Production performance and rumen bacterial community structure of Hu sheep fed fermented spent mushroom substrate from Pleurotus eryngii

doi:10.21203/rs.3.rs-2337524/v1

This study aimed to compare the slaughter performance, meat quality and rumen bacterial community structure of Hu sheep by adding raw materials( RL0), 0 (RL1), 15% (RL2), 30% (RL3) and 45% (RL4) fermented spent mushroom substrate from Pleurotus eryngii to the basal diet. The results showed that: (1) The pre-slaughter weight (PSW), carcass weight (CW) and slaughter rate (SR) of RL2 group were higher than others, but the differences between the groups were not significant (p > 0.05). (2) The contents of threonine, valerine, leucine, lysine, histidine, total essential amino acids, aspartic acid, serine, glutamic acid and arginine of the musculi longissimus thoracis in RL2 and RL3 groups were significantly higher than RL1 and RL4 (p < 0.05). (3) A total of 1202445 valid sequences were obtained from rumen of Hu sheep fed different amounts of fermented feed, and the valid sequences were clustered into 9824 Operational Taxonomic Units (OTUs). (4) α diversity analysis showed that the richness and diversity of rumen bacterial communities in Hu sheep in RL1, RL2, RL3 and RL4 groups were significantly higher than RL0 group (p < 0.05). β diversity analysis showed that the bacterial community structure was the most different between RL0 and RL3. (5) At the genus level, compared with RL1, the relative abundance of Christensenellaceae R-7 in RL3 group decreased significantly by 33.59%, the relative abundance of Prevotellaceae UCG001 in RL2, RL3 and RL4 decreased significantly by 50.41%, 62.24% and 49.17%, respectively, and the relative abundance of Ruminococcaceae NK4A214 in RL2 group increased significantly by 35.01% (p < 0.05). In summary, the addition of fermented Pleurotus eryngii mushroom bran to the basal diet of Hu sheep can significantly improve the slaughter performance, meat quality and rumen bacterial community diversity and abundance of Hu sheep.

Biological sciences/Ecology/Agri ecology

Biological sciences/Ecology/Grassland ecology

Biological sciences/Ecology/Microbial ecology

Biological sciences/Microbiology/Bacteria

Biological sciences/Microbiology/Communities

Biological sciences/Ecology

Biological sciences/Microbiology

Spent mushroom substrate from Pleurotus eryngii

Fermented feed

Production performance

Rumen bacterial community diversity

Hu sheep

The lack of feed raw materials and their cost have always been an important factor restricting the development of animal husbandry industry In recent years^[1], the development and utilization of new feed resources has become an urgent problem to be solved. China is a major producer of edible fungi, ranking first in the world in annual output ^[2]. While the edible fungus industry is developing rapidly, a large number of by-products have been produced. Spent mushroom substrate from Pleurotus eryngii is the remaining media residue after harvesting, which has a wide range of sources and low price. Studies have shown that spent mushroom substrate is rich in crude fiber, crude protein, polysaccharides, crude fat, calcium, phosphorus and other active nutrients, and is a new type of high-quality feed raw material for livestock and poultry ^{[3, 4]}. However, spent mushroom substrate is not fully utilized in the actual production process at present, only a very small part is developed for animal feed, most of them are directly incinerated or discarded as waste, which not only wastes biological resources but also causes serious environmental pollution problems. Therefore, the rational development utilization technology of spent mushroom substrate as feed can not only turn waste into treasure, but also alleviate the problem of shortage of feed resources and improve the economic benefits of animal husbandry industry, which has important ecological significance and broad market prospects.

The main components of Pleurotus eryngii mushroom substrate are crude fiber raw materials such as bagasse, wheat bran, corn flour, etc., resulting in low digestion and utilization in livestock and poultry ^{[5, 6]}. Therefore, the use of microorganisms to ferment mushroom substrate from Pleurotus eryngii is an effective way of feed utilization. After microbial fermentation, macromolecular substances such as cellulose and hemicellulose are degraded into carbohydrates, amino acids, vitamins and other nutrients that are easily digested and absorbed by the animal, improving palatability and prolonging the preservation time. Previous studies have shown that mushroom substrate feed is of great significance in the development and utilization of unconventional feed for ruminants ^[7]. Rumen is a unique digestive organ of ruminants, and there are a large number of microorganisms inside of the rumen, mainly including bacteria, fungi, archaea, protozoa, etc., of which anaerobic bacteria are the dominant microorganisms ^[8]. The feed is fermented and decomposed under the action of microorganisms after entering the rumen, which is conducive to the efficient absorption of nutrients by the animal. Henderson et al. ^[9] and Maga et al. ^[10] found that feed is the dominant factor affecting the change of rumen microbial community structure in ruminants, which in turn affects the digestion and absorption of nutrients and energy supply in livestock and poultry. Therefore, understanding the microbial community composition structure of the rumen is the key to promote feed digestion and absorption and improve animal production performance. In this study, Bacillus subtilis, Saccharomyces cerevisiae and lactic acid bacteria were used to ferment spent mushroom substrate from Pleurotus eryngii, and the effects of different amounts of fermented feed on the slaughter performance, meat quality and rumen bacterial community structure of Hu sheep were explored, and technical support was provided for the development and efficient utilization of ruminant feed resources.

1.1 Effects of fermented spent mushroom substrate on slaughter performance and meat quality of Hu sheep

It can be seen from Table 1 that with the increase of the addition of fermented feed, the PSW, CW and SR of Hu sheep increased and then decreased. Among them, the PSW, CW, SR, ST and back fat thickness (BFT) were the highest in the RL2, and the lowest in the RL1, but there was no significant difference between two groups (p > 0.05). The EMA and GR values in the RL2 and RL3 were significantly higher than those in the RL1 and RL4 (p < 0.05). Compared with RL1, the EMA of RL2 and RL3 increased by 37.23% and 42.30%, and the GR value increased by 60.00% and 66.67%, respectively. In addition, there were no significant differences in MCP, pH of the musculi longissimus thoracis at 1 h and 24 h after slaughter, DR at 24 h and 48 h after slaughter (p > 0.05) (Table S.1). The results showed that the use of fermented spent mushroom substrate of Pleurotus eryngii as the diet of Hu sheep had no significant effect on the meat quality.

Table 1

Effects of different fermented *Pleurotus eryngii* mushroom substrate addition on the slaughter performance of Hu sheep
Treatment	PSW (kg)	CW (kg)	SR (%)	ST (cm)	BFT (cm)	EMA (cm²)	GR Value (cm)
RL1	36.80 ± 6.51a	16.95 ± 3.39a	45.96 ± 1.10a	0.15 ± 0.01a	0.13 ± 0.00a	9.67 ± 0.05b	0.90 ± 0.08b
RL2	41.54 ± 1.47a	19.78 ± 1.66a	47.56 ± 2.32a	0.18 ± 0.01a	0.27 ± 0.09a	13.27 ± 2.66a	1.44 ± 0.00a
RL3	39.73 ± 3.35a	18.83 ± 1.59a	47.38 ± 0.01a	0.16 ± 0.03a	0.21 ± 0.01a	13.76 ± 3.78a	1.50 ± 0.32a
RL4	39.27 ± 3.01a	18.47 ± 1.86a	46.98 ± 1.13a	0.18 ± 0.04a	0.16 ± 0.07a	9.38 ± 1.20b	1.06 ± 0.12b
Note: A different letter in each column indicates a significant difference (p < 0.05, n = 3).

Among the essential amino acids, the contents of threonine, valerine, leucine, lysine, histidine and total amount of essential amino acids of the musculi longissimus thoracis in RL2 and RL3 were significantly higher than those in RL1 and RL4 (p < 0.05) (Table 2), the content of isoleucine was significantly higher than RL4 (p < 0.05), and the contents of methionine and phenylalanine were not significantly different between samples (p > 0.05). Compared with RL1, the contents of threonine, valerine, leucine, lysine, histidine and total essential amino acids in the RL2 significantly increased by 20.81%, 16.17%, 21.04%, 24.49%, 17.37% and 20.35%, respectively, while the RL3 increased by 20.47%, 18.86%, 20.84%, 24.49%, 19.25% and 19.97%, respectively.

Table 2

Effects of different fermented *Pleurotus eryngii* mushroom substrate addition on amino acid content of mutton (g·kg^− 1)
Items	Groups
Items	RL1	RL2	RL3	RL4
Essential amino acid
Threonine	2.98 ± 0.40b	3.60 ± 0.05a	3.59 ± 0.09a	2.80 ± 0.03b
Valerine	3.34 ± 0.44b	3.88 ± 0.22a	3.97 ± 0.07a	3.27 ± 0.07b
Methionine	1.09 ± 0.41a	1.54 ± 0.25a	1.32 ± 0.12a	1.24 ± 0.58a
Isoleucine	2.93 ± 0.43bc	3.45 ± 0.10ab	3.47 ± 0.11a	2.55 ± 0.32c
Leucine	4.99 ± 0.70b	6.04 ± 0.11a	6.03 ± 0.10a	4.66 ± 0.20b
Phenylalanine	3.28 ± 0.50a	3.72 ± 0.34a	3.72 ± 0.35a	3.07 ± 0.08a
Lysine	5.35 ± 0.81b	6.66 ± 0.10a	6.66 ± 0.11a	5.16 ± 0.16b
Histidine	2.13 ± 0.37b	2.50 ± 0.08a	2.54 ± 0.07a	2.03 ± 0.04b
Total essential amino acids	26.09 ± 3.95b	31.40 ± 0.82a	31.30 ± 0.58a	24.78 ± 0.71b
Non-essential amino acids
Aspartic acid	6.01 ± 0.76b	7.13 ± 0.07a	7.08 ± 0.09a	5.56 ± 0.22b
Serine	2.56 ± 0.29b	3.15 ± 0.13a	2.95 ± 0.10a	2.42 ± 0.04b
Glutamic acid	10.58 ± 1.21b	12.46 ± 0.07a	12.43 ± 0.11a	10.08 ± 0.12b
Glycine	5.08 ± 0.19a	4.27 ± 0.20c	4.34 ± 0.22bc	4.90 ± 0.51ab
Alanine	4.52 ± 0.34ab	4.84 ± 0.26a	4.76 ± 0.13ab	4.35 ± 0.16b
Tyrosine	2.09 ± 0.33ab	2.47 ± 0.25a	2.42 ± 0.16a	1.93 ± 0.11b
Arginine	4.32 ± 0.41b	5.31 ± 0.24a	5.24 ± 0.29a	4.24 ± 0.21b
Proline	3.56 ± 0.11a	3.26 ± 0.07ab	3.21 ± 0.13b	3.36 ± 0.28ab
Note: A different letter in each line indicates a significant difference (p < 0.05, n = 3).

Among the non-essential amino acids, the contents of aspartic acid, serine, glutamic acid and arginine of the musculi longissimus thoracis in RL2 and RL3 were significantly higher than those in RL1 and RL4 (p < 0.05), and the tyrosine content was significantly higher than that in the RL4 (p < 0.05). Compared with RL1, the contents of aspartic acid, serine, glutamic acid and arginine in RL2 were significantly increased by 18.64%, 23.05%, 17.77% and 22.92%, respectively, and by 17.80%, 15.23%, 17.49% and 21.30% in RL3, respectively.

1.2 Otu Composition And Structure Of Bacterial Community

After quality control of data obtained by the IonS5^TMXL sequencing platform, a total of 1 202 445 valid sequences were obtained from raw materials and rumen liquid of Hu sheep, with an average valid sequences of 80 163 per sample. Valid sequences are clustered as 9824 OTUs at a sequence similarity threshold of 97%. Among them, RL0 has an average of 417 OTUs, RL1 is 980, RL2 is 1081, RL3 is 1165, and RL4 is 1061. The number of OTUs in RL0 was significantly lower than others (p < 0.05). The dilution curve directly reflects whether the extracted optimized sequence depth is reasonable and indirectly reflects the species richness in the sample. It can be seen from Fig. 1 that the number of sequences extracted reaches more than 30 000, and the curves tend to be flat, indicated that the sequence reservoir volume measured by different samples can better reflect the number of bacterial community species, and the amount of sequencing data is basically reasonable.

The Venn diagram can visualize the differences and overlaps in the OTU composition of bacterial communities in different samples (Fig. 2). The results of Venn analysis showed that at the OTU level, specific bacterial OTU accounted for 14.71% (359) of the total OTU sequence number in RL0, specific bacterial OTU accounted for 8.85% (216) of the total OTU sequence number in RL1, specific bacterial OTU in RL2 accounted for 3.07% (75) of the total OTU sequence number, and specific bacterial OTU in RL3 accounted for 9.63% (235) of the total OTU sequence number. Specific bacterial OTUs in RL4 accounted for 6.06%(148) of the total OTU sequence number. In addition, the number of bacterial OTUs shared by RL0, RL1, RL2, RL3, and RL4 was 277 (11.35%).

1.3 Analysis Of Bacterial Community Diversity

The species richness and uniformity of rumen bacterial communities treated with different amounts of fermented spent mushroom substrate were evaluated by the α diversity index in the samples (cutoff = 37 136). Table S.2 shows that the α diversity index of rumen bacterial community in RL1, RL2, RL3 and RL4 was significantly higher than RL0 (p < 0.05), indicated that the species diversity of rumen bacterial community was significantly higher than raw materials. However, with the increase of the addition of fermented spent mushroom substrate of Pleurotus eryngii, the differences in Observed species index, Shannon index, Simpson index, Chao1 index and ACE index were not significant (p > 0.05).

The β diversity index could measure the degree of divergence in species diversity between two samples by Unweighted Unifrac distance. The smaller the value, the smaller the difference in species diversity between two samples. As can be seen from Fig. 3, the smallest distances of Unweighted Unifrac were RL2 and RL4, with a value of 0.343, and the largest distances were RL0 and RL3, with a value of 0.763. It can be seen that the difference in bacterial community structure between RL2 and RL4 is the smallest, and the difference between RL0 and RL3 is the largest.

1.4 Pcoa Analysis Of Bacterial Community

The results of Principal Co-ordinates Analysis (PCoA) of rumen bacterial community with different treatments based on OTU were shown in Fig. 4, and principal component 1 (PC1) and principal component 2 (PC2) explained 80.45% and 7.71% of the variance of the variables, respectively, and the cumulative contribution rate was 88.16%. PC1 clearly distinguished the bacterial community in RL0 from RL1, RL2, RL3 and RL4, and PC2 clearly distinguished between four groups, indicated that there were great differences in the bacterial community structure of raw materials and the rumen liquid of Hu sheep with different treatments.

1.5 Changes In Bacterial Community Composition And Structure

A total of 10 phyla were detected in raw materials and the rumen of Hu sheep treated with different amounts of fermentation feed, the dominant taxa were as follows: Firmicutes (42.86%-78.73%), Bacteroidetes (8.54%-48.56%), Proteobacteria (0.71%-7.49%) and Fibrobacteres (0.18%- 6.86%) (Figure S.1). Compared with RL1, the relative abundance of Firmicutes in the RL4 was significantly increased by 9.36% (p < 0.05), the relative abundance of Bacteroidetes in the RL3 was increased by 2.10%, but the difference was not significant (p > 0.05), and the relative abundance of Fibrobacteres in the RL4 was significantly increased by 68.24% (p < 0.05).

At the genus level, the dominant genera (abundance > 1%) of bacterial community of the RL0 were Lactobacillus, Prevotella 1, and Bacteroides. In the rumen of Hu sheep treated with different amounts of bran, Prevotella 1, Christensenellaceae R-7, Ruminococcaceae NK4A214, Fibrobacter, Rikenellaceae RC9, Saccharofermentans and Prevotellaceae UCG001 were all dominant genera in RL1, RL2, RL3 and RL4 (Table 3).

Table 3

Relative abundance of rumen bacterial community of Hu sheep fed with different fermented *Pleurotus eryngii* mushroom substrate addition based on genus level
Genus	Relative abundance(%)
Genus	RL0	RL1	RL2	RL3	RL4
Lactobacillus	64.88 ± 36.24a	0.10 ± 0.01b	0.05 ± 0.00b	0.09 ± 0.00b	0.10 ± 0.02b
Prevotella 1	2.64 ± 4.41b	22.71 ± 3.46a	21.48 ± 2.94a	18.78 ± 9.13a	19.88 ± 2.79a
Christensenellaceae R-7	0.51 ± 0.68c	16.67 ± 3.19a	14.60 ± 3.33ab	11.07 ± 3.24b	16.26 ± 1.75a
Ruminococcaceae NK4A214	0.12 ± 0.16c	5.97 ± 0.85b	8.06 ± 1.01a	6.13 ± 1.12b	6.51 ± 1.37ab
Fibrobacter	0.18 ± 0.29b	4.15 ± 3.10a	5.39 ± 0.95a	5.15 ± 1.07a	6.94 ± 0.33a
Rikenellaceae RC9	0.33 ± 0.50b	5.09 ± 0.16a	5.60 ± 1.02a	6.32 ± 0.78a	4.92 ± 1.43a
Saccharofermentans	0.08 ± 0.12c	2.95 ± 0.92ab	1.56 ± 0.20bc	3.08 ± 0.73ab	3.83 ± 1.65a
Prevotellaceae UCG001	0.10 ± 0.11c	4.82 ± 0.59a	2.39 ± 0.31b	1.82 ± 0.38b	2.45 ± 0.89b
Bacteroides	2.41 ± 1.17a	0.03 ± 0.01b	0.01 ± 0.01b	0.01 ± 0.00b	0.02 ± 0.01b
Ruminococcus	0.13 ± 0.19b	0.65 ± 0.72ab	0.69 ± 0.50ab	0.86 ± 0.11ab	1.74 ± 1.38a
Note: A different letter in each line indicates a significant difference (p < 0.05, n = 3).

The relative abundance of Lactobacillus and Bacteroides in RL1, RL2, RL3 and RL4 decreased significantly compared with RL0 (p < 0.05), and the relative abundance of Prevotella 1, Christensenellaceae R-7, Ruminococcaceae NK4A214, Fibrobacter, Rikenellaceae RC9 and Prevotellaceae UCG001 increased significantly (p < 0.05). Compared with RL1, the relative abundance of Christensenellaceae R-7 in RL3 decreased significantly by 33.59%, the relative abundance of Prevotellaceae UCG001 in RL2, RL3 and RL4 decreased by 50.41%, 62.24% and 49.17%, respectively, and the relative abundance of Ruminococcaceae NK4A214 in RL2 increased significantly by 35.01% (p < 0.05). In addition, the analysis of bacterial community heat map based on genus level also showed that the composition of the rumen bacterial community of raw materials and the rumen liquid of Hu sheep with different treatments changed significantly (Fig. 5).

2.1 Effects of fermented feed on slaughter performance and meat quality of Hu sheep

Slaughter performance is one of the important indicators reflecting animal production performance and economic value, among which PSW, CW and SR are important factors affecting animal economic benefits. Chu et al. ^[11] showed that adding 30% fermented mushroom by-product from Flammulina velutipes to the diet of growing‐fattening pigs can significantly improve the carcass weight and quality of pigs. The results of this study showed that after adding different proportions of fermented spent mushroom substrate from Pleurotus eryngii to the diet, the PSW, CW and SR of Hu sheep increased, but the difference was not significant (p > 0.05). Moreover, with the increase of the addition of fermented feed, the PSW, CW and SR of Hu sheep showed a trend of first increasing and then decreasing, indicated that the proportion of fermented feed to the diet of Hu sheep should not be too high. This may be due to the beneficial bacteria and bioactive substances contained in fermented spent mushroom substrate to improve the rumen environment and promote body’s digestion of feed, thereby improving the conversion rate of fermented feed and improving its slaughter performance. Adding too much fermented feed will lead to a decrease in slaugh performance, which may be due to the large amount of cellulose contained in the substrate, which stimulates rumen peristalsis, accelerates the circulation rate of chyme, so that the nutrients in the chyme are not completely absorbed and are excreted, reducing the apparent digestibility of feed nutrients ^[12]. It can be seen that Hu sheep fed with 15% of fermented spent mushroom substrate from Pleurotus eryngii had the best slaughter performance.

Amino acids are important raw materials for protein synthesis, providing a material basis for host growth, metabolism and maintenance of vital signs, and their type and content are important factors affecting muscle flavor and nutritional value, and essential amino acid content is an important indicator to measure muscle quality ^[13]. When Boutry et al. ^[14] fed newborn pigs a leucine-rich diet, they found that the rate of protein synthesis in the muscles of pigs was accelerated, and the signaling pathways related to protein synthesis were significantly enriched. The results of this study showed that the total amount of essential amino acids in RL2 and RL3 was significantly higher than that in RL1, indicated that the addition of 15% and 30% of fermented feed could significantly increase the protein content of mutton and improve the nutritional value of meat quality. Aspartic acid, glutamic acid and glycine are umami amino acids in muscles, and their content is an important indicator that affects the flavor of meat, of which aspartic acid and glutamic acid can also be used as drugs to treat some diseases, mainly used for the treatment of liver diseases, digestive tract diseases, encephalopathy, cardiovascular diseases, respiratory diseases and for improving muscle vitality, pediatric nutrition and detoxification. In this trial, the contents of aspartic acid, glutamic acid and glycine in RL2 and RL3 were significantly higher than those in RL1. It can be seen that the addition of 15% and 30% fermented mushroom substrate to the diet of Hu sheep has a significant effect on the amino acid content in muscle, which can improve the flavor of mutton and improve the nutritional value of mutton.

2.2 Effects Of Fermented Feed On The Structure Of Rumen Bacterial Community In Hu Sheep

The microorganisms in ruminant rumen mainly include bacteria, fungi and protozoa, the rumen microbial composition structure is essential for maintaining environmental homeostasis in the rumen, promoting feed digestion and absorption, and benefiting animals. The structure of rumen microbial community is affected by many factors, of which diet types, structure and feeding mode are the most important factors ^[15]. For example, Liu et al. found that the addition of yeast culture supplementation to diets changed the bacterial community composition of sheep’s rumen in the house feed ^[16]. Previous studies have shown that the Bacteroidetes and the Firmicutes are the dominant phylum in ruminant rumens ^{[17, 18]}. Evans et al. ^[19] found that Bacteroides plays an important role in the degradation of non-fibrous substances, while Firmicutes is mainly involved in the decomposition of fibrous substances. In this experiment, the dominant phylum of Hu sheep rumen was Firmicutes, Bacteroidetes and Fibrobacteres, which were basically consistent with previous studies. With the increase of the addition of fermented mushroom substrate, the relative abundances of Firmicutes and Fibrobacteres in RL4 were significantly increased by 9.36% and 68.24% (p < 0.05), respectively, compared with those in RL1. The results showed that the addition of fermented mushroom substrate from Pleurotus eryngii to the basal diet of Hu sheep could promote the growth and proliferation of Firmicutes and Fibrobacter, and was conducive to the degradation of fibrous substances in the diet.

Previous studies have shown that Prevotella is the dominant genus of ruminant rumen ^{[20, 21]}. Thoetkiattikul et al. ^[22] discovered by high-throughput sequencing of 16S rDNA that the dominant bacteria of ruminant rumens were Prevotella and Flavobacterium. In this experiment, the dominant genera in the rumen of Hu sheep were Prevotella 1, Christensenellaceae R-7, Ruminococcaceae NK4A214, Fibrobacter, Rikenellaceae RC9, Saccharofermentans and Prevotellaceae UCG001. It is not completely consistent with the results of previous studies, and it is speculated that it may be caused by differences in animal breed, age, feed structure, feeding management, etc. Prevotella is considered to be a degrading genus with a strong ability to decompose hemicellulose ^[23], and plays an important role in the degradation of crude proteins, starches, xylan, and pectin ^{[22, 24]}. The results of this experiment showed that the relative abundance of Prevotella 1 in the rumen of Hu sheep was the highest, which was consistent with the previous results, but the difference between the groups was not significant. This may be due to the fact that the diet designed for this study had similar crude protein and energy levels, so it did not have a significant effect on the relative abundance of Prevotella. Ruminococcaceae include ruminococcus flavefaciens and ruminococcus albus and are the main fibro degrading bacteria in the rumen and can produce a large amount of cellulase, hemicellulase and xylanase to degrade cellulose and hemicellulose in forage. In this study, the relative abundance of Ruminococcaceae NK4A214 in RL2 was significantly higher than that of RL1 by 35.01% (p < 0.05). The results showed that the addition of fermented spent mushroom substrate from Pleurotus eryngii to the basal diet of Hu sheep could promote the growth and reproduction of rumen and improve the degradation rate of fiber in the diet.

3.1 Preparation of spent mushroom substrate from Pleurotus eryngii

Spent mushroom substrate from Pleurotus eryngii is provided by Fujian Greenbao Group, the cultivation material formula is as follows: bagasse 13.0%, wood chips 22.2%, corn cob 26.0%, bran 18.0%, corn flour 8.8%, soybean meal 9.0%, lime 1.2%, light calcium 1.8%. Waste fungus sticks were selected after harvest the mushrooms for 1 time, the mycelium is white, fresh and mildew-free, then unbagged, crushed and set aside.

The compound microbial agent "Huojunduo" was bought from Beijing Shengyuda Biotechnology Co., Ltd. Number of viable bacteria: Bacillus subtilis ≥ 100×10⁶ CFU·g^− 1, lactic acid bacteria ≥ 10×10⁶ CFU·g^− 1, Saccharomyces cerevisiae ≥ 100×10⁶ CFU·g^− 1, total bacterial ≥ 210×10⁶ CFU·g^− 1. The formula of spent mushroom substrate mixed fermentation material is: 500 kg of fungus bran, 260 kg of fine bran, 180 kg of barley, 10 kg of brown sugar, 1 kg of fermentation fungus agent and 50 kg of water. The microbial agent was evenly sprayed on the raw materials, mixed evenly and divided into sealed bags, and fermented anaerobically at room temperature for 21 days. The nutrients before and after fermentation are shown in Table S.3.

3.2 Experimental Design

The present study used 120 Hu-breed lambs, the ratio of male to female stands at 1:1, around 60 days of age, the mean body weight was 13.50 kg (SD = 3.10) at the start of the performance test. A univariate experimental design was used and randomized into 4 groups with 3 replicates in each group and 10 Hu sheep per replicate. 0 (RL1), 15% (RL2), 30% (RL3) and 45% (RL4) of fermented Pleurotus eryngii mushroom bran were added to the basal diet respectively, and the diet formula and nutrient level were shown in Table S.4. The trial was carried out by house feeding, sheep were adapted to the diet for a period of 10 days followed by 150 days of growth performance recording. Before the test, the sheep were numbered and dewormed, the sheep house was cleaned regularly, disinfected on time, and raised by special personnel in the well-ventilated sheep house. Feed once a day at 8:00 and 17:00 and drink freely. Feed should be based on a slight surplus in the trough, and the surplus should be collected and measured daily.

3.3 Measurement Indicators And Method

3.3.1 Slaughter performance determination

At the end of the experimental period, three test sheep were randomly selected in each group, fasting for 24 h and water for 2 h before slaughter. Slaughter before weighing, cut neck and bleed, peel off fur, remove head, hooves and internal organs, and measure slaughter performance indicators, including carcass weight (CW), slaughter rate (SR), skin thickness (ST), eye muscle area (EMA), back fat thickness (GR value), etc. Carcass weight: pre-slaughter live weight to remove the weight of the head, fur, hooves, tail, and internal organs (retaining the kidneys and surrounding fat); SR(%) = CW/ weight before slaughter×100;EMA: the cross-sectional area of the superior spine-ophthalmic muscle between the 12th ~ 13th ribs, the cross-sectional contour of the eye muscle was depicted with sulfuric acid paper, and then the contour area was calculated by the accumulation meter (QCJ-2000, Shandong); GR value: The thickness of the tissue between the 12th ~ 13th ribs was measured 11 cm from the midline of the dorsal spine with a vernier caliper.

3.3.2 Meat Quality Determination

The left musculi longissimus thoracis was taken after slaughtered to determine the meat quality indicators such as meat cooking percentage (MCP), pH and drip rate (DR). MCP: peel off the muscle-attached fat, weigh with a balance with a sensitivity of 0.1 g (count as W1), put the meat sample into a polyethylene packaging bag, steam in an aluminum steamer for 30 min, take it out, and put in cool water to cool down for 1 h, dry the surface moisture with paper, weigh (count as W2), MCP(%) = W2/W1×100%. pH: The pH was measured 45 min after slaughter with a pH meter, and the average value was taken as the final result. DR: Cut 2 pieces of meat with a length of 5 cm, width of 3 cm and thickness of 2 cm, put them in plastic bags (meat samples must not contact the wall of plastic bags), tie the mouth of the bag tightly and hang them in a refrigerator at 4°C, take them out after 24 h and 48 h, absorb the moisture on the surface with absorbent paper and weigh, record as the last weight. DR (%) = (initial weight - final weight) / initial weight × 100%. The amino acid composition of the musculi longissimus thoracis was determined. After treatment with a freeze dryer before testing, smashed and passed through a 0.425 mm sieve. Referring to the method of GB 5009.168–2016, the Hitachi L-8900 automatic amino acid analyzer (Japan) was used for measurement.

3.4 Sample Determination And Data Analysis

3.4.1 DNA extraction and PCR amplification of rumen liquid

The rumen liquid of Hu sheep in the RL1, RL2, RL3 and RL4 group and raw material sample (RL0) were collected, the total DNA of the microbial genome of the samples was extracted by CTAB. DNA concentration was determined by Nanodrop and DNA quality was detected by 1.2% agarose gel electrophoresis. Dilute DNA to 1 ng·µl^− 1 using sterile water. The V4 region of the bacterial 16S rDNA gene was amplified using universal primers 515F (5'-GTGCCAGCMGCCGCGGTAA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3'). PCR products were detected using a 2.0% agarose gel for electrophoresis and bands of interest were recovered by the Gene JET Gel Recovery Kit (Thermo Scientific). The recovered products were sent to the IonS5^TMXL platform of Beijing Novogene Technology Co., Ltd. for high-throughput sequencing.

3.4.2 Statistical Analysis

After the sequencing data were quality controlled by Fast QC, Cutadapt (V1.9.1) was used to filter short sequences (< 200 bp) and low-quality sequences (q < 25). Operational Taxonomic Units (OTUs) are assigned to valid sequences with 97% similarity with the SILVA database by the Mothur method. The sample diversity index was calculated using Qiime (Version 1.9.1) and principal component analysis was performed using R. One-way ANOVA was performed by SPSS 22.0, and p < 0.05 was used as the criterion for significant difference.

Under the test conditions, the slaughter performance of Hu sheep was the best when the addition amount of fermented spent mushroom substrate in the basal diet was 15%. When the adding proportion was 15% and 30%, the protein content of mutton can be significantly increased and the nutritional value of meat quality can be improved. When the adding proportion was 15% and 45%, it can significantly promote the growth and proliferation of microorganisms such as Firmicutes, Fibrobacteres and Ruminococcaceae NK4A214, which is conducive to the degradation of fibrous substances in diet. In summary, the addition of fermented mushroom bran to the basal diet of Hu sheep can significantly improve the slaughter performance, meat quality and rumen bacterial community diversity and abundance.

Acknowledgements

Supported by Special Fund for Scientific Research on Public Causes of Fujian Province (2020R1021002), "5511" Collaborative Innovation Project of People's Government of Fujian Province and Chinese Academy of Agricultural Sciences (XTCXGC2021010, XTCXGC2021019), and Project of Fujian Academy of Agricultural Sciences (CXTD2021009-1, YDXM202205).

Author contributions statement

XSH conceived the experiments, XYH and XFY conducted the experiments, LTZ, HDH and XZC analysed the results. All authors reviewed the manuscript.

Data availability statement

The datasets used and analysed during the current study available from the corresponding author on reasonable request.

Ethics statement

All of the procedures involving animals were approved by the Animal Care and Use Committee and Ethics Committee at the Agriculture Ecology Research Institute, Fujian Academy of Agricultural Sciences (NO. PZCASFAAS21022). We obtained written informed consent to use the animals in this study from the owners of the animals. All of the experiments were performed in accordance with the recommendations in the Regulations for the Administration of Affairs Concerning Experimental Animals of the State Council of the People’s Republic of China. All of the experiments were carried out in compliance with the ARRIVE guidelines.

Additional Information

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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No competing interests reported.

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Production performance and rumen bacterial community structure of Hu sheep fed fermented spent mushroom substrate from Pleurotus eryngii

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Introduction

1 Results And Analysis