In recent years, the application of enzymes in ruminants to improve feed utilization and optimize growth performance has been identified as a valuable measure. Non-starch polysaccharidases (NSPases) have been increasingly investigated as green and harmless additives for the degradation of non-starch polysaccharides (1–3). Previous studies have shown that the main effects of enzyme supplementation in ruminant diets are: breaking chemical bonds in the diet; improving the availability of nutrients in the digestive system; and increasing total enzyme activity in the rumen (4, 5). A meta-analysis showed inconsistent effects of added enzyme preparations (cellulase, xylanase, glucanase, ferulose esterase and combinations of each of their single enzymes) (6), and the reasons for their inconsistent results may be due to the source of enzymes, the amount of enzymes added, the experimental animals and the different diets on which enzymes acted in each study. Such as: Valdes reported that the supplementation of the diet of Suffolk lambs with enzyme products containing cellulases, xylanases, proteases and α-amylases improved average daily gain and feed conversion rate (7). Sakita fed an fibrolytic enzyme product to growing lambs which increased their dietary digestible energy and ADF digestibility (8). Similar results were reported by Shi et al. (9). Moreover, numerous studies have shown that supplementing dairy cow diets with enzymes can improve lactation performance (10–12). In contrast, Peters fed a combination of xylanase and dextranase to lactating cows and found no improvement in milk production (13). And Vinyard exported exogenous enzymes did not improve in vitro ruminal fermentation in the diet of high-producing dairy cows (14). Although there are many reports on combination enzymes, there are no specific standards or bases for the application of combination enzymes and the amount to be added.

Non-starch polysaccharides refer to the cellulose, xylan, β-glucan, xyloglucan, galactomannan and pectin present in plant cell walls which are difficult to degrade. The main basal polysaccharides in the plant cell wall other than cellulose are arabinoxylan and (1,3)(1,4)-β-D-glucan (15). (1,3)(1,4)-β-D-glucan binds cellulose and other polysaccharides tightly and forms the main part of the endosperm wall (16). The rumen of ruminants acts as a natural reactor for the degradation of plant cell walls, which includes most fiber-degrading bacteria such as Ruminococcus, Clostridium, and Prevotella (17). These microbes degrade non-starch polysaccharides and then generate volatile fatty acids (VFAs). NSPases include cellulases, xylanases, β-glucanases, and mannanases, which can synergize with rumen microbes, break the cell wall and promote the degradation of the rations. Cellulases and xylanases are the most widely used exogenous enzymes in animals and play important roles in nutrient utilization. In addition, enzyme supplementation has a potential effect on the composition and metabolic capacity of rumen microbes (18, 19), which can alter the composition of microbial communities (20, 21) and improve rumen fermentation and health (22). In the actual production process, most farmers use a sloppy meat farming model to obtain higher economic benefits. Particularly in the late fattening stage, fast short-term fattening of sheep meat is achieved by increasing the proportion of concentrate in the diet. However, feeding a high proportion of concentrate accelerates rumen fermentation and produces large amounts of volatile fatty acids, which cause acidosis, inflammatory reactions and nutritional metabolism (23). Currently, various additives, including enzymes, are used to attenuate the negative effects of high concentrations in ruminants fed high-concentrate diets. Studies on the supplementation of high concentrate diets with complex NSPases in sheep have not yet been conducted.

The effect of sex has been shown to be a major determinant of differences in growth and deposition rates across body tissues and carcasses (24). Regarding feed efficiency, it is well known that males have a better ability to convert feed into weight gain and carcass component tissues than females (25). Males showed a more pronounced rate of carcass gain, deposited more carcasses and gained more weight per kg of DM consumed than females (p < 0.05), with castrated animals in an intermediate position. This is consistent with the effects of sex on growth, body composition and carcass of animals reported by Washaya (26) and Paulino et al. (26). Purwin et al. also showed that sex has a significant effect on the rate of change in body weight and relative growth rate (27). Most of the current studies have focused on males, while only few studies have been conducted on female meat ruminants; therefore there is a need to study females to improve production performance. We hypothesized that supplementation with exogenous NSPases could improve rumen fermentation and production performance in ewes.

We hypothesized that a specific combination of NSPases (i.e., cellulose, xylanase and mannanases) applied to a high-concentrate diet would break the cell wall and, improve rumen fermentation and the growth performance of sheep. This study evaluated the effects of NSPases on the intake and total apparent digestibility of nutrients, rumen fermentation, and rumen microbial composition in sheep.

Animal growth requires an optimal amount of feed, while simultaneously, improving the digestibility of feed is essential to maximize the quality of growth. Growing animals may exhibit different metabolic and digestive disorders that affect their performance and health. In this study, we aimed to improve the growth performance and health of fattening sheep by adding an exogenous complex of NSPases. It has been hypothesized that this complex enzyme primarily improves fiber digestion and maintains increased microbial activity by interacting with endogenous rumen enzymes. Positive effects on animal health and metabolism have been reported.

Supplementation with NSPases in this study had no significant effect on the digestibility of DM, OM, CP, ADF, and NDF in any of the groups, however the T2 group showed some improvement in intake and significantly increased ADG. In other words, the fact that the total apparent digestibility of nutrients did not improve does not necessarily mean that there was no effect on growth performance. Evaluation of nutrient digestibility depends not only on the total apparent digestibility, but also on the fermentation and retention times of the digesta in the rumen, absorption in the small intestine, and fermentation in the large intestine (30). In the present study, we found an increase in DMI in the T2 group, and it was assumed that supplementation with NSPases had a positive effect on the fiber degradation properties. As shown in a previous study (31), supplementation of dairy cattle rations with enzymes reduced retention time in the rumen and increased the rate of food transfer, thus increasing feed intake but not total intestinal digestibility. This was also studied by Martins et al. (11).

Supplementation with NSPases may increase the availability of the nutrients required for rumen fermentation (11, 32). In the current study, supplementation with NSPases significantly increased the ratio of acetate to propionate, mainly because of the reduced propionate concentration. Ribeiro found that the use of fibrolytic enzymes in sheep diet was consistent with the findings of this study regarding a low propionate concentration and a high acetate to propionate ratio (33). Consistent results were obtained by Miorin et al. (34) in Nellore bulls fed a diet supplemented with xylanase. With the addition of an appropriate amount of NSPases, the enzymes may function synergistically with rumen microbes, preferentially acting on structural carbohydrates and promoting the degradation of non-starch polysaccharides in the rumen (35). It did not cause the accumulation of production or negative feedback effects as in the high dose group. In addition, Nellore bulls fed a high carbohydrate ration supplemented with fibrinolytic enzymes exhibited higher rumen pH, indicating that fibrinolytic enzymes can act during high carbohydrate loading (34). This may be due to changes in microbial populations or metabolic pathways (33). Prevotella and Ruminococcus are important fiber-degrading genera in the rumen (36). Prevotella is a protein-degrading bacterium (37). In our study, the abundances of Prevotella and Ruminococcus were higher in the T2 group than in the CON group, and the NH₃-N concentration was significantly higher in the T2 group than in the CON group, presumably indirectly promoting the degradation of dietary proteins and non-starch polysaccharides by changing the abundance of related microbes. Consistent results were also obtained by Guo et al. (38). Thus, in the present study, supplementation with a medium-dose of NSP improved productive performance probably by altering the composition of the sheep rumen microbial community.

Supplementation of fattening sheep diets with high doses of NSPases significantly reduced ADG and ruminal pH, significantly increased feed conversion rate, and exhibited a quadratic curve effect of the enzyme. Similar results have been reported in other studies (3), where excess enzymes attached to the feed competed with the rumen microbial binding sites for feed pellets, thus affecting feed utilization (39, 40). It has also been reported that a negative feedback mechanism occurs when the enzyme-substrate interaction reaches a critical concentration that inhibits enzyme action (41, 42). In this study, a high percentage of concentrate resulted in rapid degradation in the rumen (43). After supplementation with high doses of NSPases, rumen fermentation was further accelerated, which tended to cause accumulation of VFAs, thus inhibiting further fermentation. Therefore, in the present study, the second view was preferred owing to the quadratic curve effect of the enzyme.

As discussed, rumen fermentation was enhanced by supplementation with exogenous NSPases, resulting in a lower pH. In the present study, the enzyme treatment tended to increase rumen NH₃-N concentration, which is in contrary to the findings of Vittorazzi et al. (10), because the present experiment was conducted after 2 h of morning feeding for rumen fluid collection, which may be related to the fermentation rate. Fiber-degrading bacteria in the rumen have a high preference for ammonia as the nitrogen source (44). In addition, the increased abundance of fiber- and protein-degrading bacteria in this experiment resulted in an increase in the overall utilization of NH₃-N for MCP in the rumen.

When exogenous enzymes improve rumen fiber digestion, changes in rumen fermentation patterns may occur, resulting in an increase in the molar proportion of acetate and decrease in the molar proportion of propionate (45). Similarly, Ribeiro reported an increase in the total VFA concentration and proportion of acetic acid in vitro and a reduced proportion of propionic acid following enzyme treatment (46). In this experiment, acetate was elevated and propionate was decreased in the T2 group, but no significant changes in the VFA profile were observed, which is consistent with the lack of an effect on total digestive tract digestibility, suggesting that supplementation with exogenous NAPases has a minor effect on rumen fiber digestion.

As a significant increase in ADG was observed, we measured the serum hormones levels in each treatment group. GH, secreted by the anterior pituitary gland, is a main regulator of body growth and can be secretes IGF-1 through the liver (47). Animal growth is influenced by the growth hormone GH-IGF1 symbiont axis, in which GH is a major regulator of development, growth, and anabolic processes. IGF1 regulates the biological actions of GH by binding to its receptor (IGF1R) (48).

In the present study, the concentrations of GH and IGF-1 significantly increased after supplementation with NSPases, suggesting that NSPase supplementation promotes an increase in ADG levels, probably by regulating the growth hormone-releasing hormone (HPGH) axis. However, the specific mechanisms and interrelationships among these effects require further investigation. However, nutrient intake and end body weight did not significantly improve in this study. As discussed earlier, the addition of enzymes accelerated the rate of feed degradation in the rumen (49), produced more available energy, and contributed to an increase in associated hormones, but did not increase the extent of feed degradation. Therefore, there is a possibility that the increase in retention time of fiber in the rumen may have counteracted the improvement in digestibility and was not sufficient to cause any improvement in intake and end body weight.

However, the data showed that the T2 group (0.3% NSPases/kg DM) increased 108 g/d, 104.87 g/d, 1.59 g/d, 3.64 g/d, and 0.9 g/d in DM, OM, CP, NDF, and ADF intake, respectively, and all of the test groups were higher than the control group. Therefore, it was speculated that it might be due to the large within-group difference that caused insignificant differences between the results of the test group and the control group. Furthermore, a meta-analysis of studies suggested that long-term serial experiments are preferable for observing the response of exogenous cellulose-degrading enzymes to dietary treatment (6). In this experiment, only a 42 d test cycle was conducted, and the possible effects on growth performance were not demonstrated. Therefore, future subsequent related studies should increase the sample size, reduce within-group variations, and conduct consecutive long-term test cycles.

Leptin is a hormone secreted by white adipose tissue that crosses the blood–brain barrier and affects host appetite and energy metabolism (50–53). It has been shown that short-chain fatty acids (SCFAs), derived from the gastrointestinal tract, enhance the release of leptin and inhibit host appetite (54). There are various pathways for the regulation of related hormones by gastrointestinal flora; for example, derived short-chain fatty acids not only regulate the release of appetite hormones related to the gastrointestinal tract and affect gastrointestinal nerve signaling, but also affect the function and integrity of the intestinal barrier (55–58). In the present study, leptin concentrations gradually increased significantly with increasing NSPases supplementation. As females have a more pronounced maturation rate than males, as well as a greater accumulation of body fat, the consumptive capacity of prehensile females decreases with increasing body weight, as fat directly affects the physical restriction of abdominal fat to the rumen, and indirectly affects food intake through the secretion of leptin by the adipocytes, which is precisely the hormone associated with reduced feeding (59). However, there were no effects until the end of the experimental period, probably because the females had not yet reached sufficient body fat content to trigger a reduction in feed intake. In this study, leptin concentration increased significantly (p < 0.05) with the amount of enzyme added, but feed intake was not affected, suggesting that enzyme supplementation during fattening did not negatively affect the experimental sheep, or that the negative effects were not manifested, possibly because sufficient body fat content had not yet been achieved to trigger a reduction in feed intake.

Because the medium-dose group showed better results, we measured rumen microbe in the CON and T2 groups. In the present study, the bacterial abundance increased in the T2 group, in which Firmicutes and Bacteroidota were the dominant phylum in the rumen of sheep, consistent with the results of previous studies (60, 61). Among them, the Bacteroidota was mainly for the energy conversion and acquisition, and the Firmicutes mainly played an essential role in the degradation of non-structural carbohydrates (62, 63). Both the Bacteroidota and Firmicutes were more abundant in the T2 group than in the CON group. This indicates that supplementation with NSPases promotes non-structural carbohydrate degradation and energy uptake. Another study reported that feeding high concentration resulted in higher abundance of Firmicutes in the rumen than feeding on high-fiber diets (64). In the present study, supplementation with NSPases resulted in a higher abundance of the Firmicutes in the T2 group than in the CON group, confirming that supplementation with NSPases increased the rate of degradation and energy conversion in the rumen and creating the illusion that the T2 group had a higher non-structural carbohydrate content than the CON group.

Interestingly, we found significantly lower abundances of Uroviricota and Proteobacteria in the T2 group than in the CON group. When the number of Proteobacteria is low, it behaves benignly; when it is more abundant, it may become a microbe that triggers inflammation, and its long-term enrichment may represent microbial instability or disease state (65). Some Proteobacteria are easily degraded and produce metabolites after death capable of destroy the mucosal barrier in the gastrointestinal tract (65). In contrast, Bifidobacteria, a major genus of Actinobacteria can avoid excessive inflammation (66). It has also been reported that the maintenance of gastrointestinal homeostasis depends on (SCFAs) as microbial metabolic byproducts (e.g., acetate, propionate, and butyrate) and that these acids affect immune responses in the intestine and other organs and tissues (67, 68). The regulatory relationship between microbial-derived fatty acids and host defense peptides was elaborated. In the present study, it was found that supplementation with NSPases not only increased the abundance of the Bacteroidota, Firmicutes and Actinobacteria, but also resulted in a significant increase in immunoglobulins, accompanied by a decrease in the abundance of viruses in rumen microbes, suggesting that the addition of appropriate amounts of NSPases to high concentrate rations can lead to antiviral effects and help improve rumen health, which may be associated with altered rumen microbes.

Among them, acetate concentration had a significant positive correlation with Prevotella and propionate had a significant positive correlation with unclassified_f_Lachnospiraceae, and both Prevotella and unclassified_f_Lachnospiraceae abundance were higher in the T2 group than in the CON group, indicating that the potential of acetate and propionate was higher in the T2 group than in the CON group, but in reality the propionate concentration was lower in the T2 group than in the CON group, presumably because the supplementation of NSPases improved in rumen health and helped rumen propionate fermentation, which led to faster uptake of propionic acid by the rumen epithelium, resulting in low measured concentrations. Moreover, propionate, as a precursor of gluconeogenesis, can be absorbed by the liver for use by the organism, ultimately leading to an increase in ADG in the T2 group. This also explains why total apparent digestibility was not improved by NSPase supplementation, while the ADG was improved. Therefore, NSPases supplementation improves rumen health, promotes rumen fermentation, and enhanced energy absorption and utilization. Previous studies have shown that enzyme preparations based on Aspergillus can increase bacterial populations (69, 70) an act synergistically with rumen microbes to break down plant cell walls and promote nutrient release (71).

Using the Wilcoxon rank-sum test of the abundance of CAZy in the CON and T2 groups, we found that with NSPases supplementation promoted the degradation of NSP, which also confirmed that NSPases supplementation helped improve rumen fermentation after feeding high concentrates.

In conclusion, supplementation with non-starch polysaccharide degrading enzymes increased average daily weight gain, improved rumen health, and enhanced immunity. Feeding with NSPases improves rumen health and promotes energy absorption and utilization by increasing immunoglobulin concentrations and decreasing rumen viral abundance.

The metagenome sequences obtained from sheep rumen fluids have been deposited in the NCBI Short Read Archive (GenBank accession number: PRJNA978373).

The animal studies were approved by Approval Letter of Biology Ethics Committee of Shihezi University. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

YX: Writing – original draft, Writing – review & editing. HS: Writing – review & editing. HG: Validation, Writing – review & editing. CN: Project administration, Writing – review & editing. SN: Writing – review & editing. QL: Resources, Writing – review & editing. CC: Writing – review & editing. WZ: Writing – review & editing.