of the genetic architecture underlying body conformation traits. Our results provide useful biological information for the improvement of growth performance and have practical applications for genomic selection in goats. [ABSTRACT FROM AUTHOR]