Effects of Low Temperature and Wind Treatment on Physiological Indexes, Rumen Microbiota, Immune Responses and Hormones in Sheep

Background: Low-temperature environments can strongly affect the normal growth and health of livestock. Previous studies have shown that cold exposure can alter the intestinal microbiota and thereby affect other traits. In winter, cold weather can be accompanied by strong winds that aggravate the effects of cold on livestock. In this study, an experiment was conducted to investigate the effect of low temperature and wind speed on physiological indexes, rumen microbiota, and immune responses in sheep. Methods: The sheep were divided into control group and test group according to their ambient temperature.Sheep in the test group were divided into four groups according to wind-speed treatment: no wind (average wind velocity less than 0.5 m/s), low wind velocity (average wind velocity of 3 m/s), medium wind velocity (average wind velocity of 4 m/s) and high wind velocity (average wind velocity of 5 m/s). Results: Average daily gain and the utilization of forage, especially soluble ber, decreased with increasing wind velocity in cold temperature (P<0.05). In rumen, the enzyme activity of cellulose degradation was also lowerwith increasing wind velocity (P<0.05). The abundance of potentially benecial bacteria showed differedamong the wind treatments (P<0.05).The large uctuations in the amount of bacteria provided a breeding opportunity forpotentially harmful bacteria (P<0.05). In addition, there were signicant decreases in the serum levels of IL-2 and IFN-γ (P<0.05) and a large increase in IL-4 level (P<0.05), which indicated that the sheep underwent immune suppressionduring the trial. The signicant increase in the activities of the antioxidant enzymes SOD, GSH-PX, and CAT (P<0.05) indicated that the production of oxygen free radicals was increased. Conclusions: The cold environment signicantly the growth of sheep and altered the composition of rumen reducing the utilization of soluble ber the rumen Furthermore, the sheep large


Background
Climate change can cause stress responses in animals [1], especially sharp decreases in temperature. In cold environments, the weight of livestock tends to be reduced. It has been found that cold exposure alters the composition of the intestinal microbiota by affecting food intake [2,3], which could cause other changes in animal phenotype.
Studies in many monogastric animal models have investigated correlations between the intestinal microbiota and host phenotype [4]. In ruminants, the rumen is a large fermentation site containing microbes. Similar to the intestinal microbiota, the rumen microbiota could interact with the host [5]. Furthermore, the host could limit the abundance and community composition of rumen microbes to maintain homeostasis [6]. In turn, the rumen microbiota composition could affect in ammation and oxidation, which can be measured by in ammatory-related markers, such as interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), and interferon-γ (IFN-γ), and markers of oxidative stress, such as malondialdehyde (MDA) and various antioxidant enzymes, respectively. The rumen epithelium could produce hormones that in uence phenotype, such as peptide-1, peptide YY, ghrelin, and leptin [7,8].
Changes in the rumen microbiota could affect metabolites and the energy supply to tissues by producing volatile fatty acids (VFAs) by fermenting carbohydrates and in ammatory reaction [7,9,10]. Cold exposure could lead to changes in the intestinal microbiota in monogastric animals and affect host oxidation and in ammation [11]. Many studies have shown that cold exposure could alter the intestinal microbiota. However, the effects of a cold environment on the rumen microbiota are unclear.
High-altitude areas in the Northern Hemisphere experience low temperatures in winter, which reduce the productivity and feed-utilization e ciency of livestock and severely constrain the economic bene ts of animal husbandry [12]. In northern China, the cold winter temperatures are always accompanied by strong northwest winds. Wind and low temperature aggravate the convection and heat dissipation of the body, imposing cold stress on livestock. In this experiment, we varied wind speed to investigate how cold exposure affects rumen microbiota and metabolism in sheep. We hypothesized that a cold environment affects the rumen microbiota and that changes to their metabolites affect host phenotype in ruminants.

Experimental animals and design
In this experiment, the test site and the corresponding test materials were provided by Linze Experimental Station of Lanzhou University, China. Twelve healthy 6-month-old crossbred hybrid ewes (small-tailed Han sheep × Hu sheep; mean body weight (BW) 30.36 ± SE 1.68 kg; not pregnant before the experiment) were randomly allocated into three groups: I, II and III (four sheep per group).
The experiment spanned 20 days, comprising 10 days for adaptation and 10 days for the trial period. The sheep were divided into a control group and treatment groups. Andthe experimental condition other than wind sheep were the same across the treatment groups (including feeding management, test time, time of use of metabolic cages, etc.). According to records of annual average wind speed in the Atlas of Natural Disaster Systems in China, the annual average wind speed in the study area is approximately 3~4 m/s; this range was used to set the wind-speed gradient. Increasing wind speed increases thermal convection, exacerbating the effect of cold stress on sheep. The sheep in the treatment groups were subjected to one of four wind-speed treatments: no wind (average wind speed or less than 0.5 m/s, low temperature (LT)), low wind (average wind speed of 3 m/s, LW), medium wind (average wind speed of 4 m/s, moderate wind (MW)) and high wind (average wind speed of 5 m/s, high wind (HW)).To reduce the in uence of diurnal temperature variation on the results, wind-speed treatment was performed from 8:00 p.m. to 8:00 a.m. of the second day, and the rest of the time was used as a rest and recovery period.During the test period, the outdoor night average temperature was -17°C and the average temperature in the sheep house was approximately 5°C.
The test was carried out in two stages. In the rst stage, the test sheep of groups I, II and III were allocated as the control group(C), no wind under low temperature(LT), and low windunder low temperature (LW), respectively. In the second stage of the test, the sheep of groups II and III were treated with MW and HW, respectively, under LT. Stage 1 spanned days 10 to 15, and stage 2 spanned day 16 to 20. There was no interval between the two phases, which spanned 5 days each (See Additional le 1: Fig.S1). This procedure ensured that the test sheep gradually adapted to cold stress, which increased from low to high, avoiding injury or death due to direct exposure to high-intensity cold stress. In addition, compared with a sudden change, a gradual change in temperature more closely resembles temperature change in the eld.
The wind treatments were carried out by industrial electric fan ( 65-1, Watson, China; maximum wind speed, 6.5 m/s). In each treatment, four fans were used to create wind in different directions to ensure a uniform wind speed. During the test period, a windproof barrier was built at the test site to avoid interference from external natural wind. The external wind speed was monitored throughout the experiment. Testo 405-v1 anemometer (testto405-v1,tmall, Germany) was used to measure the wind speed at different points in the sheepfold and calculate the average wind speed.

Feeding and management
To avoid excessive differences in wind speed among different places in the large sheepfold, two open sheep pens (2.5 × 2.5 m) were set up in the sheepfold before the experiment began and sterilized. The experimental sheep were fed twice daily (at 9:00 a.m. and 6:00 p.m.), and some residual feed remained after each feeding, indicating that the sheep had been adequately fed.
The sheep were fed complete formula pellet feed (582 Formula Feed, Yuansheng, 120 China). The feed composition and nutrient levels are shown in Table S1.Before the test period, due to the low ambient temperature, the water in the water tank provided for drinking completely froze for approximately 1 to 2 h each day. Therefore, the water tank was regularly monitored during the study and any ice removed to ensure sheep access to drinking water.

Sampling
In each treatment group, the sheep were fed in a metabolic cage from the third day to the fth day of treatment. The sheep were weighed without feeding at 8:00 a.m. before entering the metabolic cage, and the average daily gain (ADG) was calculated. In the morning of the 4th, 5th, and 6th days, all feces and urine were collected and weighed, and the daily fecal and urine volumes were recorded. The collected feces and urine were then stored at -20°C in a refrigerator. The collected fecal samples and feed samples were dried in an oven at 65°C for 24 h. The initial moisture was determined, and the dry fecal content and dry matter intake (DWI) were calculated. The dried fecal samples and feed samples were crushed into powder for testing.DWI, average daily weight gain, fecal volume, urine volume, and apparent digestibility of dry matter were measured.
On the last three days of every treatment, blood samples from all sheep were collected by jugular venipuncture into a serum separator tube andimmediately centrifuged at 3,000 g for 20 min. The serum was then stored at -40°Cfor the analysis of biochemical indicators. The levels of antioxidant enzymes, including superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT) and glutathione peroxidase (GSH-PX); total antioxidant capacity (T-AOC); and the levels of immune factors IL-2, IL-4, IL-6, and IFN-γ were measured.The kits used to measure the indexeswere purchased from Beijing Huaying Biotechnology Research Institute, Beijing, China.The timeline of the experiment was shown (See Additional le 1: Fig.S1). On the last day of each treatment, after fasting the sheep overnight, the rumen contents were carefully pumped out, separated and stored at -20°C for analysis.
Total DNA extraction from rumen uid and quanti cation PCR Total genomic DNA was extracted using the stool DNA kit (OMEGA Bio-Tek, Norcross, GA, USA) according to the manufacturer's instructions. The concentration and quality of DNA were measured using a K5800 microspectrophotometer (KAIAO, Beijing, China). Quantitative PCR was executed in triplicate using SYBR Premix Ex Taq II (RNaseH Plus) assay kit (TaKaRa Bio Inc., Kusatsu, Shiga, Japan). The reaction system and procedure followed a previous study [13]. The primer sequences were selected based on past research [14].
16S r RNA gene sequencing, data processing and functional prediction The V3-V4 region of the total microbial 16S r RNA gene was ampli ed using primers 338F(5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') that were tailed with speci c sequences and ampli ed genes. Ampli cation was performed with the following cycling conditions: 95°C for 3 min, followed by 27 cycles of 95°C for 30 s, 55°C for 30 s, and 72°Cfor 45 s, and a nal extension at 72°C for 10 min [14]. The products were separated on 2% agarose gel, and nucleotides were isolated via bead puri cation using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, USA). Each product was assembled in equimolar amounts and sequenced on the Illumina MiSeq platform (Illumina, San Diego, USA).
The sequence data from 16S rRNAMiSeq sequencing were analyzed, quality-ltered using Trimmomatic, and merged by FLASH [15,16]. The RDP classi er can quickly and accurately classify sequences into high-order taxonomy, which can provide a range of classi cation structures from the domain to genus level and accurately evaluate each stage [15]. Reads of 97% similarity were clustered into operational taxonomic units (OTUs) with ≤1% incorrect bases using UPARSE [17]. Chimeric 16S rRNA sequences were removed after CS detection [18]. QIIME can be used to analyze a microbial community and graphically display the results [19]. The functional prediction of rumen bacteria was performed based on previous work [14].

Volatile fatty acid (VFA) analysis
The rumen uid was thawed on the ice and centrifuged to obtain the supernatant, which was stored at 4°C. For SCFA analysis, a solution was prepared by mixing the supernatant and the crotonic acid at the ratio of 10:1 and then ltered and analyzed using gas chromatography (Agilent Technologies 7820A GC system, Santa Clara, USA) according to previous studies [20].

Statistical analysis
For each 16S rRNA sample, the abundances of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were estimated by calculating the means and standard errors of the mean (SEM) using oneway ANOVA with SPSS version 17.0 (SPSS Inc.; Chicago, IL, USA). For the bacterial community, we performed alpha and beta diversity analyses; the alpha diversity indexes Simpson, Shannon, and Chao were calculated, and beta diversity was explored by PLS-DA graphs. Rarefaction curves were produced in R (version 3.5.1).
The data, including BW, antioxidant indexes, immune indexes, and SCFA concentrations, were analyzed using R (3.5.0). R was used to analyze the associationsbetweenmicrobial community composition and these factors [21]. Covariance analysis was used to determine the effect of cold stress (sheep number was included as a covariate). R was used to construct graphs. In addition, we performed correlation network analysis. All data are presented as the mean ± SE, and values of P<0.05 were considered statistically signi cant.

Results
Growth performance and nutrient digestibility We measured and calculated some of the metrics to estimate the performance of the production quota. We found that the ADG of sheep signi cantly decreased (P<0.05) with increasing intensity of cold stress, and after initiating wind treatment, sheep weight began to decrease (Fig. 1a).
To study whether changes in energy intake or energyoutput were responsible for the change in BW, sheep were fed in the metabolic cage. We found that Dry matter intake (DMI) varied among the treatment groups. DMI was low in the C group and lowest in the LT group. DMI then increased with increasing wind velocity, being similar between the LW and MW groups and highest in the HW group (P< 0.05) (Fig. 1b).
The apparent digestibility of dry matter was much lower in LT and LW than C (P<0.05), whereas that in MW and HW was similar to that in C (Fig. 1c). As cold stimulation increased, the amount of crude ber (CF) in feces increased signi cantly (Fig. 1d), possibly due to the degradation of primarily carbohydrate rather than cellulose by the rumen microbiota in the MW and HW groups. The levels of metabolic energy and digestibility energy were signi cantly lower in the LT and LW groups than the other groups (See Additional le 1: Fig.S2). These data indicated that the cold environment led to weight loss in the sheep and reduced digestion of the ber in their feed.

Rumen microbiota changes with environmental changes
We used 16S rRNA gene sequence technology to analyze the abundance of rumen microbiota. The coverage index indicated that cold temperatures in uenced microbial diversity (See Additional le 2: Table S1, S2).Through partial least squares discriminant analysis (PLS-DA), we found that microbiota community structures differed among the treatments (Fig. 2a).
At the genus level, 216 taxa were identi ed. Univariate ANOVA of the bacterial abundances revealed several signi cant differences in the rumen microbes among treatments (Fig. 2). The relative abundance of Prevotellaceae_UCG-003(Bacteroidetes, P = 0.045)and Solobacterium(Firmicutes,P = 0.017)were decreased in the LT, LW, MW and HW groups compared with the C group. Furthermore, the abundance of Brachybacterium(Actinobacteria, P = 0.040), Devosia(Proteobacteria, P = 0.008), Sphingomonas(Proteobacteria, P = 0.000) and unclassi ed_f__Enterobacteriaceae(Proteobacteria,P = 0.050) were higher in the LT group than in the other groups. The abundance of Rhizobium(Proteobacteria,P = 0.007)was higher in the LT and LW groups than the other groups. In contrast, the abundance of Sphaerochaeta(Spirochaetae,P = 0.044)was decreased in the LT and LW groups compared with the other groups. Furthermore, the abundance of Pseudobutyrivibrio(Firmicutes,P = 0.039) was higher in the MW group than in the other groups. The abundance of Ruminiclostridium_1(Firmicutes,P = 0.020), Ruminococcaceae_UCG-005(Firmicutes,P = 0.044), norank_c__WCHB1-41(Verrucomicrobia,P = 0.043)was increased in the HW group relative to the other groups. Interestingly, the abundance of Lachnospiraceae_XPB1014(Firmicutes,P = 0.012)exhibited highest levels in the LT group and the lowest levels in the LW group (Fig. 2).
We used qPCR to verify the changes in some bacterial groups (See Additional le 2: Table S3). The diversity of the dominant bacteriadid not differ signi cantly (See Additional le 2: Table S4).

Bacterial function prediction and molecular pathways in the rumen
We predicted the functions of the rumen bacteria and the associated molecular pathways in sheep to assess the impact of wind treatment.  Table S7). The gene abundance did not differ signi cantly among groups. These ndings showed that LT and wind speed may affect the nervous system in the rumen.

Changes in the concentration of VFAs and cellulase activity in the rumen
Studies have shown that VFAs could provide energy to the host and participate in the host metabolism [4]. After discovering the changes in the rumen microbiota, we explored the levels of VFAs.We found that the concentration of total VFAs was signi cantly reduced in the MW and LW groups relative to the other groups (P<0.05) (Fig. 3a). Accordingly, the concentrations of acetic acid and propionic acid were decreased signi cantly in the MW and HW groups (P<0.05). However, the ratio of acetic acid to propionic acid did not markedly differ among the groups (See Additional le 2: Table S8). In addition, butyrate level was signi cantly reduced in the HW group relative to the other groups (P<0.05), whereas isobutyricacid and isovalerate levels were signi cantly increased in the MW and HW groups compared with the other groups (P<0.05).Cellulase activity in the rumen contents decreased with increasing wind speed, being signi cantly lower in the HW group than in the other groups(P<0.05) (Fig. 3b). These ndings suggested that in the cold environment, the rumen microbes reduced their digestion of CF.

Changes in in ammatory factors and antioxidant enzymes
As we expected, the contents of proin ammatory factors, such as IL-2, IL-6, and IFN-γ, in plasma were reduced in the wind-treatment groups relative to the C group (P<0.05). In contrast, the contents of antiin ammatory factors, such as IL-4, were increased in the wind-treatment groups relative to the C group (P<0.05) (Fig. 4).
We found that MDA content was signi cantly decreased in the wind-treatment groups compared with the C group (P<0.05) (Fig. 5a). In contrast, the serum concentrations of SOD, CAT and GSH-PX showed similar trends as T-AOC (Fig. 5), being increased in the wind-treatment groups relative to the C group (P<0.05). The ratio of T-AOC to MDA re ects the relationship between the body's antioxidant capacity and oxidative damage. Low-temperature treatment signi cantly increased the ratio of T-AOC to MDA in serum (P<0.05), and this ratio increased signi cantly with increasing WV (P<0.05). However,the ratio of T-AOC to MDAwas signi cantly lower in theLW group than in all of the other groupsexcept the C group, for which no signi cant difference was observed. These data showed that cold stimulation led the sheep to enter an immunosuppressive and antioxidant state.

Associations of rumen microbiota with host phenotype
We used correlation analysis to research the associations between microbiota and host phenotype (Fig. 6, Additional le 3: Table S9

Discussion
Winter in northwest China is not only cold but also subject to strong winds. We used the local temperature in this study and strictly controlled the wind speed. In the present study, we explored the whole-body and rumen responses of sheep to both cold and wind speed. When the sheep were exposed to cold temperature, DMIdecreased sharply. In contrast, Bo reported that voles exposed to cold temperature (4°C) increased their food intake [22] to maintain a constant body temperature. However, in the present study, the range of temperature was very large. The sharp decrease in BMI with cold exposure observed in the present study corresponds to the rst stage in the stress response, i.e., a panic reaction or mobilization phase, as proposed by Canadian pathologist Hans Selye. This reaction led to a decline in animal feed intake. In addition,the metabolic analyses revealed that the apparent digestibility of DM, DE, and MEdecreased sharplyupon cold exposure. Young et al concluded that each 10°Cdecrease in an environment below 20°C would cause 1.8 percentage points of DM digestibility change [23], reducing the feed-utilization e ciency of sheep. This conclusion is consistent with our results. Subsequently, wind treatment was applied to the sheep in the cold environment. Under wind exposure, the sheep increased the apparent digestibility of DM, DE, and ME. They then entered the second phase of the stress response: the adaptation phase. They generated more heat from feeding and the body to maintain a constant body temperature. As has been described in previous studies, the ADG of sheep decreased signi cantly under wind treatment and became negative [22].
The rumen microbiota change when animals are exposed to cold conditions [2]. For example, when the sheep were subjected to cold treatment, the abundance ofLachnospiraceae_XPB1014, which is highly enriched in the gut of nonalcoholic fatty liver patients [24], increased, and the abundance of Prevotellaceae_UCG003 decreased. These two bacterial taxa represent more than 1% of the bacterial community in the rumen anduse dietarysoluble ber as substrate to produce short chain fatty acids [25].Furthermore, several nitrogen-xing microbial groups, such asDevosia and Rhizobium [26], were enriched in the wind-exposed sheep, which might increase the amount of ammonia and urea produced via the rumen nitrogen cycle. As a result, the abundance of Brachybacterium increased, which uses urea to breed and degrade harmful substancessuch asphenol [27]. In addition, the abundance ofSphingomonas, which is involved in redox reactions and has a reducing effect [28], was increased under cold temperature.Large uctuations in the amount of bacteria in the rumen provide a breeding opportunity for pathogens such as unclasssi ed_f_Enterobacteriaceae [29]. Treatments with different wind velocities were applied to sheep. As WV increased, the abundances of Lachnospiraceae_XPB1014, Ruminiclostridium_1, Ruminococcaceae_UCG005 and norank_f_WCHB1-41 increased. Lachnospiraceae_XPB1014 digests soluble ber;theother two groups had abundances between 0.1% and 1% and degrade cellulose [30][31][32]. Furthermore, the abundance of Pseudomonasincreased, which might reduce oxidative stress [33]. We concluded that after cold stimulation, rumen bacteria that digest soluble ber uctuated in abundance, while the abundances of bene cial bacteria decreased and those of harmful bacteria increased.
PICRUSt1 functional prediction revealed that the rumen microbiota were regulated by the nervous system under cold temperature. Several studies have found that the gut microbiota in mice are regulated by neurons, such as VIP neurons [34]. We suspect that rumen microbes can similarly be regulated by neuronal factors. Changes in the rumen microorganisms caused changes in VFAs. Consistent with our ndings, previous studies have found that cold conditions reduce the contents of total VFA, acetate, butyrate, and valerate [12]. These reductions occur due to the lower e ciency of rumen microorganisms in fermenting soluble bers in cold conditions [35].
Some studies have shown that animals exposed to cold temperature can enter a severe inhibitory stateof the immune response [36,37].This observation is consistent with our ndings. IL-2, which could represent the level of cellular immunity [38], and IFN-γ, which is mainly involved in cellular immune-related immune responses, were decreased in the sheep exposed to cold temperature. Furthermore, the level of IL-4, which could represent the activation level of TH2 cells, was increased in these sheep. Following these changes,the content of TH2 likely increased sharply and that of TH1 likely decreased [39], which destroyed the normal dynamic equilibrium state of the two types of cells, causing the body to enter an immunosuppressive state. In addition, the serum content of the proin ammatory factor IL-6 was decreased in the cold-treated sheep in this study. Inconsistent with our results, Guo et al found that IL-6 levels increased under cold conditions [40].The difference may be due to study differences in the levels of cold stimulation in the experimental design and in the genetic backgrounds of the animals. The immune response is very complex, and proin ammatory factors are affected by many factors [34], which deserve further study.
When the sheep were exposed to cold temperature,they metabolized large amounts of nutrients to increase their bodies' heat production.During this process, the body could produce oxygen free radicals, resulting in oxidative damage [41]. However, in the present study,MDA content,which re ects the extent of cell damage [42], was decreased in the cold-exposed sheep. We speculate that the high levels of antioxidant enzymes, such as SOD [43], CAT [44], and GSH-PX [45], in plasma under cold temperature led to the degradation of the oxygen free radicals produced by sheep metabolism [46]. Studies have shown that low levels of cold stress enhance antioxidant capacity and reduce the body's exposure to the effects of oxidative stress [47]. Furthermore, studies have shown that elevated levels of antioxidant enzymes in sheep are indicative of oxidative stress, with high enzyme levels needed to avoid damage fromoxygen free radicals [45]. These observations are consistent with our ndings.
Accumulating studies have shown that microbes have signi cant associations with and can alter host phenotypes [48,49]. VFA content was strongly correlated with ADG in this study. VFAs, especially butyrate [35], can provide large amounts of energy to fuel metabolism. However, the mechanismsunderlying the associationsbetween speci c microorganisms and ADG need further study. Some studies have shown that the abundances of Prevotellaceae_UCG003 [25] and Lachnospiraceae_ XPB1014,which produce metabolites such as VFAs [50],in uence the immune response. In addition, the abundance of Sphingomonas, which is involved in reduction reactions, has been shown to be increased in the rumen of animals subjected to cold stress [51]. The increase in Sphingomonas abundance resulted in a decrease in MDA content and a positive correlation with blood hormone content, consistent with our results. However, the associations between Prevotellaceae_UCG003 and blood hormones indicative of oxidative damage deserve further study.

Conclusion
In the present study, when the sheep were exposed to the cold environment, animal growth and feed e ciency decreased signi cantly, and the fermentation of soluble bers by rumen microorganisms decreased signi cantly. In addition, sheep increased the levels of antioxidant enzymes to resist damage; however, the sheep were in astate of immunosuppression.      ber (CF) in feces. C (sheep exposed to 5°C), LT (sheep exposed to -15°C and an average wind velocity less than 0.5 m/s), LW (sheep exposed to -15°C and an average wind velocity of 3 m/s), MW (sheep exposed to -15°C and an average wind velocity of 4 m/s), HW (sheep exposed to -15°C and an average wind velocity of 5 m/s).

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