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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]