All experimental procedures in this study were performed in accordance with Guidelines for the Care and Use of Agricultural Animals in Research and Teaching and approved by the Animal Care and Use Committee of China Agricultural University (Beijing, China, permit no. CAU 20150825-2). The experimental goats were reared on a commercial farm (YiWei White Cashmere Goat Farm) located in Inner Mongolia Autonomous Region, China, 39°06’N, 107°59’E, and the study lasted from April 2016 to April 2018.
Animals, Experimental Design And Management
Twenty-four female Inner Mongolian Cashmere goats were randomly allocated to two groups (n = 12): melatonin-treated (Melatonin) and control (Control) that were balanced for age, live weight and cashmere yield. Capsules containing melatonin were subcutaneously inserted behind an ear in cashmere goats on April 30 and June 30, 2016 at a dosage of 2 mg/kg live weight, which is based on our previous studies [3, 8]. The melatonin implants were purchased from Kangtai Biotechnology Co., Ltd (Beijing, China) and can release melatonin sustainably for two months. During the experimental period, all goats were on pasture from 0800 to 1700 h daily and were housed in an open barn from 1800 to 0700 h under natural photoperiodic conditions. The grassland in the area is part of semi-desertification grassland and included primary vegetation such as Caragana stenophylla poiark, Caragana rorsninskii kom, Agriopyron cristutum gaertn, Agriopyron cristutum schut, Alium polyrhizum turcz, Artemisia frigidi willd, Artemisia ordosica praschen, Stipa breviflora griseb, Haloxylon ammodendron bunge. The additional supplementary concentrates were provided to the goats from January to June, which was 0.275 kg/d per goat in January, gradually increased to 0.4 kg/d in April and subsequently increased to 0.55 kg/d in May and June to meet the needs of the animals during gestation and subsequent lactation. The supplementary concentrate consisted of 70% corn and 30% concentrate feed and was purchased from a local feed company (Baotou Jiuzhoudadi Biotech Company, Baotou, China). The does were mated in October, kidded in March and weaned in July. The experimental cashmere goats were combed on April 30, 2017 and April 30, 2018. The time when melatonin implants were inserted, and the estimated release period of melatonin are shown in Fig. 1.
Blood samples were collected one day before melatonin treatment and then were collected monthly on May, June, July, August, September 2016. In addition, blood samples were collected on April 30 and September 30, 2017. Blood samples were taken from a jugular vein of each goat via plain vacutainer tubes under low red-light conditions (< 3 lux) at night between 2300 and 2400 h. The blood samples were allowed to coagulate at room temperature before centrifugation at 3000 × g for 20 min. Serum was collected and stored at − 20 °C until analysis of melatonin concentrations. Total cashmere fleece weight of each goat was recorded at combing in April 2017 and April 2018. Before combing, cashmere samples within an area of 10 cm × 10 cm were shorn from the left midside of each goat close to the skin for the measurement of fibre staple length and diameter. Skin biopsies of each goat were obtained from the right midside flank region one day before melatonin treatment and then were collected monthly on May, June, July, August, September 2016. In addition, skin biopsies were collected on April and September 2017. Skin biopsies were excised as described by Duan  for analysis of hair follicle parameters.
Serum melatonin concentration was determined in duplicate by radioimmunoassay using a commercial RIA kit (BAR-3300, LDN, Nordhorn, Germany) which had a sensitivity of 2.3 pg/ml and intra- and inter-assay CVs of 9.7%~13.4% and 8.0%~13.3%, respectively.
Cashmere Fibre Measurements
Cashmere fibre staple length was measured with a ruler as described by Yang  and cashmere fibre diameter was measured by an optical microscopic projection method as described by Duan . Cashmere fibre density was determined by measuring the active secondary hair follicle density in the skin obtained on September when the active secondary hair follicle numbers reach their seasonal maximum and agree with the cashmere fibre numbers.
Hair Follicle Parameters
Skin samples were embedded in paraffin after dehydration via a series gradient of ethanol solutions, and sections were sliced into three 5 µm sections and then stained by a modified Sacpic method . The sub-epidermal level immediately below the sebaceous glands was selected for carrying out microscopical observations. In this study, primary hair follicles and secondary hair follicles were evaluated separately. They are easily distinguished from each other based on their specific characteristics as described by Yang . Hair follicles were classed as active or inactive according to their size, shape, staining and the presence or absence of an inner root sheath. The basic criterion for distinguishing an active from an inactive follicle is the presence of a fibre and inner root sheath cells. In an active follicle stained with the Sacpic technique a yellow stained fibre is surrounded by a distinct bright red-stained inner root sheath, whereas non-active follicles were identified by the absence of these features and the presence of a quiescent hair germ or a fibre with a brush end.
All statistical analysis was performed with SAS statistical software (version 9.2; SAS Institute Inc., Cary, NC, USA). The significance of differences in cashmere production (cashmere weight, fibre staple length, fibre diameter and fibre density) between melatonin-treated and control cashmere goats was analyzed by the independent sample t-tests procedure. Statistical analysis of melatonin concentrations and hair follicle parameters (follicle density, ratio of secondary to primary follicles, percentage of active hair follicles) were performed with the repeated measures ANOVA procedure, and data are presented as least squares means ± SEM. Melatonin treatment was between-subject effect and time was within-subject effect. The difference was considered significant at P < 0.05 and a tendency was declared at 0.05 < P < 0.10.