Effects of Mobile Sheepfold and Supplementary Feeding on Growth Performance, Serum Indicators and Gut Microbiota in Natural Grazing Gangba Sheep

Simple Summary

Gangba sheep live in high-altitude areas characterized by cold weather and strong winds. To relieve environmental stress, and the grass shortage that comes with it, the protective effects of designed mobile sheepfolds and supplementary feeding on grazing Gangba sheep against extremely harsh natural conditions were examined. The results showed that mobile sheepfolds can effectively block wind and improve nighttime living conditions, making the environment more suitable for Gangba sheep breeding. This helps reduce environmental stress on the sheep and supports better welfares. Supplementary feeding supplies sufficient nutrition, which promotes weight gain and improves growth performance. Changes with sheepfolds also influence the gut microbial community, with more bacteria related to efficient energy use becoming dominant. These findings suggest that the microclimate modification has systemic metabolic effects mediated, at least in part, by intestinal microbial remodeling. Overall, combining wind-protected housing with proper nutritional support is a simple and practical way to improve the growth, health, and environmental adaptability of Gangba sheep living in extreme high-altitude regions.

Abstract

High-altitude grazing animals are continuously exposed to strong wind and low temperature, which challenge physiological homeostasis and energy metabolism. Improving living conditions and nutritional supplementation are two commonly used strategies. In this study, sixty 7-month-old Gangba sheep (initial body weight (BW) 21.00 ± 1.90 kg) were allocated to a 42-day trial with four groups (open-air sheepfold, mobile sheepfold, open-air sheepfold + supplementary feeding, mobile sheepfold + supplementary feeding) to investigate their effects on growth performance, serum parameters and gut microbiota in naturally grazing Gangba sheep. Mobile sheepfolds increased the temperature–humidity index (THI) and reducing the wind chill index (WCI) (p < 0.05). The sheep with mobile sheepfold showed higher serum total antioxidant capacity and lower levels of heat shock proteins HSP70 and HSP90 (p < 0.05), indicating alleviated stress. Supplementary feeding markedly increased final BW and average daily gain (p < 0.05). The interaction between sheepfold type and feeding supplementation showed increasing IgA levels in the open-air sheepfold with supplementary feeding group and increasing IL-4 levels in the mobile sheepfold with supplementary feeding group, while TNF-α concentrations were reduced in all three treatment groups (p < 0.05). Meanwhile, KB and FFAs were increased in the open-air sheepfold with supplementary feeding group but decreased in the mobile sheepfold with supplementary feeding group (p < 0.05). The mobile sheepfold also increased the Bacillota-to-Bacteroidota ratio, suggesting improved microbial community structure. Functional predictions showed enrichment of reductive acetogenesis and reduction in aerobic chemoheterotrophy and sulfur-related respiration pathways (p < 0.05). Moreover, key microbial genera were significantly correlated with THI and WCI (p < 0.05). Collectively, these results demonstrated that mobile sheepfold together with feeding supplementation improve stress responses, serum immune and lipid metabolic indicators, and potentially altered gut microbial composition and function, providing insights into host–microbiota interaction in extreme high-altitude environments.

1. Introduction

The Gangba sheep is a distinctive local breed distributed in Gangba County, Tibet, inhabiting the Qinghai–Tibet Plateau at an average altitude of about 5000 m, which encompasses one of the largest alpine grasslands in the world [1]. Gangba County lies at the northern foothills of the Himalayas and is characterized by a typical high-altitude plateau climate, featuring low temperatures, limited precipitation, large diurnal temperature fluctuations, long cold seasons, and short warm periods [2,3]. Due to forage shortages, Tibetan sheep—including the Gangba variety—under natural grazing often experience nutritional stress [4]. In the absence of human intervention, grazing Gangba sheep with insufficient feed supply are highly vulnerable to the harsh plateau environment, such as strong winds and low temperatures, resulting in reduced growth efficiency and compromised health [5]. Therefore, creating a comfortable living environment and ensuring adequate nutritional intake are crucial for improving livestock production efficiency and animal welfare in this region.

In open-air sheepfolds, environmental factors such as temperature, relative humidity and wind speed can constrain animal growth. Sheep grazing at high altitude are frequently exposed to low temperatures and strong winds that may impose cold stress. Cold stress has been reported to increase maintenance energy expenditure and lipid mobilization, thereby impairing growth performance and productivity in sheep [6]. Although Gangba sheep are well adapted to cold high-altitude environments, breed-specific thermal comfort thresholds have not yet been established. Therefore, the general thermal comfort ranges reported for sheep were used as a reference baseline for environmental evaluation in the present study. The average night temperature during warm seasons at Gangba County ranges from 10 °C to 15 °C, lower than the suggested thermal comfortable zone of goat and sheep, potentially affecting the health and growth of natural grazing Gangba sheep. Windbreaks can improve the microclimate for grazing sheep, reduce heat loss and increase productivity [6,7]. The wind chill index (WCI), which integrates wind speed and temperature, strongly influences the productivity of grazing sheep [8]. When the WCI is between 300 and 350, sheep are only slightly affected by cold winds, but when it exceeds 450, under severe cold stress, the mortality of lambs may increase [7,8]. Thus, improving the ambient temperature and wind speed in the sheepfold is essential for the production and health of Gangba sheep.

Information regarding supplementation with agricultural by-products in sheep remains limited. However, studies in other small ruminants, such as goats, have shown that such supplementation may improve growth performance under nutrient-restricted grazing conditions [9]. It has been reported that feeding made from corn straw and wet distiller grains, as well as agricultural waste products, does not substantially affect weight gain and meat quality in beef cattle [10]. Highland barley, a key crop on the Qinghai–Tibet Plateau with a 270,000-hectare planting area, makes up 90% of China’s total output and is both a staple feeding for Tibetans and is used for unique highland barley liquor [11]. Barley grain generates substantial agricultural by-products, including highland barley straw and highland barley distillery grains, which can be used as nutritional supplements, and provides high-quality supplementary feeding for grazing cattle and sheep.

Given the severe environmental constraints and nutritional deficits faced by naturally grazing sheep in Gangba County, strategies integrating housing improvement and targeted supplementation may represent practical approaches to enhance animal resilience and productivity under plateau conditions. However, the combined effects of microclimate modification and nutritional supplementation on physiological adaptation and gut microbial ecology in high-altitude sheep remain poorly understood. We therefore hypothesized that improving the nighttime microclimate through mobile sheepfolds, together with supplementary feeding based on local agricultural by-products, would alleviate cold-induced physiological stress, enhance antioxidant and immune status, and beneficially modulate gut microbial communities, thereby improving growth performance in Gangba sheep. Accordingly, the objectives of this study were to evaluate the effects of these integrated interventions on growth performance, serum antioxidant and immune indices, and fecal microbiota composition in naturally grazing Gangba sheep, providing mechanistic insights and practical guidance for sustainable livestock production on the Qinghai–Tibet Plateau.

2. Materials and Methods

The study was conducted from July to August at the Mende Village, Gangba County, Shigatse (Tibet, China). The natural pasture is located at an altitude of 4700 m, free from fertilizers and pesticides, with a longitude of 88.418° E and a latitude of 28.262° N. The Gangba sheep were allowed to graze freely during the day on the natural grasslands at Mende Village.

2.1. Preparation of Mobile Sheepfolds

The mobile sheepfolds were assembled with galvanized steel tubes welded together to form columns. The columns were connected into the shape of a house using a nut structure. A polyester silk base cloth with a waterproof coating was fixed to the house frame through ropes, and vents were installed. This design protected the sheep from wind and its size could be adjusted based on the sheep needing. Specifically, the smallest unit was designed with 4 m (length) × 2 m (width) × 1.8 m (height) for 5–10 Gangba sheep (Figure 1A,B). For this study, 3 units were used per group, totaling 12 units.

2.2. Ingredients and Nutrient Levels of Supplementary Feeding

The supplementary feeding was formulated to meet the NRC (2007) requirements for sheep, using locally sourced agricultural by-products, including highland barley distillers’ grains, sea buckthorn seed meal, and highland barley straw, along with a vitamin–mineral premix. The exact composition and nutrient levels of this supplement are detailed in

Table 1. Ingredients and nutrient levels of feed.

Item	Content
Ingredient, %
Corn	10.39
Wheat	10.51
Highland barley distiller’s grains	34.02
Spray-dried corn germ meal	10.15
Sea buckthorn seed meal	3.38
Molasses (sugarcane)	1.98
Highland barley straw	14.88
Murphy dregs	11.91
NaCl	0.55
Stone	1.35
Premix 1	0.68
Urea	0.20
Total	100
Nutrition level 2
Crude protein, %	11.27
Moisture content, %	44.10
Crude fat, %	1.91
Ash, %	5.61
Neutral detergent fiber, %	34.9
Acid detergent fiber, %	18.16
Gross energy, kcal/kg	4223

¹ Each kilogram of feed contains vitamin A: 325,000 IU, vitamin D3: 75,000 IU, vitamin E: 1500 IU, niacinamide: 320 mg, copper: 200 mg, iron: 1500 mg, manganese: 1300 mg, zinc: 2100 mg, iodine: 18 mg, selenium: 12.5 mg, cobalt: 6 mg, calcium: 13%, total phosphorus: 1.0%, sodium chloride: 25%. ² Measured values.

.

2.3. Experimental Design and Management

A 2 × 2 factorial design was employed in this study, using 60 male 7-month-old Gangba sheep with an average body weight (BW) of 21.00 ± 1.90 kg under natural grazing conditions. The primary factors were sheepfold type (canvas-covered mobile sheepfold vs. open pen) and supplementary feeding (with vs. without). The sheep were evenly assigned to 4 groups (15 animals per group, housed in three pens with five animals per pen): open-air sheepfold, mobile sheepfold, open-air sheepfold + supplementary feeding, and mobile sheepfold + supplementary feeding. The trial lasted for a total of 42 days; during the day (09:00–18:00), all sheep grazed together in the pasture of Mende Village. At night (18:00–09:00), they were housed in their respective sheepfolds. Sheep in the open-air sheepfold + supplementary feeding group and mobile sheepfold + supplementary feeding had free access to supplementary feed at night, and feed intake was recorded at the pen level.

2.4. Monitoring of Environmental Parameters

The temperature, relative humidity (RH), ammonia concentration, and wind speed were recorded every 3 h from 18:00 to 09:00 the following day in all groups using a remote environmental monitoring system equipped with integrated temperature, humidity, wind speed, and ammonia sensors (DYF-C360, Dayufeng Co., Ltd., Dalian, China). These parameters were used to evaluate the living environment of Gangba sheep. In addition, the temperature–humidity index (THI) and WCI were calculated using the following formulas (T, temperature; RH, relative humidity; and V, wind speed):

THI = (1.8T + 32) − (0.55 − 0.55RH/100) × [(1.8T + 32) − 58]

(1)

WCI = (10.45 + √V) × (33 − T)

(2)

2.5. Growth Performance and Sample Collection

All sheep were weighed on days 1 and 42, with the final BW recorded at the end of the trial to evaluate growth performance. Average daily gain (ADG) was calculated for each animal as (final BW − initial BW) divided by the number of experimental days (42 days). BW was measured individually before the morning feeding using a calibrated electronic livestock scale (Shenghui platform scale, Jinhua, China) to minimize variation due to gut fill. Before 09:00 on day 42, venous blood was collected from 6 Gangba sheep per group (two animals randomly selected from each pen; n = 6) using sterile plain vacuum blood collection tubes without anticoagulant or additives. The blood collection tubes were placed at a 45° angle for 30 min, then centrifuged at 3000× g for 20 min to collect serum. Meanwhile, feces from the corresponding sheep were collected directly from the rectum using sterile gloves by gentle rectal massage, temporarily stored with dry ice, and then promptly transferred to −80 °C for preservation.

2.6. Determination of Serum Indicators

The total antioxidant capacity (T-AOC, JN24358X), catalase (CAT, JN24314), glutathione peroxidase (GSH-Px, JN24284X), superoxide dismutase (SOD, JN24918X), malondialdehyde (MDA, JN24318X), total cholesterol (TC, JN24772X), triglycerides (TG, JN24770X), ketone bodies (KB, JN65546), and free fatty acids (FFAs, JN62835) in serum were measured using colorimetric kits (Shanghai Jining Institute of Bio-Engineering, Shanghai, China). Serum insulin (JN21254), interleukin-4 (IL-4, JN21766), IL-6 (JN21752), tumor necrosis factor-alpha (TNF-α, JN20906), immunoglobulin A (IgA, JN82299), IgG (JN82597) and IgM (JN20997) were measured using enzyme-linked immunosorbent assay kits, with the specific method selected according to the characteristics of each analyte (Shanghai Jining Institute of Bio-Engineering).

2.7. Fecal 16s rDNA Gene Sequencing Analysis

The microbial total DNA was extracted from fresh sheep fecal samples using the DNA extraction kit (Omega, Bio-tek, Norcross, GA, USA) and strictly following the kit’s operating procedures [12]. After extraction, the fecal microbial DNA was detected for its quantity and quality using 2% agarose gel electrophoresis and the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Specific primers with barcodes were synthesized for the V3–V4 hypervariable region of bacterial 16S rDNA for PCR amplification. The bacterial gene-specific primers were 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The ABI GeneAmp^® 9700 PCR instrument (Applied Biosystems, Foster City, CA, USA) was used, along with the TransGen AP221-02 system and PCR reaction system. The analysis was completed by Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Majorbio, Shanghai, China). The microbial DNA samples were subjected to detection, PCR, purification, library construction, and Miseq sequencing. All sequencing data were analyzed on the Majorbio I-Sanger Cloud Platform (https://cloud.majorbio.com/ (accessed on 14 March 2025)). Operational taxonomic units (OTUs) were clustered at a 97% similarity threshold for downstream analysis. Alpha diversity indices, including Ace, Chao1, Shannon, and Simpson indices, were calculated from the OTU table using Mothur software (version 1.30.1). Prior to diversity analysis, samples were normalized to an equal sequencing depth to minimize bias caused by uneven sequencing effort. Then, we utilized R language (version 3.3.1) to analyze the OTUs across different groups, and generated corresponding charts based on the statistics of shared and unique species in each group in a Venn diagram. Principal coordinate analysis (PCoA) was performed using the R package Vegan (version 2.5.3) based on Bray–Curtis distance. Additionally, microbial functional profiles were inferred using BugBase (phenotypic traits via Greengenes) and Functional Annotation of Prokaryotic Taxa (FAPROTAX, ecological functions, version 1.2.1).

2.8. Statistical Analysis

Data were analyzed using analysis of variance (ANOVA) with the general linear model in SAS 9.4 software (SAS Institute, Cary, NC, USA). Pens were considered the experimental units for feed intake and environmental parameters (n = 3 per treatment), whereas individual animals were treated as the experimental units for growth performance, serum biochemical indices, and microbiota analyses (n = 6 per treatment for sampled animals). The model included sheepfold type (open-air sheepfold or mobile sheepfold), supplementary feeding (with or without), and their interaction as sources of variation. When the factorial analysis indicated significant differences or trends among groups, further pairwise comparisons were performed using the contrast test. Results with p < 0.05 were considered statistically significant, while 0.05 ≤ p < 0.10 indicated a tendency. Spearman’s rank correlation analysis was performed to evaluate monotonic associations between microbial relative abundances and environmental parameters (THI and WCI) using GraphPad Prism (version 9.5; GraphPad Software, San Diego, CA, USA). Correlation coefficients were calculated, and statistical significance was determined at p < 0.05.

3. Results

3.1. Effects of Mobile Sheepfolds on Housing Environmental Parameters

The use of mobile sheepfolds altered nighttime temperature to varying degrees but had limited impact on relative humidity (Figure 1C,D). Specifically, mobile sheepfolds significantly increased temperature at 21:00, 03:00, and 06:00 compared with the open-air sheepfold groups in both mobile sheepfold treatments (with or without supplementary feeding) (all p < 0.05), while no significant differences were observed for relative humidity at any recorded time point. Meanwhile, mobile sheepfolds significantly increased ammonia concentration at all nighttime moments (18:00, 21:00, 00:00, 03:00, 06:00, and 09:00) and significantly reduced wind speed compared with the open-air sheepfold groups (all p < 0.05; Figure 1E,F). Correspondingly, THI values at 21:00, 03:00, 06:00, and the average THI were significantly higher, whereas WCI values at all time points and the average WCI were significantly lower in the mobile sheepfold groups relative to the open-air sheepfold groups (all p < 0.05; Figure 1G,H).

3.2. Effects of Mobile Sheepfold and Supplementary Feeding on Growth Performance

Supplementary feeding significantly increased the final BW of Gangba sheep in both open-air and mobile sheepfold groups (p < 0.001,

Table 2. Effects of sheepfold type and supplementary feeding on growth performance of natural grazing Gangba sheep.

Items	Sheepfold Type and Supplementary Feeding				SEM	p-Value
	Open-Air Sheepfold	Mobile Sheepfold	Open-Air Sheepfold + Supplementary Feeding	Mobile Sheepfold + Supplementary Feeding		Sheepfold	Supplementary Feeding	Interaction
Initial BW, kg	21.41	21.18	21.41	21.68	1.13	0.952	0.973	0.851
Final BW, kg	22.42 b	22.89 b	24.18 a	25.05 a	0.34	0.170	<0.001	0.686
ADG, g/d	47.86 d	64.52 c	90.05 b	104.05 a	6.40	0.013	<0.001	0.838
ADFI, g/d	--	--	643.01	669.52	--	--	--	--

BW, body weight; ADFI, average daily feed intake, ADG, average daily gain. ^a–d Values within a row with no common superscripts differ significantly (p < 0.05). --, not determined.

). Moreover, compared with the open-air sheepfold group, the remaining three groups (mobile sheepfold group, open-air sheepfold + supplementary feeding group and mobile sheepfold + supplementary feeding group) exhibited a higher ADG (p < 0.05). In addition, the increases in ADG were greater in the mobile sheepfold + supplementary feeding group than those in the mobile sheepfold group (p < 0.05).

3.3. Effects of Mobile Sheepfold and Supplementary Feeding on Serum Antioxidant Indicators and Heat Shock Proteins

Sheepfold type significantly affected serum T-AOC concentration (p < 0.05,

Table 3. Effects of sheepfold type and supplementary feeding on serum antioxidant indicators and heat shock proteins of natural grazing Gangba sheep.

Items	Sheepfold Type and Supplementary Feeding				SEM	p-Value
	Open-Air Sheepfold	Mobile Sheepfold	Open-Air Sheepfold + Supplementary Feeding	Mobile Sheepfold + Supplementary Feeding		Sheepfold	Supplementary Feeding	Interaction
T-AOC (U/mL)	12.93 b	18.52 a	12.62 b	18.58 a	1.15	0.020	0.955	0.936
MDA (nmol/mL)	12.04	14.09	14.53	12.98	0.64	0.845	0.596	0.176
GSH-Px (U/mL)	306.86	269.61	305.80	308.86	13.17	0.524	0.477	0.453
T-SOD (U/mL)	3.94	4.56	4.25	4.50	0.27	0.429	0.817	0.740
CAT (U/mL)	76.98	91.92	87.93	74.96	3.09	0.875	0.632	0.035
HSP70 (pg/mL)	512.27 a	374.33 b	459.84 a	304.17 c	28.86	<0.001	0.073	0.787
HSP90 (pg/mL)	2398.06 a	1979.54 b	2179.71 a	1565.57 c	115.49	0.005	0.067	0.556

T-AOC, total antioxidant capacity; MDA, malondialdehyde; GSH-Px, glutathione peroxidase; T-SOD, total superoxide dismutase; CAT, catalase; HSP70, heat shock protein 70; HSP90, heat shock protein 90. ^a–c Values within a row with no common superscripts differ significantly (p < 0.05).

). Sheep in the mobile sheepfold groups (with or without supplementary feeding) exhibited higher T-AOC levels than those in the two open-air sheepfold groups (p < 0.05). In terms of HSP70, sheepfold type significantly affected serum HSP70 concentration (p < 0.05), and supplementary feeding had a tendency (p = 0.073). Specifically, sheep in the mobile sheepfold groups (with or without supplementary feeding) exhibited lower HSP70 levels than those in the two open-air sheepfold groups (p < 0.05). Moreover, supplementary feeding further reduced serum HSP70 in the mobile sheepfold group (p < 0.05). Similarly, sheepfold type significantly affected serum HSP90 concentration (p < 0.05), and supplementary feeding had a tendency (p = 0.067). Specifically, sheep in the mobile sheepfold groups (with or without supplementary feeding) exhibited lower HSP70 levels than those in the two open-air sheepfold groups (p < 0.05). Moreover, supplementary feeding further reduced serum HSP90 in the mobile sheepfold group (p < 0.05).

3.4. Effects of Mobile Sheepfold and Supplementary Feeding on Serum Immune and Lipid Metabolism Indicators

Supplementary feeding significantly altered serum IgA levels (p < 0.05,

Table 4. Effects of sheepfold type and supplementary feeding on serum immune and lipid metabolism indicators of natural grazing Gangba sheep.

Items	Sheepfold Type and Supplementary Feeding				SEM	p-Value
	Open-Air Sheepfold	Mobile Sheepfold	Open-Air Sheepfold + Supplementary Feeding	Mobile Sheepfold + Supplementary Feeding		Sheepfold	Supplementary Feeding	Interaction
IgA (μg/mL)	24.42 b	28.31 ab	29.93 a	28.11 ab	0.08	0.196	0.034	0.024
IgG (μg/mL)	34.44	43.40	43.73	55.73	2.63	0.011	0.009	0.687
IgM (μg/mL)	1277.45	1632.87	1503.17	1685.59	67.70	0.011	0.161	0.377
IL-4 (pg/mL)	36.59 b	35.60 b	39.03 b	47.04 a	1.36	0.084	0.002	0.030
IL-6 (pg/mL)	102.84	101.19	102.66	119.49	7.56	0.486	0.407	0.398
TNF-α (pg/mL)	197.60 a	146.48 bc	158.51 b	134.47 c	3.12	0.0006	<0.001	0.042
TG (μg/mL)	508.10	592.86	600.00	632.38	35.51	0.242	0.636	0.295
TC (μg/mL)	1520.64	1527.00	1715.17	1534.63	86.16	0.483	0.417	0.452
KBs (mmol/L)	1.82 b	1.64 bc	2.46 a	1.48 c	0.04	0.014	<0.0001	0.0002
FFAs (μmol/L)	706.49 b	583.74 c	946.45 a	592.02 c	14.54	0.0004	<0.0001	0.0007
INS (μIU/mL)	29.99	29.97	27.46	25.78	1.49	0.691	0.127	0.699

IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M; IL-4, interleukin-4; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α; TG, triglycerides; TC, total cholesterol; KBs, ketone bodies; FFAs, free fatty acids; INS, insulin. ^a–c Values within a row with no common superscripts differ significantly (p < 0.05).

), and a significant interaction effect between sheepfold type and supplementary feeding was observed (p < 0.05). Specifically, serum IgA concentrations were significantly higher in the remaining three groups than in the open-air sheepfold group. Both sheepfold type and supplementary feeding significantly altered serum IgG levels (p < 0.05). Specifically, serum IgG concentrations were significantly higher in the three other groups than in the open-air sheepfold group, and they were highest in the mobile sheepfold + supplementary feeding group (p < 0.05). Additionally, sheepfold type significantly altered serum IgM levels (p < 0.05). Specifically, serum IgM concentrations were significantly higher in the three other groups than in the open-air sheepfold group. In terms of serum IL-4 levels, supplementary feeding significantly altered (p < 0.05), and a significant interaction effect between sheepfold type and supplementary feeding was observed (p < 0.05). Specifically, serum IL-4 concentrations in the mobile sheepfold + supplementary feeding group were significantly higher than those in each of the other three groups (p < 0.05). As for serum TNF-α concentrations, both sheepfold type and supplementary feeding significantly altered (p < 0.05), and a significant interaction between these two factors was observed (p < 0.05). Specifically, sheep in the mobile sheepfold groups (with or without supplementary feeding) had significantly lower TNF-α levels than those in the open-air sheepfold group (p < 0.05). Moreover, supplementary feeding also significantly reduced the serum level of TNF-α (p < 0.05). For lipid metabolism indicators, both sheepfold type and supplementary feeding significantly altered serum KB and FFA concentrations (p < 0.05), with a significant interaction observed (p < 0.05). Specifically, sheep in the open-air sheepfold + supplementary feeding group exhibited markedly higher serum KB and FFA levels than those in each of the other three groups (p < 0.05).

3.5. Fecal Microbial Community Structure and Functional Prediction

There were no significant differences for alpha diversity indexes, including Ace, Shannon, Chao1, and Simpson index (Figure 2A–D). Principal coordinate analysis (PCoA) (R = 0.50, p = 0.001) revealed that the mobile sheepfold significantly altered the microbiota composition and structure (Figure 2E). At the phylum level, compared with the two open-air sheepfold groups, the mobile sheepfold groups (with or without supplementary feeding) showed a significantly higher relative abundance of Bacillota and a higher Bacillota-to-Bacteroidota ratio, along with significantly lower abundances of Bacteroidota and Verrucomicrobiota (Figure 2F–J). At the genus level, the mobile sheepfold groups (with or without supplementary feeding) exhibited significantly higher relative abundances of Turicibacter, Candidatus_Saccharimonas, norank_f_norank_o_RF39, unclassified_f_Ruminococcaceae, and norank_f_norank_o_Clostridia_UCG-014, while the abundances of NK4A214_group, Akkermansia, Rikenellaceae_RC9_gut_group, and norank_f_UCG-010 were significantly lower (Figure 3A–L).

In addition, a Spearman correlation analysis between the top 20 genera of fecal microbiota and THI/WCI indexes showed that THI was positively correlated with the relative abundance of norank_f__norank_o__Clostridia_UCG-014 (R = 0.62; p < 0.01), unclassified_f__Ruminococcaceae (R = 0.52; p < 0.01), norank_f__norank_o__RF39 (R = 0.60; p < 0.01), and Candidatus_Saccharimonas (R = 0.55; p < 0.01) (Figure 3M). Conversely, THI was negatively correlated with the relative abundance of norank_f__UCG-010 (R = −0.59; p < 0.01), Rikenellaceae_RC9_gut_group (R = −0.56; p < 0.01), Akkermansia (R = −0.44; p = 0.03), and NK4A214_group (R = −0.65; p < 0.01). Additionally, WCI was positively correlated with the relative abundance of norank_f__UCG-010 (R = 0.71; p < 0.01), Rikenellaceae_RC9_gut_group (R = 0.71; p < 0.01), Akkermansia (R = 0.55; p < 0.01), and NK4A214_group (R = 0.70; p < 0.01). However, WCI was negatively correlated with the relative abundance of norank_f__norank_o__Clostridia_UCG-014 (R = −0.73; p < 0.01), norank_f__Eubacterium_coprostanoligenes_group (R = −0.45; p = 0.03), unclassified_f__Ruminococcaceae (R = −0.60; p < 0.01), norank_f__norank_o__RF39 (R = −0.64; p < 0.01), Candidatus_Saccharimonas (R = −0.57; p < 0.01), and Turicibacter (R = −0.54; p < 0.01).

In addition, the results of BugBase analysis showed that the use of a mobile sheepfold increased the proportions of phenotypes Contains Mobile Elements, Gram-positive and Facultatively Anaerobic, while it decreased the proportions of Gram-negative, Forms Biofilms and Aerobic phenotypes (Figure 4A–F). The FAPROTAX results showed that the use of mobile sheepfold increased the mean proportion of Reductive Acetogenesis, and decreased the mean proportions of Aerobic Chemoheterotrophy, Sulfate Respiration and Respiration of Sulfur Compounds (Figure 4G–J).

4. Discussion

Environmental conditions play a central role in livestock health and productivity, directly influencing growth performance and animal welfare [13]. Under cold conditions, animals must increase metabolic activity to maintain body temperature, which inevitably raises energy expenditure [14]. When low temperature is combined with strong wind, heat loss becomes more severe, further increasing metabolic burden and reducing productive efficiency [15,16]. In the present study, nighttime conditions in Gangba County were characterized by low temperature and high wind speed, with WCI frequently exceeding 450. Under such circumstances, sheep housed in open-air sheepfolds were exposed to substantial heat loss, which may have contributed to impaired metabolic status and reduced growth performance. In contrast, mobile sheepfolds effectively reduced WCI and increased the THI, which was associated with a more favorable nighttime environment and lower maintenance energy expenditure for thermoregulation, thereby allowing more dietary energy to be partitioned toward growth and physiological functions rather than heat production. Although ammonia concentrations were higher in mobile sheepfolds than in open-air sheepfolds, the values remained below the recommended safety threshold of 25 ppm [17], indicating that air quality did not pose a health risk. Together, these findings suggest that mobile sheepfolds provide a more suitable microenvironment for naturally grazing Gangba sheep, helping to maintain physiological stability and promote growth. In parallel, supplementary feeding significantly improved ADG and final BW, which is consistent with previous reports showing that agricultural by-products can effectively support growth in grazing goats and cattle [9,10]. These results further support that nutritional supplementation is associated with improved nutrient supply and performance maintenance, especially under harsh environmental conditions [18]. Although the improvement in ADG with supplementation was modest, additional nutrients under high-altitude conditions are likely utilized not only for growth but also for maintenance metabolism, thermoregulation, and immune function. Moreover, the supplementary feed was primarily composed of low-cost local agricultural by-products, which not only enhanced body energy reserves for overwinter survival but also supported adequate ADFI. Feed intake directly determines the supply of fermentable substrates available to the rumen and intestinal microbiota, thereby influencing microbial fermentation, short-chain fatty acid production, and overall energy harvest efficiency. Thus, stable or increased ADFI under mobile sheepfold and supplementation conditions likely contributed to improved feed efficiency, enhanced ADG, and the favorable microbial configurations observed in this study, indicating that supplementation is both biologically effective and economically justified in this production system.

Serum biochemical indices further supported the beneficial effects of mobile sheepfolds and supplementary feeding. Serum antioxidant capacity is commonly used as an indicator reflecting the ability of the organism to counteract oxidative stress [19,20], while heat shock proteins (HSPs) are widely considered markers of environmental and physiological stress [21]. Sheep housed in mobile sheepfolds exhibited higher total antioxidant capacity and lower concentrations of HSP70 and HSP90, indicating reduced oxidative and physiological stress under improved housing conditions. As molecular chaperones that are upregulated during environmental and metabolic stress, HSPs reflect the systemic energetic and inflammatory burden experienced by the host [6,21]. Therefore, their reduction under improved microclimatic conditions may signify lower maintenance energy demands and attenuated stress responses, which could indirectly favor more stable gut microbial activity and more efficient nutrient utilization. This indicates that mobile sheepfolds function not only as physical shelters but also as an important means of improving physiological resilience in grazing sheep. Immune-related parameters showed similar trends. Both mobile sheepfolds and supplementary feeding significantly influenced serum immunoglobulins and cytokines. The highest TNF-α levels were observed in sheep housed in open-air sheepfolds, indicating a stronger inflammatory response under harsher environmental exposure [22]. This response was clearly alleviated when mobile sheepfolds and supplementary feeding were applied. TNF-α is a key pro-inflammatory cytokine involved in initiating systemic inflammatory cascades, whereas cytokines such as IL-4 and IgA are associated with anti-inflammatory regulation and mucosal immune protection. Therefore, coordinated changes in these cytokines may reflect shifts in immune balance in response to environmental and nutritional stressors [23]. Reduced inflammatory activation and oxidative stress are expected to decrease maintenance energy expenditure, thereby improving metabolic efficiency and supporting growth. The significant interaction effects observed for IgA, IL-4, and TNF-α further suggest that improvements in housing and nutrition act synergistically to regulate immune homeostasis. This interpretation is consistent with previous studies demonstrating that improved housing conditions can modulate cytokine expression profiles and mitigate stress-related inflammation in livestock [24].

Changes in lipid metabolism indicators provided additional insight into energy utilization. Although TG levels were not significantly altered, a general increasing tendency was observed under mobile sheepfold and supplementary feeding conditions. Notably, the highest concentrations of ketone bodies and free fatty acids occurred in the open-air sheepfold with supplementary feeding group. This pattern suggests greater reliance on lipid mobilization in sheep exposed to cold and wind, despite adequate nutrient supply, with ketone bodies acting as key metabolic intermediates. Given that triglycerides and cholesterol are widely used as indicators of lipid storage and transport, whereas ketone bodies and free fatty acids reflect lipid mobilization and oxidation, and insulin regulates systemic energy metabolism [25,26,27,28], these changes collectively indicate altered energy partitioning among treatments. By reducing wind exposure, mobile sheepfolds may decrease the need for excessive lipid breakdown, thereby conserving energy for growth and body weight gain. These metabolic patterns are consistent with a shift from survival-oriented energy mobilization toward more efficient nutrient utilization for productive purposes.

At the microbial level, Bacillota and Bacteroidota dominated the intestinal community and showed clear shifts in response to housing conditions. These two phyla are generally associated with fiber fermentation and carbohydrate–protein metabolism, respectively [29,30]. A higher Bacillota-to-Bacteroidota ratio is often associated with enhanced energy storage, whereas a lower ratio may reflect increased metabolic demand or inflammatory activity [31]. 16S rRNA gene sequencing results revealed that mobile sheepfolds were strongly associated with microbial community structural differences. They increased the abundance of Bacillota, decreased that of Bacteroidota, and correspondingly raised the Bacillota-to-Bacteroidota ratio—indicating a potential shift toward microbial configurations related to improved energy harvest. Further correlation of the top 20 gut genera across four groups with THI and WCI pinpointed 11 genera whose abundance was associated with mobile sheepfolds and supplementary feeding. Classification of these differential genera revealed that the use of mobile sheepfolds increased the abundance of five genus-level microorganisms belonging to Bacillota, including Turicibacter, Candidatus_Saccharimonas, norank_f_norank_o_RF39, unclassified_f_Ruminococcaceae, and norank_f_norank_o_Clostridia_UCG-014. These genera have been previously reported to participate in fiber degradation and short-chain fatty acid production, which may enhance host energy harvest while also improving epithelial barrier integrity and modulating immune and inflammatory responses [32,33]. Therefore, the enrichment of these taxa may collectively contribute to improved metabolic efficiency and immune homeostasis, thereby linking microbial restructuring with the enhanced antioxidant capacity and growth performance observed in the host. Conversely, genera more abundant in open-air sheepfolds, such as the NK4A214_group and norank_f_UCG-010 of Bacillota can degrade cellulose and produce SCFAs [34]. Members of Bacteroidota and Verrucomicrobiota, including Rikenellaceae_RC9_gut_group, Bacteroides, and Akkermansia, are known to be involved in polysaccharide utilization, lipid metabolism, and intestinal barrier function [35,36,37,38]. Overall, these changes in microbial composition reflect adaptive shifts in the intestinal microbiome under different housing conditions rather than direct causal effects, adjusting community structure to meet host demands. This scenario was corroborated by BugBase and FAPROTAX predictions. Mobile sheepfolds increased the relative abundance of Gram-positive and Facultatively Anaerobic phenotypes (consistent with the rise in Bacillota), together with a higher prevalence of Contains Mobile Elements, reflecting enhanced genomic plasticity for rapid adaptation [39,40]. FAPROTAX further revealed a significant increase in Reductive Acetogenesis, an anaerobic carbon-fixing pathway associated with enhanced acetate yield and energy recovery. As acetate is the predominant volatile fatty acid and a major oxidative energy substrate in ruminants, increased acetate availability can be readily utilized for ATP production and lipogenesis, thereby supporting tissue accretion and growth performance. Meanwhile, Gram-negative taxa, biofilm-formers, and sulfate/sulfur respiration showed reduced abundance. These functional shifts may collectively contribute to lower membrane-associated inflammation and reduced toxic H₂S production, potentially favoring a more stable intestinal microenvironment [41]. Such reductions in intestinal inflammatory load may decrease systemic immune activation and maintenance energy costs, thereby allowing greater nutrient allocation toward growth and productive performance.

Taken together, the present findings show that mobile sheepfolds and supplementary feeding act in a complementary manner. By improving nighttime microclimate and nutritional status, they were associated with improvements in growth performance, antioxidant capacity, immune balance, and lipid metabolism, while simultaneously accompanying shifts in gut microbiota structure and function toward a more energy-efficient configuration. Importantly, these microbial alterations appear to functionally mediate host physiological adaptation by enhancing energy harvest and reducing inflammatory stress, thereby strengthening the conceptual link between intestinal ecology and systemic metabolism. The coordinated responses observed at physiological, metabolic, and microbial levels highlight the importance of integrating environmental management with nutritional strategies. Therefore, the combined use of mobile sheepfolds and supplementary feeding represents a practical and biologically sound approach for improving the productivity and health of naturally grazing Gangba sheep. Notably, sampling was mainly performed at the end of the trial, and temporal changes in gut microbiota and host physiology were not systematically assessed. Future longitudinal studies with multiple time points would further clarify their long-term dynamics and effects on animal performance. In addition, although highland barley distillery grains were selected due to their local availability and low cost, comparative evaluations with alternative commercial supplements and formal cost-effectiveness analyses were beyond the scope of this study and warrant further investigation.

5. Conclusions

This study confirmed that the mobile sheepfold with night-microclimate improvement and supplementary feeding with local agricultural by-products have positive effects on natural grazing Gangba sheep. The mobile sheepfold was associated with improved intestinal microbial structure under more favorable temperature–humidity and wind chill conditions, while the supplementary feeding enhances weight gain. They collectively improved growth performance, serum immunity and lipid metabolism, providing a solution and foundation for the efficient production of Gangba sheep on the Qinghai–Tibet Plateau, as well as the sustainable development of ecological animal husbandry in alpine grasslands.