The study was carried out at the College of Veterinary Medicine and Animal Science (FAMEZ) of the Federal University of Mato Grosso do Sul (UFMS), Campo Grande, MS, Brazil (20° 26′ 50′′ S, 54° 50′ 21′′ W, altitude 417 m a.s.l.). The annual and regular rainfall is 1,625 mm (ranging from 1500 to 1750 mm), with a dry period of less than four months, corresponding to a water deficit of 350 to 500 mm.
All procedures involving animals in this study followed the guidelines of the National Council for the Control of Animal Experimentation and approved by the Ethics Committee in the Use of Animals of the Federal University of Mato Grosso do Sul (Protocol n. 805/2016). Study is reported in accordance with The ARRIVE (Animal Research: Reporting of in vivo Experiments) guideline25.
Thirty-six Nellore steers with an average initial weight of 250.0 kg and approximately 15 months of age were used. Animals were identified with plastic earrings, submitted to parasite control, and vaccinations according to the sanitary calendar. All animals were kept in Marandu grass pasture (Brachiaria brizantha Stapf cv. Marandu). The experimental area consisted of four paddocks (4.0 hectares each), with two paddocks per treatment with nine animals each, according to a completely randomized design.
Two supplements were formulated (Table 3), containing different protein source (extruded urea and soybean meal) with 41% and 26% of crude protein based on dry matter, respectively, for different seasons (Table 4). Supplements comprised mineral, grounded corn, and soybean meal or extruded urea. Nellore steers were supplemented with 0.45% of body weight (BW) during the rainy season (summer) and 0.7% of BW in the dry season (transition period summer–winter). The extruded urea used was Amireia-200® (Pajoara Ind. e Comércio Ltda. Campo Grande—Mato Grosso do Sul State, Brazil).
To evaluate the skin, a 1.0 cm2 trichotomy was performed in the left lateral region at the average height of the side of each steer. The fur coat thickness (CT) was measured from the perpendicular distance between the fur insertion points in the epidermis to the fur coat surface with a digital electronic caliper (model 799A-6/150, Starrett brand).
Skin biopsy26 was performed at three different times: summer (December), autumn (April), and winter (July). For the morphological evaluation of the skin, after asepsis with alcohol-iodine, a transverse fragment, with 1 cm in diameter, was collected from the left dorsal region (same site of the trichotomy), with the aid of a “Punch” type pourer, after administration of local anesthetic (2% lidocaine hydrochloride), subcutaneously. After removing the skin fragment, the perforation in the epidermis was sutured with a surgical stitch. Skin samples were fixed in 10% formaldehyde.
The samples were sent to the Histology Laboratory (FAMEZ/UFMS), dehydrated in ascending series of alcohol (70, 90, and 100%), treated with xylene and infiltrated in paraffin. The small blocks formed were cooled for 10 min to become more consistent and to facilitate microtomy. Three skin fragments from each animal were obtained by transverse incisions, and serial cuts (7 µm thick) were performed in each block. After that, they were deparaffinized and stained with hemotoxylin-eosin27.
The tissue sections were employed for quantifying the epithelial thickness (measured in linear µm) and dermal thickness (also measured in linear µm). For the sweat glands, we assessed their area (in µm2), depth (measured in linear µm), and the total count of sweat glands. Furthermore, we determined the number of hair follicles by utilizing the Image Analysis System (specifically, Image Pro-Express v.6.0) in conjunction with a micrometric scale, which was integrated with a light microscope from the Olympus brand (model BX40) using a 10 × objective.
The study of the morphology of the skin samples, as a function of the nutritional treatment, was carried out by scanning electron microscopy (SEM) in a JEOL equipment, model JSM 6380LV, at Physics Institute (INFI/UFMS). Samples in their original form (skin) were dispersed on double-sided carbon tape for fixation on top of a copper stub. The set was taken to the interior of the sputtering chamber (Denton Vacuum, Desk III model) for the deposition of a thin layer of gold on the surface of the dust particles to obtain a better resolution of the SEM image and an increase in conductivity. Sample power to avoid the effects of electronic charging that degrade image resolution. For the analysis, a voltage of 15 kV, working distance (WD) 12 mm and spot size 20 were used. In the context of observational analyses, measurements were collected, and images were captured at a magnification level of 2500 × to facilitate comparative morphological assessments.
The data analysis followed a completely randomized design implemented within a split-plot framework. In this setup, protein sources were designated as the primary plot, and the different time points across the year were assigned to the subplot. This analysis was carried out using PROC GLM within the SAS statistical package (SAS University Edition, Sas Institute Inc., Cary, CA, USA). To compare the means of different protein sources, we applied the F test, while for comparing means across different seasons, the Dunnett test was employed, with the commencement of summer serving as the control (initial sampling). Statistical significance was determined at a 5% level.
Additionally, Pearson’s correlation coefficients between variables were calculated using the PROC CORR procedure within SAS (SAS University Edition, Sas Institute Inc., Cary, CA, USA).
