Antibiotics have been widely used since the 1940s to build the immunocompetence of livestock against infectious diseases and as growth promoters (GPs). However, long term use of in-feed antibiotics as GPs will lead to the development of drug resistance bacteria, which can further be transferred to humans and be potential threatens to human health (1). With increasing concerns toward the use of antibiotics in the ruminant feed industry, more emphasis has been given to increase public awareness on the issue, disease prevention, and the use of other natural GPs, such as direct-fed microbials (DFMs) (2).

DFMs are mono or mixed cultures of live microbes that exert beneficial health effects by improving gastrointestinal tract microbial balance when fed to the host (3). Aspergillus oryzae culture (AOC) is one of the most common DFM product and has been widely used as a feed additive in ruminant production (4–6). Studies have shown that AOC can regulate rumen microbiota (7), improve fiber digestibility (8). Supplementation of Aspergillus oryzae (AO) in diets of lactating cows increased milk production, feed efficiency, and tolerance to heat stress in some (9) but not all (10, 11) studies. However, reports about the effects of AOC on the rumen microbial community and characteristics of sheep are relatively scarce. The effective dosage of AOC in the production of ruminants such as sheep has not yet been reported. Most of the additive doses reported in existing studies are less than 10g, and the effects were different. As a nutritional additive, AOC is an excellent protein raw material. Therefore, increasing the addition amount in production may produce better results.

The rumen microbiome includes various microbes, such as bacteria, fungi, protozoa, archaea, and bacteriophages. The rumen microbiome plays a crucial role in shaping digestion physiology and ruminant production, and its potential utility for health and performance manipulation (12). A key reason for this is that rumen microbes could help ruminants transform fibrous and nonfibrous plant material into meat and milk. Therefore, regulating ruminal microbes and fermentation could improve feed efficiency in ruminants, that means a lot especially as the costs of forage and cereals have been continuously increased in recent years.

In the sheep industry, the sheep should be fed by balanced diets which contain all kinds of nutrients even some small feed additives to improve nutrients digestion and absorption, promote ruminal fermentation and hematological parameters, ultimately enhance the growth performance and productivity of sheep (13). Hematological indicators could reflect the health status of animals. To assess the impact of additives such as AOC on sheep health, hematological indicators may be timelier and more effective. Nowadays, Hu sheep husbandry has gradually become large-scale and intensive production (14), which has brought about some new problems, especially the high incidences of nutritional and metabolic diseases. The disorder of rumen microbiota is one of the causes of these diseases. Therefore, the use of DFMs to stabilize the rumen environment while ensuring the efficient operation of the digestive capacity of the gastrointestinal tract is being considered as the best strategy to solve the problem of antibiotics being disabled.

The DFMs feed additives with dual characteristics of nutritional and non-nutritional have improved the utilization rate of nutrition digestion and regulated the health of animals (15), especially AOC. However, whether AOC could increase the rumen digestibility, shape rumen microbiome, improve the health status of Hu sheep have not been reported. Here, the objective of the present study was to investigate the effects of supplementation with different amounts of AOC on the degradation rate of various feeds for 24 h, rumen fermentation parameters, microbial community, and blood cell composition of Hu sheep.

All experimental protocols were approved by the animal protection committee of Gansu Province, China (2005-12), and the experimental procedures used in this study were in line with the University's animal research guidelines.

In the present study, AOC supplementation increased the 24 h degradation rate of DM, and 40 g/d AOC increased the DM degradation rate of alfalfa hay and corn straw by 18.53% and 18.08%, respectively. The main reason for this is that AOC contains high activity enzyme series (23), including neutral protease activity (1020 IU/g), alkaline protease activity (770 IU/g), and cellulase activity (482 IU/g), so that it can significantly increase the activity of microcrystalline cellulase in alfalfa meal and corn straw, therefore accelerating fiber cracking (24) and microbial colonization in feed, and thus significantly increasing the DM degradation rate of alfalfa hay and corn straw at 24 h. Previous studies also obtained similar results. Newbold et al. (25) and Tricarico et al. (26) found that AO extract can increase the DM intake of lactating cows and heifers, and feeding 5 g/d of AO in vitro can improve the DM degradation rate of hay and barley in the artificial rumen for 24 h. Gomez-Alarcon et al. (9) conducted experiments on cows at dry and lactation stages and showed that feeding 3 g/d of AO could improve the fiber degradation rate and digestibility of the total digestive tract. In addition, Sun et al. (27) reported that AOC supplementation had a positive effect on rumen MCP in dairy cows, which may be due to the improvement in microbial activity. The results indicated that AOC could stabilize the rumen fermentation environment, ensure the growth of beneficial bacteria, increase the number of total anaerobic bacteria, and reduce the number of protozoa; therefore, more rumen bacteria can survive, which is conducive to the fermentation and degradation of nutrients in the rumen, thus improving the degradation rate of DM at 24 h. The predicted functions of rumen bacteria were also proved that the carbohydrate and lipid metabolism were improved in the AOC treatments, which indicated that the greater degradation rate of DM in the rumen could be advantageous for bacterial metabolism.

In the present study, feeding AOC significantly increased the content of TVFAs, including butyrate, butyrate, isovalerate, and valerate. Aditionally, the ratio of acetate to propionate decreased in the AOC-40 g/d treatment. Combined with the results of the DM degradation analysis, the increasing DM degradation in 24 h of alfalfa hay, corn stalk, and corn germ meal led to the greater TVFA content in the AOC supplementary treatments (20 g/d and 40 g/d). The same results have been reported previously: AOC could improve the rumen TVFA content, promote the decomposition of fiber, improve the rumen fermentation efficiency, and produce VFAs (5, 24). Gomezalarcon et al. (9) reported that TVFA content tended to be higher and that the molar ratio of A/P was lower (2.64 vs. 3.01) in high concentrate diets. Jouany et al. (28) found that feeding AOC could significantly reduce acetic acid content and increase propionic acid and butyric acid content. The unaffected acetate propionate concentration among treatments may be caused by the diet used in the present study. Isovalerate is a growth factor for some types of fibrolytic bacteria in the rumen. The increase in isovalerate may improve the microbial community structure to raise the DM degradation rate, which matched the results of the present experiment.

The TVFA affected the pH in the rumen after feeding. The dynamic change in the rumen pH was caused by the quick fermentation of the fast-degrading carbohydrates in the rumen. The rate of VFA production was quicker than the absorption of rumen epithelium and outflow rates; therefore, the accumulation of VFA decreased the pH. Afterward, the fermentation drops made VFA output balance with absorption, and the pH recovered to the normal range. The rumen pH value was not affected by AOC supplementation in the present study for either the mean, maximum, or minimum pH. These results were supported by Higginbotham et al. (6) and Fondevila et al. (23), who found that the AOC did not affect rumen pH in lactating dairy cows or sheep. However, some researchers reported that feeding 1 g/d, 2 g/d, 4 g/d, and 6 g/d AOC produced an effect, but without a clear trend for dose dependency in cattle (29), while feeding 27 g/d of AOC tended to lower the rumen pH (30). The different results were related to the different supplementation amounts of AOC, the basic diet (the ratio of concentrate to roughage), and ruminant species, all of which could affect the response of rumen pH to AOC supplementation. The dynamic trend of rumen pH after ingestion is to first decrease and then increase, returning to a normal physiological level. The ratio of concentrate to roughage in the diet used in the present study was 80 to 20, which is a typical diet for fattening sheep. The presence of a large amount of concentrate could make the rumen pH less affected by AOC. Rumen pH was mainly determined by the number of feeds converted into VFA yield by rumen microorganisms in the rumen after feeding. Interestingly, the dynamic changes of rumen pH in 24 h in the present study showed that the recovery of the pH value after intake was quicker in the AOC supplementation treatments than in the control treatment (from 5.6 to 6.4). This may be related to the greater DM degradation rate at 24 h in the AOC treatments, especially in the AOC-40g/d group. The rumen pH fluctuates within the range of 5.0 ~ 7.5, while the optimal pH of AO cellulase activity is 5.5 ~ 6.0. The pH after feeding AOC could provide a suitable acid-base environment for AOC cellulase to exert its maximum effect.

The α-diversity indices of all treatments were not significantly different in the present study. Combined with the results of rumen fermentation and pH, AOC did not affect the rumen bacterial diversity, which indicated that the rumen microbial flora maintains a better steady- state after AOC supplementation. Other study reported that the rumen bacterial community was changed after feeding with DFMs (31). The inconsistent results among studies were likely due to differences in diet treatments, dietary starch contents, and experimental animals.

Our results indicated that AOC significantly positively regulates the abundance of Kiritimatiellaeota. Although few data on sheep rumen have described Kiritimatiellaeota, these families and their classes are relevant to the metabolic body weight, dry matter intake, and residual feed intake of sheep (32). In addition, Kiritimatiellaeota has been reported to be saccharolytic and derive energy via fermentation (33). In the present study, AOC supplementation prompted an increase in Kiritimatiellaeota abundance, which may lead to an increase in DM digestibility and changes in rumen fermentation parameters, especially TVFA. Due to a lack of information on Kiritimatiellaeota, there was no significant difference at the genus level of rumen bacteria. The present study did not measure the DMI of the sheep because we used an in situ experimental design. Perhaps Kiritimatiellaeota in feeding regulation provides a new target in follow-up fattening experiments. In the present study, the AOC-40 g/d supplementation group and the CON treatment had different bacterial community structures in the rumen of Hu sheep. The bacterial community structure of various individuals was also different. The variation in the present study was the dosage of AOC. Due to the stability of the microbial community in the rumen (34), the results of β-diversity were also understandable.

Hematological parameters such as blood cell content are typically auxiliary indexes used to monitor health conditions or metabolic processes of animals (13). The blood cell analysis results of the sheep in the present study showed that all indicators were within the normal reference range (35), and AOC supplementation did not affect the blood cell composition, indicating that the experimental sheep remained healthy and free from disease. Previous studies have shown that AO and its culture have different effects on serum biochemical indices such as phosphorus, urea nitrogen, and alkaline phosphatase activity (36, 37), while the effect on blood cell composition has not been studied. The effects of nutritional additives on hematological parameters in sheep (13) and Nile tilapia (38) were usually insignificant, which were consistent with our results.

The supplementation of Hu sheep with AOC could improve feed DM degradation and increase the energy supply of TVFAs in the rumen. Based on the feed conditions of the present study, supplementation 40 g/d of AOC could increase the production efficiency of sheep while higher level have to further investigate.

The data presented in the study are deposited in the NCBI repository, accession numbers SRR17532854, SRR17532869, and PRJNA795832.

The animal study was reviewed and approved by the Animal Protection Committee of Gansu Province, China (2005-12).

LG and DZ collected the sample, analyzed the data, and drafted the manuscript. RD, FaL, and TR collected the sample. FeL presented the idea of this manuscript, supported the funding, analyzed the conclusions, and revised the manuscript. All authors contributed to the article and approved the submitted version.

This research was financially supported by the Natural Science Foundation of Gansu (China), Grant Number 20JR10RA299.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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