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Article

Growth Performance and Rumen Microbiota of Sheep Respond to Cotton Straw Fermented with Compound Probiotics

by
Peiling Wei
1,
Mingxuan Guan
1,
Xuhui Liang
2,
Kaixin Yuan
2,
Ning Chen
3,
Yuxin Yang
2 and
Ping Gong
1,*
1
Xinjiang Academy of Animal Science, Urumqi 830057, China
2
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China
3
Institute of Animal Husbandry and Veterinary Medicine, Xinjiang Academy of Agricultural Reclamation Sciences, Shihezi 832000, China
*
Author to whom correspondence should be addressed.
Fermentation 2025, 11(5), 244; https://doi.org/10.3390/fermentation11050244
Submission received: 24 March 2025 / Revised: 13 April 2025 / Accepted: 15 April 2025 / Published: 29 April 2025
(This article belongs to the Special Issue Application of Fermentation Technology in Animal Nutrition: 2nd Edition)
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Abstract

:
To develop cotton straw as a feed resource through biological fermentation, it was fermented using compound probiotics (Bacillus subtilis, Saccharomyces cerevisiae, and Lactobacillus plantarum) and subsequently fed to sheep after the nutrients and hygienic indices of the fermented cotton straw (FCS) were analyzed. Sixty sheep were randomly assigned to five groups: a control group (CON); a low-proportion fermented cotton straw group (LFC, with FCS comprising 14.5% of the diet); a high-proportion fermented cotton straw group (HFC, with FCS comprising 29.0% of the diet); a compound microbial group (MIC, containing Bacillus licheniformis, Bacillus subtilis, and yeast); and a microbial-enzymatic preparation group (MEY, containing compound probiotics and enzymes such as cellulase, xylanase, β-glucanase, amylase, and protease). The trial lasted seven weeks and was divided into two stages: stage 1 (weeks 1–4, days 1–28) and stage 2 (weeks 5–7, days 29–49). Body weight and daily feed intake were registered, and blood and rumen fluid samples were obtained at day 28 and day 49 of the feeding trial. Fermentation significantly increased the crude protein content of cotton straw while reducing neutral detergent fiber (NDF) and acid detergent fiber (ADF) (p < 0.05). Additionally, fermentation reduced the residues of aflatoxin B1, vomitoxin, zearalenone, and free gossypol in the treatment groups (p < 0.05). LFC possessed the lowest value of feed-to-gain ratio (F/G) among all groups. Serum indices related to antioxidant capacity and utilization of fat and protein increased in the treatment group (p < 0.05). Rumen microbiota were separated between different groups (p < 0.05). LFC and HFC enhanced the abundance of Prevotella. These findings could provide conclusions that fermented cotton straw has the tendency to enhance the growth performance of sheep by increasing the abundance of bacteria related to utilization of protein, carbohydrate, and other nutrients such as Prevotella, in which the LFC group has the best fast-fattening (about 50 d) effect.

1. Introduction

Cotton is a crucial source of natural fiber for the global textile industry [1]. Cotton cultivation spans over 30 million hectares across 85 countries and regions, with more than half participating in the global cotton textile trade [2]. Cotton straw is the main by-product of cotton; however, vast amounts of cotton straw are burned, which not only causes the waste of resources but also aggravates environmental pollution because of the SO2 and NO2 produced [3]. Cotton straw is rich in nutrients, and its crude protein (6.61%), calcium (0.34%), and phosphorus (0.15%) contents are higher than those of corn straw, rice straw, and wheat straw; hence, it possesses the potential to be a high-quality feed resource. Efficient use of cotton straw in animal husbandry could reduce carbon emissions and provide conditions for the production of green agricultural products, which is in line with the concept of green ecological development of animal husbandry [4,5]. However, cotton straw is hard in texture and poor in taste, so direct-feeding efficiency is unsatisfactory. Straw could be treated by physical methods such as steam explosion, chemical methods such as ammoniation, and biological methods such as fermentation then fed to cattle and sheep [6,7,8]. Among these methods, the bacterial-enzyme synergistic biological fermentation not only enhances the nutritional value of feed but also improves its palatability, addressing the limitations of physical and chemical methods. The application of probiotics as fermentative inoculants in feed production has been shown to markedly enhance both feed quality and the antioxidant capacity of the resulting product [9,10,11,12]. Research indicates that aerobic bacilli consume free oxygen in the intestinal tract during their growth and metabolic processes, creating an anaerobic environment that supports the growth, reproduction, and metabolism of anaerobic probiotics like lactic acid bacteria [13]. Lactic acid bacteria, such as Lactobacillus plantarum, exhibit strong antibacterial activity against a wide range of foodborne pathogens [14]. Yeasts, including Saccharomyces cerevisiae, inhibit mold growth in feed [15]. Additionally, cellulase and protease produced by probiotics enhance nutrient utilization, while enzymes that degrade mycotoxins and gossypol safeguard animal health [10,16,17]. Therefore, this study seeks to develop cotton straw as a novel feed resource through biological fermentation and to evaluate its feeding effects in sheep. The findings aim to offer theoretical and practical insights into the development and utilization of innovative feed resources for ruminants.

2. Materials and Methods

2.1. Production of Fermented Cotton Straw

The probiotics employed in the biological fermentation process, including Bacillus subtilis, Lactobacillus plantarum, and Saccharomyces cerevisiae, were preserved and supplied by our research team.
The cotton straw was provided by Tumushuke Wanzhiyang Farmers Professional Cooperative. The inoculation ratio of Lactobacillus plantarum/Bacillus subtilis/Saccharomyces cerevisiae = 2:1:1 was used to produce fermented cotton straw (inoculum dose: 1% of the total weight of cotton straw). The fermentation of straw was carried out using a mixed inoculum containing Bacillus subtilis (≥1.0 × 109 CFU/g), Lactobacillus plantarum (≥0.5 × 109 CFU/g), and Saccharomyces cerevisiae (≥1.0 × 109 CFU/g). The microbial strains were preserved and provided by the Sheep Genetic Improvement and Germplasm Innovation Team of the College of Animal Science and Technology, Northwest A&F University. The cotton straw fermented by activated probiotics was the treatment group (T), the straw fermented by inactivated probiotics (autoclaved, 127 °C) was the control group (C), and the cotton straw without treatment was the raw material (R). Feed samples were randomly collected on days 0, 14, 28, 42, and 56. The first aliquot of samples was for sensory evaluation and measurement of feed hygienic indices, and the second aliquot of the samples was used for measurement of nutritional components. After evaluation of feed hygiene and the nutrient level of the fermented cotton straw (FCS), large-scale production of fermented cotton straw was made for a growth trial of sheep.

2.2. Experiment Animals and Design

The trial was conducted at the Tumushuke Wanzhiyang Farmers Professional Cooperative in Xinjiang, China (39.91 N 79.51 E). The feeding period was 11 weeks in total, including 2 weeks for transition, 2 weeks for adaptation, and 7 weeks for the growth trial. The growth trial was divided into 2 stages: stage 1 was week 1–week 4 (day 1–day 28), and stage 2 was week 5–week 7 (day 29–day 49). Sixty 4-month-old sheep (Duolang sheep♂ × Karakul sheep♀) in healthy condition (BW 28.82 ± 3.35 kg) were randomly divided into 5 groups (12 sheep/group). Each group consisted of four replicates, with three sheep per replicate housed in the same sheep shed, sharing one pen per replicate. The groups were CON (control group), LFC (low-proportion fermented cotton straw group, where FCS accounted for 14.50% of the diet), HFC (high-proportion fermented cotton straw group, where FCS accounted for 29.00% of the diet), MIC (microbial group) and MEY (mixture of microbial and enzymatic preparation group). The sheep were provided by Tumushuke Wanzhiyang Farmers Professional Cooperative. The probiotics used in the MIC group were developed with the participation of the Sheep Genetic Improvement and Germplasm Innovation Team of the College of Animal Science and Technology, Northwest A&F University, while the formulation used in the MEY group was provided by Guangdong Vtr Bio-Tech Co., Ltd. Before the start of the trial, all sheep pens were thoroughly disinfected, and the sheep were dewormed and numbered. Animals were fed twice a day at 8:00 and 18:00, and all the animals were provided with free access to feed and water.

2.3. Diets

All diets were formulated according to the feeding standards of meat-producing sheep and goats. There are 3 kinds of diet in this study shown in Table 1: a basal diet for the CON group, a low-proportion fermented cotton straw diet for the LFC group, and a high-proportion fermented cotton straw diet for the HFC group. On the basis of the CON group, the MIC group was given compound probiotics (2% of the daily feed); the MEY group was given an enzyme and Saccharomyces cerevisiae combination preparation (1% of the daily feed) on the basis of the MIC group. The MIC used in this study was developed by our research team and subsequently commercialized by First Bio-technology Co., Ltd. (Xianyang, China). The composition of MIC included Bacillus licheniformis (≥1.0 × 108 CFU/g), Bacillus subtilis (≥5.0 × 107 CFU/g), and Saccharomyces cerevisiae (≥1.0 × 108 CFU/g). Regarding the MEY compound preparation, it contained Saccharomyces cerevisiae (dry matter) at ≥1.0 × 109 CFU/g, with enzyme activities as follows: cellulase ≥ 3000 U/g, xylanase ≥ 2000 U/g, β-glucanase ≥ 15,000 U/g, amylase ≥ 20,000 U/g, and protease ≥ 2000 U/g, along with various other fermentation-derived metabolites. This preparation was developed by VTR Bio-Tech Co., Ltd. (Zhuhai, China).

2.4. Feed Proximate Analyses and Measurement of Hygienic Indices

Analyses performed on feed samples were as follows: (1) The crude protein content of the diet was tested using the Kjeldahl method [18]. (2) The Van Soest detergent fire analysis method was used for the determination of neutral detergent fiber (NDF) and acid detergent fiber (ADF) [19]. (3) The contents of free-gossypol (FG) were determined by the aniline method [20]. (4) Aflatoxin B1 (AFB1), vomitoxin (DON) and zearalenone (ZEN) were measured by ELISA kit (Xiamen lunchangshuo Biotechnology Co., Ltd., Xiamen, China). (5) The pH of the rumen fluid was immediately measured using a calibrated pH meter (Mettler Toledo Instruments Co., Ltd., Shanghai, China).

2.5. Feed Trial

The body weight of each sheep was assessed before morning feeding on day 1, day 28, and day 49 of the growth trial, and the feed intake was registered daily in order to calculate the ratio of feed to gain (F/G). Blood samples were collected from the jugular vein, 10 mL for each sheep, and centrifuged with 3000 r/min for 15 min to collect serum on day 28 and day 49 of the growth trial for the serum indices measurement. The contents of total cholesterol (TC), triacylglycerol (TG), urea (UREA), acetyl coenzyme A (AcCoA), beta-hydroxybutyric acid (BHBA), malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (T-AOC) were measured by the kits (Sino-UK Institute of Biological Technology, Beijing, China).

2.6. Rumen Microbiota Diversity

Rumen fluid samples were collected by the oral intubation method from each sheep on day 28 and day 49 of the growth trial for the rumen microbial diversity measurement. The microbial DNA extraction was carried out using the E.Z.N.A.® soil DNA kit (Omega Bio-tek, Norcross, GA, USA). Nucleic acid quantifier NanoDrop2000 (Life Technologies, 109, Carlsbad, CA, USA) was used to determine the concentration and purity of the extracted DNA, while its quality was assessed using 1% agarose gel electrophoresis. The DNA samples that met the requirements were subjected to MiSeq sequencing. The bacterial 16S rRNA gene universal primers 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were used to amplify the original sequence. The PCR products were pooled with equal molar amounts from different samples. Sequencing libraries were generated using NEXTFLEX® Rapid DNA-Seq (Bioo Scientific, Austin, TX, USA) Kit. Fastp(https://github.com/OpenGene/fastp accessed on 26 August 2024 version 0.19.6) software was used to control the quality of the raw sequences, and FLASH (http://www.cbcb.umd.edu/software/flash accessed on 26 August 2024, version 1.2.11) software was used for splicing. The DADA2 plug-in in the QIIME2 process was used to denoise the optimized sequence after quality control. Sequences after DADA2 denoising were often referred to as ASVs (amplicon sequence variants). Follow-up data analysis based on ASVs was performed using the free online platform of the Majorbio Cloud Platform (www.majorbio.com) from Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China).

2.7. Statistical Analyses

All the data were preliminarily processed by EXCEL (Version 2021, Microsoft, Redmond, WA, USA). The data of feed nutrients, feed hygienic indices, growth performance, serum indices, and rumen fluid pH of sheep were analyzed using one-way analysis of variance by R software (Version 4.1.3, R Core Team, Vienna, Austria). The normality of the data was checked with the “qqPlot” function in “car” package, and the homogeneity of variance was checked by the “bartlett. test” function in the “stat” package. The “aov” function was subjected to perform ANOVA, and the “Duncan. test” function in the “agricolae” package was used to perform post hoc comparisons. Sequencing data were analyzed on the Majorbio platform (https://cloud.majorbio.com/). The test results were expressed in the form of “Mean ± SD”; p < 0.05 indicated significant differences.

3. Results

3.1. Effect of Fermented Cotton Straw with Compound Probiotics

Nutrient levels of cotton straw before and after fermentation are presented in Table 2. At the end of the fermentation, the contents of CP in both the treatment group and the control group were significantly higher than that of the raw material (p < 0.05), among which the treatment group exhibited a higher level. The levels of NDF and ADF in the treatment group and the control group were significantly lower than that of the raw material (p < 0.05), among which the treatment group exhibited a lower level.
The residues of aflatoxin B1 (AFB1), vomitoxin (DON), zearalenone (ZEN), and free gossypol (FG) are shown in Figure 1. The amounts of AFB1 residue in the raw material, control group, and treatment group were 0.388 ppb, 0.260 ppb, and 0.281 ppb, respectively. Compared with the raw material, the treatment group decreased by 27.58% (p < 0.05) (Figure 1A); the amounts of DON residue in the raw material, control group, and treatment group were 245.203 ppb, 212.863 ppb, and 119.998 ppb, respectively. Compared with the raw material, the treatment group reduced by 51.06% (p < 0.05) (Figure 1B); the amounts of ZEN residue in the raw material, control group, and treatment group were 2.750 ppb, 2.218 ppb, and 2.145 ppb, respectively. Compared with the raw material, the treatment group dropped by 22.00% (p < 0.05) (Figure 1C); the amounts of FG residue in the raw material, control group, and treatment group were 203.825 mg/kg, 141.520 mg/kg, and 110.413 mg/kg, respectively. Compared with the raw material, the treatment group decreased by 45.83% (p < 0.05) (Figure 1D).

3.2. Effects of Different Diets on Growth Performance of Sheep

The growth performance of sheep under different diets is presented in Table 3. At the beginning of the trial, the average weight of sheep in each group was similar, with no significant differences observed (p < 0.05). During stage 1 and the entire trial period, the dry matter intake (DMI) in the HFC group was significantly lower than that of the other groups (p < 0.05). In stage 2, the DMI in the HFC group remained significantly lower than that of the CON, MIC, and MEY groups (p < 0.05). Additionally, in stage 2, the LFC group showed a tendency to increase the average daily gain (ADG) and to reduce the feed-to-gain ratio (F/G).

3.3. Effects of Different Diets on Serum Biochemical Parameters of Sheep

The serum biochemical parameters of the sheep are presented in Table 4. In stage 1, the AcCoA levels were significantly increased in the MIC and LFC groups (p < 0.05). Growth hormone (GH) levels differed significantly among the groups (p < 0.05). The MIC, LFC, and HFC groups all exhibited higher GH levels compared to the control group, with the LFC group showing the highest value. Both MIC and LFC groups increased the SOD level (p < 0.05), and LFC enhanced the T-AOC level (p < 0.05). Among the groups, LFC had the highest levels of AcCoA, GH, SOD, and T-AOC. Furthermore, both MIC and LFC decreased the MDA level, with MIC showing the lowest value.
In stage 2, all treatment groups significantly reduced the levels of TC, TG, UREA, BHBA, and MDA (p < 0.05), with the MEY group showing the lowest values. Additionally, all treatment groups significantly increased the levels of AcCoA, GH, SOD, and T-AOC (p < 0.05), with the MEY group showing the highest levels.

3.4. Effects of Different Diets on Rumen Fluid pH of Sheep

The rumen fluid pH values for the different groups are presented in Table 5. In stage 1, the MIC and HFC groups significantly increased the pH values (p < 0.05), with the pH fluctuating between 6.15 and 6.66. In stage 2, no significant differences were observed between the groups, with pH values ranging from 6.60 to 6.86.

3.5. Effects of Different Diets on Rumen Microbiota Diversity of Sheep

All 120 rumen fluid samples were subjected to 16S rRNA sequencing. An amount of 7,549,256 original sequences were obtained with 2,264,776,800 bp. After denoising by DADA2, a total of 6,679,844 optimized sequences were obtained with 2,794,570,466 bases (average sequence length of 418 bp). After smoothing according to the minimum number of sample sequences, 24,350 effective sequences for each sample were obtained. The statistics of the taxonomy annotation results were as follows: 24 phylum (phyla), 45 classes (class), 104 orders (order), 173 families (family), 374 genera (genus), 863 species (species), and 18,684 amplicon sequence variants (amplicon sequence variant, ASV). Subsequent microbial diversity data analyses were based on the ASVs dataset. The rarefaction curve showed that all the samples were sequenced with sufficient depth and reached the plateau (Figure 2).
The indices related to the alpha diversity of rumen microflora are listed in Table 6. In stage 1, no significant difference was observed. All treatment groups had the tendency to elevate the levels of Sobs, Ace, and Chao 1, and HFC had the most obvious impact. MIC and HFC tended to enhance the levels of Shannon and Shannoneven, among which HFC and MIC had the highest levels. In stage 2, MIC and MEY enhanced the level of coverage (p < 0.05). LFC and HFC enhanced the levels of Ace and Chao 1 (p < 0.05). All treatment groups tended to increase the levels of Shannon and Shannoneven.
The PCoA analysis based on the Bray–Curtis distance algorithm was conducted to assess the beta-diversity of rumen microbiota (Figure 3). Significant separation of rumen microbiota was observed between different groups of sheep in both stages (p = 0.001).

3.6. Effects of Fermented Cotton Straw on the Composition of Rumen Microbiota

In stage 1 (W1–W4), the four most abundant bacterial phyla were Firmicutes, Bacteroidota, Desulfobacterota, and Patescibacteria (Figure 4A). Firmicutes and Bacteroidota exhibited the highest abundance in the CON and HFC groups, respectively. In the treatment groups, the abundance of Firmicutes decreased, while that of Bacteroidota increased. In stage 2, Bacteroidota, Firmicutes, Spirochaetota, and Patescibacteria were the four most abundant bacterial phyla (Figure 4B). Bacteroidota showed the highest abundance in the LFC group.
The results in Figure 4C illustrated that the top five most abundant genera were Rikenellaceae_RC9_gut_group, Prevotella, NK4A214_group, Christensenellaceae_R-7_group, and norank_f_F082. Rikenellaceae_RC9_gut_group and Prevotella exhibited the highest abundance in the LFC and HFC groups, respectively. The relative abundance of NK4A214_group and Christensenellaceae_R-7_group decreased in the treatment groups, with the lowest levels observed in the HFC and LFC groups. The composition of rumen microbiota at the genus level in stage 2 (W4–W7) is shown in Figure 4D. The top five genera were Prevotella, Rikenellaceae_RC9_gut_group, norank_f_F082, NK4A214_group, and norank_f_Bacteroidales_RF16_group. The relative abundance of Prevotella increased in all groups except for the MIC group, with the highest abundance in the LFC group. The abundance of Rikenellaceae_RC9_gut_group decreased, with the lowest level observed in the HFC group. As for NK4A214_group, its abundance increased in all groups except for the MEY group, with the MIC group showing the highest level. The abundance of norank_f_Bacteroidales_RF16_group decreased in all groups except for the MIC group.

4. Discussion

4.1. Effects of Fermented Cotton Straw with Compound Probiotics

The fermentation trial demonstrated that the crude protein (CP) content of cotton straw significantly increased, while the acid detergent fiber (ADF) and neutral detergent fiber (NDF) contents significantly decreased (p < 0.05). Consequently, the nutritional quality of the cotton straw was markedly improved. These findings are consistent with previous studies [21,22,23]. Biological fermentation has been shown to reduce NDF and ADF levels in straw-based feeds while enhancing protein content, thereby facilitating the efficient utilization of feed resources [24,25,26,27]. Additionally, biological fermentation significantly reduced the residues of AFB1, DON, ZEN, FG (p < 0.05). Thus, the hygiene and safety of fermented cotton straw were ensured, enabling subsequent feeding trials.

4.2. Effects of Fermented Cotton Straw on Growth Performance of Sheep

The indicators of livestock growth performance mainly include body weight (BW), dry matter intake (DMI), average daily gain (ADG), and ratio of feed-to-gain (F/G). Among them, F/G is the important indicator for judging the growth performance of livestock. Based on the effects of fermented cotton straw on the growth performance of sheep, it was demonstrated that fermented cotton straw tends to improve the feed conversion ratio (FCR) of sheep, which is similar to the previous finding [28]. Fermented straw improved the apparent digestibility of livestock; this may be why fermented straw can enhance the growth performance of livestock [29]. For example, lactobacillus-driven feed fermentation has been shown to significantly improve ADG and F/G in pigs. Similarly, feeding livestock with whole-plant corn silage inoculated with lignocellulose-degrading bacteria can significantly increase ADG, DM intake, and F/G. Comparable findings have been observed in the enzyme- and bacteria-driven fermentation of buckwheat straw and alfalfa fed to Tan sheep [11,12,30]. These results align with our experimental findings, demonstrating that fermentation of cotton straw with probiotic complexes can enhance the growth performance of sheep.

4.3. Effects of Fermented Cotton Straw on Serum Biochemical Parameters of Sheep

Serum biochemical indicators reflect the health status of animals. TG and TC are used to evaluate lipid utilization in the body, with lower levels indicating better fat utilization. In this study, the TG and TC levels in the treatment group decreased, which is consistent with the findings of Khattab [31]. UREA is an indicator of feed protein utilization and body protein metabolism in sheep [32]. In stage 2, the UREA levels in the CON group were higher, suggesting enhanced utilization of feed protein [33]. Butyric acid is produced during the fermentation of rumen microorganisms, and it undergoes oxidation in rumen epithelial cells to produce beta-hydroxybutyric acid (BHBA). As a biomarker, BHBA plays a critical role in the development of rumen epithelial metabolic function. Moreover, BHBA is a major component of ketone bodies, reflecting ketone body production in the blood [24,34]. In this study, the BHBA content was significantly lower in the treatment group, indicating a lower risk of ketoacidosis in the animals. Acetyl-CoA is a key intermediate linking various metabolic processes, including the tricarboxylic acid cycle, amino acid metabolism, and fatty acid metabolism [35,36]. The somatotropin axis plays a critical role in the regulation of animal growth, primarily involving growth hormone-releasing factor (GRF), growth hormone (GH), and insulin-like growth factor (IGF). GH is the primary hormone responsible for regulating growth and development [37].
The increase in reactive oxygen species (ROS) leads to oxidative stress and cell membrane damage, impairing cell homeostasis, structure, and function. This includes the loss of cell membrane integrity, energy depletion, alterations in signal transduction pathways, and the triggering of apoptosis, among other effects [38]. Antioxidant capacity indicators mainly include T-AOC, SOD, and MDA. T-AOC encompasses various antioxidant enzymes and related biomolecules that can scavenge free radicals in specific organs or organisms, reflecting the functional status of the body’s antioxidant system [39]. SOD catalyzes the disproportionation of superoxide ions, removing harmful substances such as free radicals produced during animal metabolism. Thus, it serves as an important indicator of an animal’s antioxidant capacity [40]. MDA is a compound formed from the peroxidation of fatty acids and is often used as a marker of ROS. It has toxic effects on cells, primarily by binding to and oxidizing DNA, causing the cross-linking of nucleic acid bases and reacting with other cellular amine groups [41,42]. In this research, MIC, MEY, LFC, and HFC significantly enhanced the antioxidant capacity of sheep (p < 0.05), which is consistent with the findings of previous research [43,44].

4.4. Effects of Fermented Cotton Straw on Rumen pH and Microbiota Diversity of Sheep

The normal pH range of rumen fluid is between 5.50 and 7.50. If the pH is too high, the absorption of volatile organic acid (VFAs) by the rumen epithelium can be impaired. Conversely, if the pH is too low, it hinders the fermentation of feed in the rumen [45]. In the present study, the pH of rumen fluid in the test sheep remained within the normal range, indicating that it did not adversely affect fermentation processes.
Rumen microbial diversity includes both alpha diversity and beta diversity. Regarding alpha diversity, indices such as Sobs, Chao1, and ACE reflect the richness of the rumen microbial community. In stage 1, LFC, HFC, MIC, and MEY tended to enhance the richness of the rumen microbiota, which is consistent with the findings of Mamuad [46] and Zhang [47]. However, the community richness of rumen microbiota for MEY and MIC group got lower in stage 2. The Shannon index, which reflects the diversity of the rumen microbiota, showed a tendency to increase in the LFC, HFC, MIC, and MEY groups, consistent with the results reported by Zhang [47] and Rabee [48]. Shannoneven reflects evenness of rumen microbiota. The result illustrated that Shannoneven tended to get higher in each treatment group. Coverage means species coverage of rumen microbiota. With higher coverage level in the treatment group, the probability that the sequence was not detected in the sample of the treatment group is lower, which is similar to the research of Chen [49]. As for beta diversity, fermented cotton straw and probiotics preparation resulted in significant separation of rumen microbiota between different groups.
Bacteroidota and Firmicutes were the dominant bacterial phyla in the rumen microbiota of sheep. Bacteroidota primarily degrade non-fibrous carbohydrates, while Firmicutes are involved in cellulose degradation, which is consistent with the previous finding [11,50,51]. Prevotella play a crucial role in the utilization of nutrients such as xylan, pectin, and protein. An increased abundance of Prevotella spp. leads to the enhanced expression of associated glycoside hydrolases (GHs) in the rumen. Prevotella is recognized for its strong cellulolytic activity and ability to ferment carbohydrates into short-chain fatty acids (SCFAs), playing a crucial role in ruminal protein and carbohydrate metabolism [52]. In the present study, LFC, HFC, and MEY promoted the growth and proliferation of Prevotella, thereby increasing the utilization of xylan, pectin, and protein in sheep, consistent with previous studies [12,53,54]. The increased abundance of bacteria associated with protein and carbohydrate utilization may explain the higher feed utilization efficiency in the LFC, HFC, MIC, and MEY groups compared to the CON group, with the F/G ratio in the LFC group being significantly lower than that in the control group (p < 0.05). Therefore, fermented cotton straw and probiotic supplementation may improve nutrient utilization in sheep by increasing the abundance of bacteria involved in protein and carbohydrate metabolism, thereby enhancing growth performance.

5. Conclusions

In summary, the diet containing a low proportion of fermented cotton straw (14.50% of total feed) yielded the best short-term fattening effect in sheep (approximately 50 days). Additionally, it improved the utilization of fat and protein, as well as the antioxidant capacity in sheep. Fermented cotton straw enhanced sheep’s production performance by increasing the abundance of bacteria involved in the utilization of protein, carbohydrates, and other nutrients, such as Prevotella. The fermentation of cotton stalks can efficiently convert agricultural waste into animal feed, promoting sustainable agricultural development.

Author Contributions

Conceptualization, P.W., P.G., Y.Y.; methodology, P.W., M.G., X.L., N.C.; formal analysis, K.Y., X.L., M.G.; writing—original draft preparation, P.W., M.G.; project administration, P.G., Y.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the “Two Areas” Science and Technology Development Program Project (2023LQ02002), the Key Technology R&D Program of Xinjiang Groups (2020AB016), and the China Agriculture Research System (CARS-39-12).

Institutional Review Board Statement

The animal study was approved by the Protocol Management and Review Committee of the Feed Research Institute of Xinjiang Academy of Animal Sciences (Approval No.: 2022017) on 16 July 2022. The study was conducted in accordance with the local legislation and institutional requirements.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study.

Data Availability Statement

Raw Illumina sequencing data used in this study have been deposited in the Sequence Read Archive (SRA) database of NCBI under Bioproject No. PRJNA964488 (link: https://www.ncbi.nlm.nih.gov/bioproject/PRJNA964488 accessed on 1 May 2023).

Acknowledgments

Thanks to all participants for their advice and support of this study.

Conflicts of Interest

All authors declare no conflicts of interest.

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Fermentation 11 00244 g001
Figure 1. Hazardous substances residues in different groups. T, treatment group, cotton straw fermented by activated probiotics for 56 d; C, control group, cotton straw fermented by inactivated probiotics for 56 d; R, raw material, cotton straw without treatment. (A) Aflatoxin B1, AFB1 (p < 0.001); (B) vomitoxin, DON (p < 0.001); (C) zearalenone, ZEN (p = 0.004); (D) free gossypol, FG (p < 0.001). (n = 6). Note: In the same row, values with different letter superscripts mean significant differences (p < 0.05).
Figure 1. Hazardous substances residues in different groups. T, treatment group, cotton straw fermented by activated probiotics for 56 d; C, control group, cotton straw fermented by inactivated probiotics for 56 d; R, raw material, cotton straw without treatment. (A) Aflatoxin B1, AFB1 (p < 0.001); (B) vomitoxin, DON (p < 0.001); (C) zearalenone, ZEN (p = 0.004); (D) free gossypol, FG (p < 0.001). (n = 6). Note: In the same row, values with different letter superscripts mean significant differences (p < 0.05).
Fermentation 11 00244 g001
Fermentation 11 00244 g002
Figure 2. Rarefaction curve of rumen microflora. (A) Stage 1 (W1-W4); (B) stage 2 (W5-W7) (n = 12).
Figure 2. Rarefaction curve of rumen microflora. (A) Stage 1 (W1-W4); (B) stage 2 (W5-W7) (n = 12).
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Fermentation 11 00244 g003
Figure 3. Beta-diversity of rumen microflora. (A) Stage 1 (W1-W4); (B) stage 2 (W5-W7) (n = 12).
Figure 3. Beta-diversity of rumen microflora. (A) Stage 1 (W1-W4); (B) stage 2 (W5-W7) (n = 12).
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Fermentation 11 00244 g004
Figure 4. The composition of rumen microflora. (A) Stage 1 (W1-W4) for phylum level; (B) stage 2 (W5-W7) for phylum level; (C) stage 1 (W1-W4) for genus level; (D) stage 2 (W5-W7) for genus level (n = 12).
Figure 4. The composition of rumen microflora. (A) Stage 1 (W1-W4) for phylum level; (B) stage 2 (W5-W7) for phylum level; (C) stage 1 (W1-W4) for genus level; (D) stage 2 (W5-W7) for genus level (n = 12).
Fermentation 11 00244 g004
Table 1. The composition of diet (dry matter basis) %.
Table 1. The composition of diet (dry matter basis) %.
ItemsGroups 2
CONLFCHFC
Ingredients
Cotton leaf43.5029.0014.50
Fermented cotton straw014.5029.00
Silage corn16.9016.9016.90
Corn21.7021.7021.70
Soybean meal9.359.359.35
Cottonseed meal5.755.755.75
CaHPO40.800.800.80
NaHCO30.500.500.50
NaCl0.500.500.50
Premix 11.001.001.00
Total100.00100.00100.00
Nutrient levels 3
CP14.6213.6512.66
CF19.1421.8723.35
EE4.363.612.65
Ca0.810.930.98
P0.460.490.50
1 The premix was Cu 200 mg, Zn 586 mg, Mn 360 mg, Fe 765 mg, vitamin A 100,000 IU, vitamin D 50,000 IU, and vitamin E 380 IU. 2 CON: control group with basal diet; LFC: low-proportion fermented cotton straw diet (FCS accounted for 14.50% of the diet) group; HFC: high-proportion fermented cotton straw diet (FCS accounted for 29.00% of the diet) group. 3 CP, crude protein; CF, crude fiber; EE, ether extract; Ca, calcium; P, phosphorus.
Table 2. Changes of nutrient components of cotton straw with fermentation time (DM basis, n = 6), %.
Table 2. Changes of nutrient components of cotton straw with fermentation time (DM basis, n = 6), %.
Items 1Sampling Timep-Value
Day 0Day 14Day 28Day 42Day 56
Control
CP6.74 ± 0.02 b6.87 ± 0.14 b6.92 ± 0.03 b7.20 ± 0.14 a7.18 ± 0.08 a0.001
NDF73.28 ± 2.14 a67.58 ± 3.14 b61.79 ± 1.70 c64.65 ± 2.63 bc64.15 ± 1.57 bc<0.001
ADF62.41 ± 1.69 a56.49 ± 2.68 ab51.04 ± 1.29 b54.03 ± 1.93 b53.27 ± 0.84 b<0.001
Treatment
CP6.74 ± 0.02 b7.92 ± 0.06 a8.07 ± 0.05 a8.30 ± 0.18 a8.20 ± 0.01 a<0.001
NDF73.28 ± 2.14 a65.96 ± 5.32 b66.14 ± 2.01 b61.05 ± 2.37 c60.68 ± 1.23 c<0.001
ADF62.41 ± 1.69 a57.19 ± 1.25 b56.87 ± 1.11 b52.43 ± 1.42 c51.54 ± 0.30 c<0.001
1 CP, crude protein; NDF, neutral detergent fiber; ADF, acid detergent fiber. Note: In the same row, values with different letter superscripts mean significant differences (p < 0.05).
Table 3. Effect of different diets on growth performance of sheep (n = 12).
Table 3. Effect of different diets on growth performance of sheep (n = 12).
Items 1Groups 2p-Value
CONMICMEYLFCHFC
BW (kg)
Initial BW28.77 ± 3.3028.73 ± 2.9429.03 ± 4.2628.43 ± 4.0629.15 ± 2.380.988
Final BW38.41 ± 4.4239.12 ± 3.2939.55 ± 4.1439.26 ± 4.0238.45 ± 3.160.933
DMI (g/d)
W1–W41226.05 ± 60.07 a1246.93 ± 33.49 a1222.94 ± 33.29 a1188.84 ± 41.00 a1118.70 ± 43.88 b0.007
W5–W71334.50 ± 108.05 a1365.82 ± 75.24 a1315.80 ± 77.55 a1276.57 ± 77.47 ab1155.61 ± 107.32 b0.041
W1–W71273.50 ± 77.03 a1298.94 ± 51.71 a1263.56 ± 45.67 a1227.22 ± 50.34 a1134.85 ± 71.19 b0.014
ADG (g/d)
W1–W4243.21 ± 72.08265.59 ± 18.05263.12 ± 46.57239.35 ± 50.67225.00 ± 28.710.720
W5–W7146.43 ± 36.43152.98 ± 26.26162.70 ± 31.07208.33 ± 32.16153.57 ± 17.680.059
W1–W7200.87 ± 34.07216.32 ± 14.51219.18 ± 31.32225.78 ± 34.29193.75 ± 22.090.494
F/G
W1–W45.36 ± 1.454.71 ± 0.244.74 ± 0.685.16 ± 1.225.03 ± 0.610.803
W5–W79.55 ± 2.399.15 ± 1.818.30 ± 1.586.21 ± 0.757.54 ± 0.220.056
W1–W76.43 ± 0.746.02 ± 0.355.83 ± 0.655.53 ± 0.855.89 ± 0.420.681
1 BW, body weight; DMI, dry matter intake; ADG, average daily gain; F/G, the ratio of feed to gain. 2 CON: control group with basal diet; MIC: added compound probiotics (2% of the feed amount) on the basis of CON; MEY: added enzyme and yeast combination preparation (1% of the feed amount) on the basis of MIC; LFC: low-proportion fermented cotton straw (FCS accounted for 14.50% of the diet) group; HFC: high-proportion fermented cotton straw (FCS accounted for 29.00% of the diet) group. Note: In the same row, values with different letter superscripts mean significant differences (p < 0.05).
Table 4. Effect of different diets on serum biochemical parameters of sheep (n = 12).
Table 4. Effect of different diets on serum biochemical parameters of sheep (n = 12).
Items 1Groups 2p-Value
CONMICMEYLFCHFC
W1–W4 mmol/L
TC1.34 ± 0.331.13 ± 0.351.25 ± 0.501.00 ± 0.401.52 ± 0.660.085
TG0.31 ± 0.140.28 ± 0.120.38 ± 0.160.28 ± 0.130.44 ± 0.350.224
UREA5.10 ± 1.834.49 ± 1.445.07 ± 1.414.43 ± 1.266.01 ± 1.530.089
AcCoA36.76 ± 6.99 bc43.76 ± 2.11 a33.75 ± 3.94 c48.50 ± 8.32 a40.13 ± 3.16 b<0.001
BHBA0.30 ± 0.180.30 ± 0.060.41 ± 0.150.29 ± 0.110.36 ± 0.170.210
GH (ng/mL)4.91 ± 0.36 d5.98 ± 0.38 b4.46 ± 0.50 e6.52 ± 0.49 a5.51 ± 0.53 c<0.001
SOD (U/mL)64.50 ± 16.08 bc73.94 ± 4.53 a58.84 ± 3.18 c75.85 ± 2.70 a67.92 ± 2.10 b<0.001
MDA (nmol/mL)4.34 ± 0.63 b3.13 ± 0.72 d4.99 ± 0.38 a3.50 ± 0.93 cd4.05 ± 0.70 bc<0.001
T-AOC (U/mL)8.14 ± 1.19 bc9.34 ± 0.62 ab7.65 ± 1.10 c10.99 ± 1.73 a8.86 ± 0.42 b<0.001
W5–W7 mmol/L
TC1.47 ± 0.41 a0.60 ± 0.32 bc0.41 ± 0.23 c0.69 ± 0.32 bc0.77 ± 0.32 b<0.001
TG0.30 ± 0.10 a0.13 ± 0.09 bc0.09 ± 0.06 c0.17 ± 0.10 b0.16 ± 0.08 b<0.001
UREA5.69 ± 1.25 a3.57 ± 1.11 cd3.03 ± 0.72 d4.00 ± 1.04 bc4.51 ± 1.01 b<0.001
AcCoA29.98 ± 7.25 d56.21 ± 3.55 b66.00 ± 4.21 a50.66 ± 5.25 c53.25 ± 5.66 bc<0.001
BHBA0.33 ± 0.11 a0.18 ± 0.08 b0.14 ± 0.09 b0.21 ± 0.10 b0.17 ± 0.06 b<0.001
GH (ng/mL)3.49 ± 0.58 d8.10 ± 0.47 b9.32 ± 0.39 a7.59 ± 0.35 c8.48 ± 0.54 b<0.001
SOD (U/mL)52.30 ± 2.66 d84.06 ± 2.71 b89.16 ± 2.81 a78.10 ± 3.93 c81.64 ± 3.11 b<0.001
MDA (nmol/mL)5.58 ± 0.91 a2.11 ± 0.44 c1.25 ± 0.40 d2.85 ± 0.22 b2.48 ± 0.30 c<0.001
T-AOC (U/mL)6.64 ± 0.46 d13.74 ± 1.49 ab14.65 ± 1.03 a11.61 ± 1.04 c12.84 ± 1.56 bc<0.001
1 TC, total cholesterol; TG, triglyceride; AcCoA, acetyl coenzyme A; BHBA, beta-hydroxybutyric acid; GH, growth hormone; SOD, superoxide dismutase; MDA, malonaldehyde; T-AOC, total antioxidant. 2 CON: control group with basal diet; MIC: added compound probiotics (2% of the feed amount) on the basis of CON; MEY: added enzyme and yeast combination preparation (1% of the feed amount) on the basis of MIC; LFC: low-proportion fermented cotton straw (FCS accounted for 14.50% of the diet) group; HFC: high-proportion fermented cotton straw (FCS accounted for 29.00% of the diet) group. Note: In the same row, values with different letter superscripts mean significant differences (p < 0.05).
Table 5. Effect of different diets on rumen fluid pH of sheep (n = 12).
Table 5. Effect of different diets on rumen fluid pH of sheep (n = 12).
ItemsGroups 1p-Value
CONMICMEYLFCHFC
W1–W46.20 ± 0.33 b6.60 ± 0.17 a6.15 ± 0.26 b6.18 ± 0.24 b6.66 ± 0.16 a<0.001
W5–W76.60 ± 0.166.71 ± 0.146.86 ± 0.286.77 ± 0.356.73 ± 0.450.111
1 CON: control group with basal diet; MIC: added compound probiotics (2% of the feed amount) on the basis of CON; MEY: added enzyme and yeast combination preparation (1% of the feed amount) on the basis of MIC; LFC: low-proportion fermented cotton straw (FCS accounted for 14.50% of the diet) group; HFC: high-proportion fermented cotton straw (FCS accounted for 29.00% of the diet) group. Note: In the same row, values with different letter superscripts mean significant differences (p < 0.05).
Table 6. Effects of different diets on alpha-diversity of rumen microflora of mutton sheep (n = 12).
Table 6. Effects of different diets on alpha-diversity of rumen microflora of mutton sheep (n = 12).
ItemsGroups 1p-Value
CONMICMEYLFCHFC
W1–W4
Coverage0.9975 ± 0.0080.9974 ± 0.00100.9977 ± 0.00100.9977 ± 0.00100.9973 ± 0.00060.731
Sobs1069.00 ± 191.391146.42 ± 198.491113.83 ± 189.021089.50 ± 184.171177.75 ± 124.180.588
Ace1090.05 ± 195.661168.28 ± 204.111132.97 ± 196.881108.59 ± 189.311200.35 ± 125.380.594
Chao11089.71 ± 195.931167.73 ± 207.021131.35 ± 197.691106.45 ± 189.511199.29 ± 124.660.596
Shannon5.86 ± 0.496.00 ± 0.335.86 ± 0.485.87 ± 0.336.04 ± 0.360.693
Shannoneven0.841 ± 0.0480.864 ± 0.0280.834 ± 0.0490.841 ± 0.0300.854 ± 0.0420.735
W5–W7
Coverage0.9977 ± 0.0007 c0.9996 ± 0.0003 a0.9990 ± 0.0005 b0.9981 ± 0.0006 c0.9982 ± 0.0007 c<0.001
Sobs1033.00 ± 221.07910.17 ± 145.13992.00 ± 170.441121.33 ± 232.481103.92 ± 167.430.057
Ace1052.12 ± 225.00 ab912.39 ± 145.05 b998.69 ± 172.23 ab1135.65 ± 235.56 a1117.79 ± 170.72 a0.041
Chao11049.41 ± 225.04 ab911.22 ± 144.92 b997.21 ± 172.15 ab1134.51 ± 235.19 a1115.17 ± 169.25 a0.041
Shannon5.76 ± 0.485.79 ± 0.405.95 ± 0.416.04 ± 0.436.02 ± 0.280.316
Shannoneven0.832 ± 0.0420.850 ± 0.0400.864 ± 0.0430.861 ± 0.0350.861 ± 0.0250.237
1 CON: control group with basal diet; MIC: added compound probiotics (2% of the feed amount) on the basis of CON; MEY: added enzyme and yeast combination preparation (1% of the feed amount) on the basis of MIC; LFC: low-proportion fermented cotton straw (FCS accounted for 14.50% of the diet) group; HFC: high-proportion fermented cotton straw (FCS accounted for 29.00% of the diet) group. Note: In the same row, values with different letter superscripts mean significant differences (p < 0.05).
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Wei, P.; Guan, M.; Liang, X.; Yuan, K.; Chen, N.; Yang, Y.; Gong, P. Growth Performance and Rumen Microbiota of Sheep Respond to Cotton Straw Fermented with Compound Probiotics. Fermentation 2025, 11, 244. https://doi.org/10.3390/fermentation11050244

AMA Style

Wei P, Guan M, Liang X, Yuan K, Chen N, Yang Y, Gong P. Growth Performance and Rumen Microbiota of Sheep Respond to Cotton Straw Fermented with Compound Probiotics. Fermentation. 2025; 11(5):244. https://doi.org/10.3390/fermentation11050244

Chicago/Turabian Style

Wei, Peiling, Mingxuan Guan, Xuhui Liang, Kaixin Yuan, Ning Chen, Yuxin Yang, and Ping Gong. 2025. "Growth Performance and Rumen Microbiota of Sheep Respond to Cotton Straw Fermented with Compound Probiotics" Fermentation 11, no. 5: 244. https://doi.org/10.3390/fermentation11050244

APA Style

Wei, P., Guan, M., Liang, X., Yuan, K., Chen, N., Yang, Y., & Gong, P. (2025). Growth Performance and Rumen Microbiota of Sheep Respond to Cotton Straw Fermented with Compound Probiotics. Fermentation, 11(5), 244. https://doi.org/10.3390/fermentation11050244

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