Effects of Flammulina velutipes mushroom residues on growth performance, apparent digestibility, serum biochemical indicators, rumen fermentation and microbial of Guizhou black goat

Abstract

Introduction
The primary objective of the current study was to evaluate the effects of Flammulina velutipes mushroom residue (FVMR) in a fermented total mixed ration (FTMR) diet on the fattening effect and rumen microorganisms in Guizhou black male goats.

Methods
A total of 22 Guizhou black male goats were allocated into two groups using the Randomized Complete Block Design (RCBD) experimental design. The average initial weight was 22.41 ± 0.90 kg and with 11 goats in each group. The control group (group I) was fed the traditional fermentation total mixed ration (FTMR) diet without FVMR. Group II was fed the 30% FVMR in the FTMR diet.

Results
The results showed that compared with group I, the addition of FVMR in the goat diet could reduce the feed cost and feed conversion ratio (FCR) of group II (p < 0.01). Notably, the apparent digestibility of crude protein (CP), acid detergent fiber (ADF), neutral detergent fiber (NDF), and dry matter (DM) were higher in group II (p < 0.01). The levels of growth hormone (GH), immunoglobulin A (IgA), and immunoglobulin M (IgM) in group II were higher than that of group I (p < 0.01), which the level of glutamic oxalacetic transaminase (ALT) and interleukin-6 (IL-6) was noticeably lower than that of group I (p < 0.01). 30% FVMR in FTMR diets had no effect on rumen fermentation parameters and microbial composition at the phylum level of Guizhou black male goats (p > 0.05). However, at the genus level, the relative abundance of bacteroidal_bs11_gut_group, Christensenellaceae_R-7_group and Desulfovibrio in group II was lower than in group I (p < 0.05), and the relative abundance of Lachnospiraceae_ND3007_group was higher than in group I (p < 0.01).

Discussion
In conclusion, the results of the current study indicated that 30% FVMR in the FTMR diet improves rumen fermentation and rumen microbial composition in Guizhou black male goats, which improves growth performance, apparent digestibility, and immunity.

Full text

Frontiers in Microbiology 01 frontiersin.org
Eects of Flammulina velutipes
mushroom residues on growth
performance, apparent
digestibility, serum biochemical
indicators, rumen fermentation
and microbial of Guizhou black
goat
YongLong
1,2, 3,4, WenXiao
2,3, YanpinZhao
2,3, ChaoYuan
2,
DefengWang
2, YangYang
2, ChaozhiSu
2, PramotePaengkoum
4
and YongHan
1,2*
1 Guizhou University of Traditional Chinese Medicine, Guiyang, China, 2 Institute of Animal Husbandry
and Veterinary Sciences, Guizhou Academy of Agricultural Sciences, Guiyang, China, 3 Key Laboratory
of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, Ministry of
Education, Guizhou University, Guiyang, China, 4 School of Animal Technology and Innovation,
Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima,
Thailand
Introduction: The primary objective of the current study was to evaluate the
eects of Flammulina velutipes mushroom residue (FVMR) in a fermented total
mixed ration (FTMR) diet on the fattening eect and rumen microorganisms in
Guizhou black male goats.
Methods: A total of 22 Guizhou black male goats were allocated into two groups
using the Randomized Complete Block Design (RCBD) experimental design. The
average initial weight was 22.41 ± 0.90 kg and with 11 goats in each group. The
control group (group I) was fed the traditional fermentation total mixed ration
(FTMR) diet without FVMR. Group II was fed the 30% FVMR in the FTMR diet.
Results: The results showed that compared with group I, the addition of FVMR
in the goat diet could reduce the feed cost and feed conversion ratio (FCR) of
group II (p < 0.01). Notably, the apparent digestibility of crude protein (CP), acid
detergent fiber (ADF), neutral detergent fiber (NDF), and dry matter (DM) were
higher in group II (p < 0.01). The levels of growth hormone (GH), immunoglobulin
A (IgA), and immunoglobulin M (IgM) in group II were higher than that of group
I (p < 0.01), which the level of glutamic oxalacetic transaminase (ALT) and
interleukin-6 (IL-6) was noticeably lower than that of group I (p < 0.01). 30% FVMR
in FTMR diets had no eect on rumen fermentation parameters and microbial
composition at the phylum level of Guizhou black male goats (p > 0.05). However,
at the genus level, the relative abundance of bacteroidal_bs11_gut_group,
Christensenellaceae_R-7_group and Desulfovibrio in group II was lower than in
group I (p < 0.05), and the relative abundance of Lachnospiraceae_ND3007_
group was higher than in group I (p < 0.01).
Discussion: In conclusion, the results of the current study indicated that 30%
FVMR in the FTMR diet improves rumen fermentation and rumen microbial
composition in Guizhou black male goats, which improves growth performance,
apparent digestibility, and immunity.
OPEN ACCESS
EDITED BY
Anusorn Cherdthong,
Khon Kaen University, Thailand
REVIEWED BY
Benjamad Khonkhaeng,
Rajamangala University of Technology Isan,
Thailand
Ilias Giannenas,
Aristotle University of Thessaloniki, Greece
*CORRESPONDENCE
Yong Han
shhuang1@gzu.edu.cn
RECEIVED 01 December 2023
ACCEPTED 08 January 2024
PUBLISHED 24 January 2024
CITATION
Long Y, Xiao W, Zhao Y, Yuan C, Wang D,
Yang Y, Su C, Paengkoum P and Han Y (2024)
Eects of Flammulina velutipes mushroom
residues on growth performance, apparent
digestibility, serum biochemical indicators,
rumen fermentation and microbial of
Guizhou black goat.
Front. Microbiol. 15:1347853.
doi: 10.3389/fmicb.2024.1347853
COPYRIGHT
© 2024 Long, Xiao, Zhao, Yuan, Wang, Yang,
Su, Paengkoum and Han. This is an open-
access article distributed under the terms of
the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction
in other forums is permitted, provided the
original author(s) and the copyright owner(s)
are credited and that the original publication
in this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 24 January 2024
DOI 10.3389/fmicb.2024.1347853

Long et al. 10.3389/fmicb.2024.1347853
Frontiers in Microbiology 02 frontiersin.org
KEYWORDS
apparent digestibility, growth performance, rumen fermentation, rumen
microorganisms, serum biochemical indicators
1 Introduction
Mushroom residue is the main by-product le aer the production
of edible fungi, and its components are mainly wood chips, cottonseed
hulls, corn cobs, rice straw, and sugarcane residue. For every kilogram
of fresh edible mushroom produced, about 5 kilograms of mushroom
residue products will beproduced (Gao et al., 2021). In 2020, the
output of mushroom residue in China reached 132–203 million tons.
At present, the primary uses of mushroom residue are the
reproduction of edible fungi, the development of animal feed,
composting, the development of biomass energy, soil improvement,
and restoration, sewage treatment, extraction of biologically active
substances and raw materials for the food industry (Guo etal., 2022;
Leong et al., 2022). e nutrients in the mushroom residue aer
harvesting edible fungi have not been completely degraded and the
contents of crude protein (CP), ether extract (EE), crude ber (CF),
and amino acids are relatively high (Estrada et al., 2009). e
mushroom sticks produce many fungal mycelium and benecial
bacteria during edible mushroom growth. During the growth process
of mycelium, enzymatic hydrolysis can produce a variety of sugars
(water-soluble dietary ber), polyphenolic compounds (quercetin,
catechin, gallate, caeate, etc.), compounds with anti-cancer activity
(Flammulina velutipes polysaccharides, fungal immunomodulatory
proteins, steroid compounds, monoterpenes, etc.) have lipid-lowering,
antioxidant and anti-cancer eects on animals (Hertog etal., 1993;
Yeh etal., 2014; Naja etal., 2019). In addition, the characteristic
mushroom fragrance of mycelia can improve the palatability of feed
and stimulate the appetite of livestock (Koutrotsios etal., 2014). A
previous study by Kim etal. (2011) indicated that 10% fermented
oyster mushroom residue in the diet could enhance Holstein calves’
growth performance and blood biochemical indicators were not
aected. Huang etal. (2023) found that the incorporation of 15%
Pleurotus eryngii fermentation residues in Hu sheep diets had the best
eect on improving production performance, rumen microbial
composition, and rumen microbial abundance of Hu sheep. Mandale
etal. (2023) considered that supplementing 10% mushroom residues
in Berari goat diets could reduce feed costs and improve growth
performance in Berari goats. Relevant studies have shown that the
incorporation of FVMR in goat diets could enhance apparent
digestibility, slaughter performance, nitrogen biological value, and
nitrogen deposition rate. At the same time, the meat quality of goats
was also signicantly improved (Meng etal., 2016, 2017).
Guizhou black goat is one of the three typical goat breeds in
Guizhou, China, which have strong survival ability due to the special
ecological environment of karst landform and harsh natural selection.
Guizhou black goat has always been famous for its high meat quality
and low cholesterol. It has been listed as a local excellent varieties
protection list in Guizhou Province. It has the characteristics of
excellent meat quality, good gregariousness, wide feeding range,
walking ability, and strong stress resistance, and an annual inventory
of about 500,000.
In current research, there are relatively few research reports on the
application of mushroom residue as feed in animal production and
research reports on feeding goats with FVMR are even rarer. In the
previous single-cage feeding study, our research group concluded that
the chemical composition of FVMR is: dry matter (92.67%), crude
protein (10.83%), crude ash (10.94%), neutral detergent ber
(53.79%), ether extract (2.79%), calcium (3.19%), and phosphorus
(0.43%). e incorporation of FVMR (30, 40, 50, 60%) in diets
through fermentation treatment could change the feed quality, and
aect the feeding behavior, rumination behavior and diarrhea rate of
Guizhou black male goats. Finally, the 30% FVMR in fermented total
mixed diet (FTMR) group had the best eect in improving the feed
quality and feeding behavior of Guizhou black male goats, promoting
rumination activity, and reducing diarrhea rates (Long etal., 2022). It
was hypothesized that long-term feeding of 30% FVMR in FTMR
diets may have many positive eects on the production performance,
rumen fermentation, serum biochemical indicators, and rumen
microorganisms of Guizhou black male goats. erefore, the objective
of the current study was to conduct a more comprehensive explanation
on the eects of 30% FVMR in FTMR diets on the production
performance, rumen fermentation, serum biochemical indicators, and
rumen microorganisms of Guizhou black male goats. In addition,
FVMR may ultimately achieve the goal of reducing feeding costs by
replacing some of the more expensive roughage.
2 Materials and methods
2.1 Animal ethics statement
e experiment was conducted at the Maiping Experimental Base
of the Guizhou Provincial Institute of Animal Husbandry and
Veterinary Medicine. All animals were meticulously conducted in
accordance with animal welfare guidelines and were subject to
stringent regulatory oversight by the Experimental Animal Ethics
Committee of Guizhou University in Guizhou, China
(EAE-GZU-2021-E024).
2.2 Materials, animals, diets, and
experimental design
Flammulina velutipes mushroom residue (e dregs le aer the
Flammulina velutipes mushroom are picked aer about 10 days of
growth. At this time, the length of the Flammulina velutipes mushroom
residue was about 15–20 cm.): was provided by Xuerong Biotechnology
Co., Ltd. (No. 2 Flammulina velutipes Factory) (Bijie, China).
White-rot fungi were purchased from the Beijing Microbiological
Culture Collection Center (Beijing, China). e mixed fermentation
agent (lactic acid bacteria; yeast; Bacillus licheniformis; Enterococcus

Long et al. 10.3389/fmicb.2024.1347853
Frontiers in Microbiology 03 frontiersin.org
faecalis; Bacillus subtilis; etc., number of viable bacteria
≥5 × 1,011 CFU/g) was acquired from Luoyang Oukebaik
Biotechnology Co., Ltd. (Luoyang, China). e mixed enzyme
preparation (Contains cellulase ≥7,000 U/g, β-glucanase ≥12,000 U/g,
xylanase ≥30,000 U/g, and pectinase ≥3,000 U/g) was acquired from
CJ Youtel Biotechnology Co., Ltd. (Shanghai, China).
The FTMR was reconfigured according to the optimal FVMR
addition ratio formulation determined in previous research by
Long etal. (2023a). Twenty-two healthy, 5–6 months old, Guizhou
black male goats with 22.41 ± 0.90 kg live body weights were used
in this experiment. The goats were allocated into two groups using
the Randomized Complete Block Design (RCBD) experimental
design, with each group consisting of 11 test duplicates. The
control group (group I) was fed with traditional FTMR without
FVMR according to the feed formula. The fed group II was made
into FTMR by adding 30% FVMR according to the designed
formula and adding white-rot fungi, mixed starter, and mixed
enzyme preparation according to the product instructions and
mixed evenly, adjusting the moisture content to 42%, put it into
fermentation bags (70 cm × 130 cm) (Guizhou Weilai Technology
Co., Ltd. Guiyang, China) for room temperature fermentation and
seal for 15 days. In the feed quality testing test, the same method
was used but smaller fermentation bags (35 cm × 50 cm) were used
to produce FTMR. A total of 12 fermentation bags were prepared,
with 6 replicates in each group. The entire feeding experimental
timeline spanned 75 days, comprising a 15-day pre-trial period
and a 60-day period dedicated to the collection of data and
samples. The composition and nutritional levels of the basic diet
formulated according to NRC (2007) nutritional requirements are
shown in Table1.
In this experiment, each goat was kept individually in a separate
fully automatic precision-feeding metabolic cage. A total of 22 fully
automatic precision-feeding metabolic cages were used in this
experiment. e fully automatic precision feeding metabolic cage
system consists of automatic weighing, automatic drinking water,
automatic temperature, humidity sensing, hydrogen sulde, and safety
sensing systems. Each metabolic cage (2 m2) is made of stainless steel,
and each cage was strictly sterilized before the experiment and
vaccinated and dewormed following the farm’s standard operating
procedures. During the pre-test and formal test phases, the diet
proportion was the same for every test group. e test goats were fed
on time at 9:00 and 17:00 every day. Before each feeding, ensure that
the remaining feed content in the trough during the second feeding is
approximately 10% of the previous feed.
2.3 Growth performance and economic
benefits
During the experiment, the remaining feed was cleaned before
each feeding. In addition, the fully automatic precision feeding system
automatically transmitted the average daily gain (ADG) of the goats
to the computer at regular intervals every day. e experimenter
recorded the weight of the remaining feed before each feeding and
calculated the dry matter intake (DMI), feed conversion ratio (FCR),
feed weight gain cost and weight gain benet according to the method
of Long etal. (2022).
2.4 Sample collection and apparent
digestibility
e duration of each digestibility trial was 12 days, including
7 days of adaption and 5 days of total collection of feces. Before the
morning feeding from day 70 to 75, the total feces of each male goat
were collected, weighed, and recorded. e manure samples were
combined in equal proportions, at a ratio of 20% relative to the weight
of fresh manure. Subsequently, the nitrogen was then stabilized by
adding 10% diluted sulfuric acid, and the samples were kept in a
refrigerator at −20°C (Li etal., 2022).
e drying, crushing, sieving, and preservation processes of feed
and feces samples were carried out according to the method of Yan g
etal. (2018). e dried samples were utilized for the analysis of various
components, including dry matter (DM, method No. 930.15), crude
protein (CP, method No. 976.05), calcium and phosphorus (method
935.13), ether extract (EE, method No. 973.18) according to the
Association of ocial Analytical Chemists (AOAC, 2000). Neutral
TABLE1 Composition and nutrient levels of diets.
Item Group
I II
Ingredients (DM)
Corn 28.00 21.50
Wheat bran 7.00 5.00
Rice bran 9.10 10.00
Soybean meal 43 4.50 5.00
Rapeseed meal 3.00 3.38
NaCl 0.50 0.51
1% composite premixa0.46 0.50
Urea 1.64 1.11
Cane molasses – 3.00
Peanut vine 45.80 20.00
Flammulina velutipes
mushroom residue
– 30.00
Tot al 100.00 100.00
Chemical compositionb
(Air-dry basis)
Dry matter (DM) 80.73 75.59
Neutral detergent ber
(NDF)
39.56 36.14
Crude protein (CP) 20.82 19.08
Ether extract (EE) 3.09 4.47
Calcium (Ca) 0.58 1.36
Total phosphorus (P) 0.50 0.71
Metabolizable energy (ME),
MJ/kg
11.61 10.32
I, Traditional FTMR without FVMR diets; II, 30% FVMR in FTMR diets.
a Premix provides per kg of ration: Vitamin A 10,000 IU, Vitamin D 15,000 IU, Vitamin E
200 IU, Iron 1,000–7,500 mg, Zinc 5,000–3,750 mg, Copper 140–150 mg, Iodine 5–200 mg,
Manganese 600–3,000 mg, Cobalt 4–40 mg, Selenium 3.5–10 mg, calcium 4–20% and
phosphorus 2–8%.
b e chemical composition is measured value.

Long et al. 10.3389/fmicb.2024.1347853
Frontiers in Microbiology 04 frontiersin.org
detergent ber (NDF) in feed was determined according to the
method of Van Soest etal. (1991) without thermostable α-amylase but
uses sodium sulte and NDF is expressed without residual ash. e
endogenous indicator acid-insoluble ash (AIA) was used to calculate
apparent nutrient digestibility (e method was to quantify
digestibility) according to the method of Van Keulen and
Young (1977).
2.5 Rumen fluid collection, rumen
fermentation parameters, and rumen
microbial testing
On the morning of the end of the experiment, 2 h aer the
morning feeding, the rumen uid was collected with a negative
pressure collector, and the catheter was inserted into the oral
esophagus until the middle of the rumen. To prevent contamination,
the rumen uid collected initially was discarded. About 50 mL of
rumen uid was collected from each goat. e rumen uid was ltered
through 4 layers of gauze, and the pH (PHS-3E, Shanghai Leici
Instrument Co., Ltd., Shanghai, China) value of the rumen uid was
detected immediately. e concentration of NH3-N was measured by
a microplate reader (ELX800, BioTek Instrument Co., Ltd., USA)
following the method of Paengkoum and Han (2009).
Gas chromatography (GC) was employed to detect VFA. Take an
appropriate amount of rumen uid sample and place it in a 2 mL
centrifuge tube. Samples were extracted in 50 μL of 15% phosphoric
acid with 100 μL of 125 μg/mL 4-methyl valeric acid solution as IS
(e IS was used to correct for injection variability between samples
and minor changes in the instrument response) and 400 μL ether.
Subsequently, the samples were centrifuged at 4°C for 10 min at
12000 rpm aer vortexing for 1 min, and the supernatant was
transferred into the vial before GC–MS analysis (Han etal., 2018).
e GC–MS analysis was performed on a trace 1,300 gas
chromatograph (ermo Fisher Scientic, USA). e GC–MS was
tted with a capillary column Agilent HP-INNOWAX (30 m × 0.25 mm
ID × 0.25 μm), and helium was used as the carrier gas at 1 mL/min. e
injection was made in split mode at 10:1 with an injection volume of
1 μL and an injector temperature of 250°C. e ion source and
interface temperature were 300°C and 250°C, respectively. e
column temperature was programmed to increase from an initial
temperature of 90°C, followed by an increase to 120°C at 10°C/min,
to 150°C at 5°C/min, and nally to 250°C at 25°C/min, which was
maintained for 2 min (total run-time of 15 min). Mass spectrometric
detection of metabolites was performed on ISQ 7000 (ermo Fisher
Scientic, USA) with electron impact ionization mode. Single ion
monitoring (SIM) mode was used with an electron energy of 70 eV
(Hsu etal., 2019; Zhang etal., 2019).
2.6 Serum biochemical indicators
At the end of the experiment, blood was collected from the jugular
vein before feeding. 10 mL of blood was collected from each goat. e
serum was separated by a low-speed centrifuge and stored in a − 20°C
refrigerator until testing. Immunoglobulin M (IgM), Immunoglobulin
G (IgG), Immunoglobulin A (IgA), Interleukin-2 (IL-2), Interleukin-6
(IL-6), Tumor Necrosis Factor-α (TNF-α), Interferon-gamma-γ
(INF-γ), Glutamic oxalacetic transaminase (ALT), Alanine
aminotransferase (AST), and Growth Hormone (GH) were measured
with a microplate reader (ELX800, BioTek, USA) according to the
detailed steps of the ELISA kit (Haoyuan Biotechnology Co., Ltd.,
Yibin, China).
2.7 Rumen microbiome detection and
analysis
Rumen uid samples were snap frozen and stored at −80°C aer
collection. Bacterial DNA was isolated from the Rumen uid using a
MagPure Soil DNA LQ Kit (D6356-02, Magen, Hilden, Germany)
following the manufacturer’s instructions. DNA concentration and
integrity were measured by a NanoDrop2000 spectrophotometer
(ermo Fisher Scientic, Waltham, MA, USA) and agarose gel
electrophoresis. e V3-V4 hypervariable region of the bacterial 16S
rRNA gene was amplied by PCR using primers (343F,
5′-TACGRAGCAGCAG-3′, 798R: 5′-AGGGTATCTAATCCT-3′) in
a 25 μL reaction. e resulting sample barcode was included in the
reverse primer, and both primers were individually ligated to Illumina
sequencing adapters.
Gelatin electrophoresis was used to visualize the Amplicon
quality. Using Agencourt AMPure XP beads (Beckman Coulter Co.,
USA), PCR products were puried, and the Qubit dsDNA kit was
used to quantify the results. Subsequently, the concentrations were
adjusted to prepare for the sequencing process. Sequencing was
carried out on an Illumina NovaSeq6000 platform, employing two
paired-end read cycles, each with a length of 250 bases. (Illumina Inc.,
San Diego, CA; OE Biotech Company, Shanghai, China).
e format of the raw sequencing data was FASTQ. en the
Cutadapt soware was used to preprocess the obtained data, detect
and cut o the joints. Aer completion, DADA2 and QIIME2 were
used to lter the obtained data, denoise, merge, detect, and cut o
chimeric reads. e methods are based on Callahan etal. (2016) and
Bolyen et al. (2019) respectively. At last, the soware outputs the
representative reads and the ASV abundance table. e representative
read of each ASV was selected using the QIIME2 package. All
representative reads were annotated and blasted against Silva database
Version 138 (or unite) (16 s/18 s/ITS rDNA) using q2-feature-classier
with the default parameters. e microbial diversity of rumen uid
samples was analyzed by alpha diversity analysis, and the Chao1,
Simpson, and Shannon index values were calculated according to the
methods of Hill etal. (2003) and Chao and Bunge (2002). e Unifrac
distance matrix performed by QIIME soware was used for
unweighted Unifrac Principal coordinates analysis (PCoA) and
phylogenetic tree construction. e 16S rRNA gene amplicon
sequencing and analysis were conducted by OE Biotech Co., Ltd.
(Shanghai, China).
2.8 Statistical analysis
All the original data obtained in this experiment were rst sorted
and recorded using Excel 2021. e data’s normal distribution was rst
ascertained using the Shapiro–Wilk test. Finally, the recorded data
soware was used for statistical analysis through SPSS 26.0. Data
analyzes were one-way ANOVA and multi-covariate ANOVA with

Long et al. 10.3389/fmicb.2024.1347853
Frontiers in Microbiology 05 frontiersin.org
general linear model (GLM module). e Duncan’s test and the LSD
method were used to conduct multiple comparisons and signicant
dierence tests. All results were expressed as means and standard error
of the mean (SEM). In the analysis, a signicance level of p < 0.05 was
regarded as statistically signicant, and p < 0.01 as highly signicant.
3 Results
3.1 Growth performance and apparent
digestibility
e DMI, FCR, and feed weight gain cost of group Iwas higher
than that of group II (p < 0.01). Compared with group I, the FBW
(Average nal body weight), TWG (Average total weight gain), and
weight gain benet of group II were increased, respectively, by 4.99,
10.89, and 10.82%, and each goat of group II earned 17.35 CNY
(Chinese Yuan) more than the group I. ere was no signicant
dierence in ADG between the two groups; however, in comparison to
Group I, the ADG of group II was also increased by 10.83% and the
feed cost of group II was reduced by 25% (Table2). In contrast to group
I, the apparent digestibility of DM, CP, and NDF in group II were
increased, by 13.84, 7.07, and 7.74% (p < 0.01). e apparent digestibility
of EE did not dier between the two groups (p > 0.05) (Table3).
3.2 Rumen fermentation parameters
e pH level in Group II was higher than that in Group I(p < 0.05).
NH
3
-N, TVFA (Total volatile fatty acids), and A/P were unaected by
the addition of FVMR (p > 0.05). e proportion of acetic acid in
TVFA in group Iwas higher than that in group II (p < 0.01), w hile
there was no dierence in the proportions of propionic acid, butyric
acid, isovaleric acid, isobutyric acid, valeric acid, and caproic acid in
TVFA between the two groups (p > 0.05) (Table4).
3.3 Serum biochemical indicators
It can bedepicted from Table5 that adding FVMR to the feed has
an impact on the serum biochemical indicators of black goats. e
FVMR in the FTMR diet had no eect on IgG, IL-2, TFN-α, or IFN-γ
(p> 0.05). Moreover, feeding black goats at a rate of FVFM in the
FTMR resulted in higher serum IgG, TFN-α, and IFN-γ levels than
group I. In addition, the level of IL-2in group II was lower than that
of group I. But there is no signicance (p > 0.05). e GH, IgM, and
IgA level in group II was higher than in group I(p < 0.01). FVFM in
the FTMR to feed black goats could reduce serum IL-6 and ALT
levels (p < 0.01), Similar, it could also reduce AST levels (p < 0.05)
(Table5).
3.4 Microbiota composition
e results of the Venn analysis showed that at the ASV level,
specic bacterial ASV accounted for 39.50% (1480) of the total ASV
sequence number in group I, specic bacterial ASV accounted for
39.90% (1495) of the total ASV sequence number in group II
(Figure1A). In addition, the number of bacterial ASVs shared by
groups Iand II was 772 (22.22%). It was concluded by the PCoA
(Figure1B) and NMDS (Figure1C) diagrams that the distribution of
samples in group II is relatively concentrated, and the samples have
better repeatability. PCoA obtained dierent contribution rates
through dierent analysis methods, indicating that although the
TABLE2 Eects of dierent diets on the performance and economic
benefits of Guizhou black male goats.
Item Group SEM p-value
I II
DMI, kg/d 0.97a0.86b0.01 <0.01
IBW, kg 22.10 22.73 0.90 0.746
FBW, kg 30.27 31.78 0.97 0.462
TWG, kg/hd 8.17 9.06 0.30 0.143
ADG, g 136.18 150.93 4.95 0.143
FCR 7.14a5.82b0.20 <0.01
Live goat
real-time
price, CNY/
kg a
40 40 – –
Feed cost,
CNY/kg
2.72 2.04 – –
Feed weight
gain cost,
CNY/kg
21.89a16.44b1.02 <0.01
Weight gain
benet, CNY/
one
160.38 177.73 5.83 0.143
p < 0.05, signicant; p < 0.01, extremely signicant. SEM, standard error of the mean.
Dierent superscript letters indicate signicant dierences. e data description method of
this paper’s rest of the tables is the same.
I, Traditional FTMR without FVMR diets; II, 30% FVMR in FTMR diets.
a Live goat real-time price and feed raw material prices (For example, the FVMR price is 120
CNY per ton) are the current price in Guizhou Province, China.
ADG = (end of test weight − beginning of test weight)/number of feeding days; FCR = DMI/
ADG; DMI (kg/d) = [feeding amount (kg) × diet DM (%) − leover (kg) × leover DM (%)]/
[number of black goats per group × number of trial days (d)]; Feed weight gain cost (CNY/
kg) = total feed intake × feed unit price (CNY)/total weight gain; Weight gain benet (CNY/
one goat) = (real-time price of live black goat − feed weight gain cost) × total weight gain.
Kg/one goat, Kilograms per black goat; CNY, Chinese Yuan; CNY/kg, CNY per kg; CNY/
one, CNY per black goat; IBW, Average initial body weight; FBW, Average nal body weight;
TWG, Average total weight gain; Feed weight gain cost, Feed weight gain cost assume cost of
gaining 1 kg BW by each animal.
TABLE3 Eects of diets on apparent digestibility of Guizhou black male
goats (%).
Item Group SEM p-value
I II
DM 72.49b82.52a1.59 <0.01
EE 72.23 73.70 0.91 0.462
CP 75.29b80.61a0.95 <0.01
NDF 65.85b70.95a0.84 <0.01
p < 0.05, signicant; p < 0.01, extremely signicant. SEM, standard error of the mean.
Dierent superscript letters indicate signicant dierences.
I, Traditional FTMR without FVMR diets; II, 30% FVMR in FTMR diets.
DM, dry matter; EE, ether extract; CP, crude protein; NDF, neutral detergent ber; ADF, acid
detergent ber.

Long et al. 10.3389/fmicb.2024.1347853
Frontiers in Microbiology 06 frontiersin.org
dierence in microbial colonies between the two groups of samples
was not signicant, there were still certain dierences.
In the two diagrams of Circos (Figure1D) and Phylogenetic
(Figure1E), the distribution of dierent ASVs of the three main
dominant colonies at the phylum level could bevisually observed. In
the Phylogenetic diagram, we can also visually observe the
evolutionary distance of dierent ASVs. From the Circos and
Phylogenetic diagrams, it was concluded by that the detected rumen
microorganism ASV in dierent test groups is mainly distributed in
Bacteroidota. In the alpha_diversity analysis (Figure 2), Chao1,
Shannon, Simpson, goods_coverage, observed-species, PD-whole-tree
and ACE indexes were similar and did not reach a signicant level
between the two experimental groups (p > 0.05). e goods_coverage
between the two groups was close to 1, indicating that the sequencing
depth is reasonable. e species distribution is uniform and diverse
with high reliability, covering all species, therefore, microbial sample
data can befurther analyzed.
At the phylum level, the dominant bacteria are Bacteroidota,
Firmicutes and Proteobacteria (Figure3A). However, the dierences in
levels of Bacteroidota, Firmicutes and Proteobacteria were not signicant
(p> 0.05) (Table6). At the genus level, Prevotella, Muribaculaceae, and
F082 were the main dominant genera (Figure3B). e incorporation
of 30% FVMR in Guizhou black male goats FTMR diets could change
the colony composition of rumen microorganisms at the genus level
(Table7). e inclusion of 30% FVMR decreased Bacteroidales_BS11_
gut_group, Desulfovibrio, and Christensenellaceae_R-7_group (p< 0.05),
while increasing Lachnospiraceae_ND3007_group when compared to
the control group (p< 0.01).
All microbial data were used in the LEfSe analysis, which
correctly identied the important bacterial groups connected to the
two groups. Figure4 depicts a representative structural cladogram of
major microbiota showing the relative abundance of species within
this group. From the Cladogram and LDA graphs, it can befound
that there is 1 colony (g_Odoribacter) with high abundance in group
Iand 7 colonies (For example, g_Lachnospiraceae_ND3007_group,
g_Anaerovibrio, and g_Allobaculum, etc.) with high abundance in
group II (Figure4).
3.5 Correlation analysis
Correlations between microbiome composition and rumen
fermentation parameters and digestibility are shown in Figure5.
e relative abundance of Rikenellaceae_RC9_gut_group exhibited
a negative correlation with acetic acid, TVFA, and propionic acid
(p < 0.05). Similarly, the relative abundance of F082,
Ruminococcaceae_UCG_002, Prevotellaceae_UCG-001, and
[Eubacterium]_coprostanoligenes_group in the rumen were higher.
In addition, the content of acetic acid, propionic acid, butyric acid,
TABLE4 Eects of dierent diets on ruminal fermentation parameters of
Guizhou black male goats.
Item Group SEM p-value
I II
pH 6.34b6.58a0.06 0.030
NH3-N, mg/dL 12.31 12.76 0.93 0.832
TVFA, mmol/L 54.53 48.12 6.41 0.640
VFA,a mol/100 mol
Acetic acid 70.13a67.93b0.43 <0.01
Propionic acid 13.92 14.47 0.47 0.569
Butyric acid 11.87 13.50 0.58 0.166
Isobutyric acid 1.65 1.68 0.13 0.928
Isovaleric acid 1.21 1.26 0.10 0.810
Valeric acid 1.01 0.98 0.03 0.667
Caproic acid 0.20 0.17 0.02 0.376
A/P 5.20 4.79 0.27 0.460
p < 0.05, signicant; p < 0.01, extremely signicant. SEM, standard error of the mean.
Dierent superscript letters indicate signicant dierences.
I, Traditional FTMR without FVMR diets; II, 30% FVMR in FTMR diets.
a e data of each VFA individual in the table, respectively, represent the proportion of this
individual in TVFA.
NH3-N, ammonia nitrogen; TVFA, total volatile fatty acids; VFA, volatile fatty acids; A/P,
acetic acid / propionic acid.
TABLE5 Eects of dierent diets on serum biochemical indexes of
Guizhou black male goats.
Item Group SEM p-value
I II
GH, ng/mL 2.89b4.13a0.17 <0.01
IgG mg/mL 4.21 4.63 0.13 0.102
IgM mg/mL 1.01b1.49a0.06 <0.01
IgA μg/mL 208.44b251.43a7.17 <0.01
IL-2 pg/mL 943.64 909.84 35.72 0.644
IL-6 pg/mL 157.80a133.35b3.86 <0.01
TFN-α pg/mL 225.68 238.54 7.73 0.415
IFN-γ pg/mL 500.17 560.94 18.94 0.110
ALT pg./mL 671.02a601.71b11.41 <0.01
AST mmol/L 319.78a285.82b7.25 0.016
p < 0.05, signicant; p < 0.01, extremely signicant. SEM, standard error of the mean.
Dierent superscript letters indicate signicant dierences.
I, Traditional FTMR without FVMR diets; II, 30% FVMR in FTMR diets.
IgM, Immunoglobulin M; IgA, Immunoglobulin A; IgG, Immunoglobulin G; IL-6,
Interleukin-6; IL-2, Interleukin-2; TNF-α, Tumor Necrosis Factor-α; INF-γ, Interferon-
gamma-γ; ALT, Glutamic oxalacetic transaminase; AST, Alanine aminotransferase.
TABLE6 Eects of dierent diets on the relative abundance of ruminal
bacterial communities at the phylum level (average relative abundance
≥0.1% for at least one treatment) of Guizhou black male goats (%).
Item Group SEM p-value
I II
Bacteroidota 68.24 63.20 1.67 0.135
Firmicutes 21.12 23.24 1.02 0.309
Proteobacteria 9.29 12.55 0.94 0.083
Desulfobacterota 0.69 0.41 0.07 0.057
Spirochaetota 0.20 0.15 0.02 0.094
Fibrobacterota 0.12 0.13 0.03 0.787
Deferribacterota 0.11 0.10 0.01 0.648
I, Traditional FTMR without FVMR diets; II, 30% FVMR in FTMR diets.

Long et al. 10.3389/fmicb.2024.1347853
Frontiers in Microbiology 07 frontiersin.org
and TVFA will bereduced. e apparent digestibility of EE was
positively correlated with the relative abundance of uncultured ora
(p < 0.05) and negatively correlated with the relative abundance of
Bacteroidales_BS11_gut_group, Prevotellaceae_UCG-003, and
Lachnospiraceae_UCG-004 (p > 0.05). e A/P ratio was shown to
benegatively related to the relative abundance of Bacteroidales_
BS11_gut_group and other genera (p < 0.05). e digestibility of
DMD, IgA, IgM, NH3-N, and ADFD was positively correlated with
the relative abundance of Lachnospiraceae_ND3007_group and
negatively correlated with the relative abundance of Bacteroidales_
BS11_gut_group. In addition, [Eubacterium]_coprostanoligenes_
group relative abundance changes were negatively correlated with
IgG, while Ruminococcus abundance changes were negatively
correlated with TFN-α (p < 0.05). ere is a positive correlation
between the relative abundance of Muribaculaceae and changes in
ALT (p < 0.05).
4 Discussion
4.1 Eects of growth performance and
apparent digestibility
Flammulina velutipes mushroom residue has high moisture
content and also high in cellulose, which easily contaminated by fungi
and bacteria if not treated in time. It will begin to decompose aer
2–3 days and produce harmful compounds aer 1 week (Kim etal.,
2007). e mushroom residue treated by microbial fermentation can
prolong the shelf life and improve the feed’s palatability and nutritional
value (Gao etal., 2008; Kwak etal., 2008). Although feed nutritional
value and palatability data were not presented in this study, previous
behavioral studies have conrmed that feeding FVFM in the FTMR
could improve feed quality and palatability (Long etal., 2023b). In the
breeding process, animal growth performance directly determines
FIGURE1
Eects of dierent diets on rumen microorganisms of Guizhou black male goats. (A–E) Are ASV-Venn, Principal coordinate analysis (PCoA) non-metric
multidimensional scale analysis (NMDS), ASV-Circos, and ASV- Phylogenetic diagram analysis of Iand II.
FIGURE2
Eects of dierent diets on alpha diversity index of rumen microorganisms in Guizhou black male goats. (A–E) Represents the Chao1 index, Shannon
index, Simpson index, goods_coverage index, and ACE index, respectively.

Long et al. 10.3389/fmicb.2024.1347853
Frontiers in Microbiology 08 frontiersin.org
economic benets to a large extent, so animal ADG and FCR are the
key research indicators. Huang etal. (2021) found that the ADG of
Liuyang black goats fed with 65% mixed concentrate +35% oyster
mushroom cha and whole plant rice co-fermented material increased
by 18.33%, and FCR decreased to 5.20. In this study, the ADG of the
Guizhou black male goats increased by 10.83% and the FCR was
signicantly lower than that of the control group. Wealso found that
the cost of feeding the experimental group containing 30% FVMR in
FTMR diets was 25% lower than that of the control group, and the
economic benet of each goat increased by 10.82%. e ndings of
this study agree with those of Huang etal. (2021) and Kang etal.
(2022). However, it is inconsistent with the research results of Guo
etal. (2015) that adding FVMR to the diet had no signicant eect on
the growth performance of Boer goats.
Feed digestibility is positively correlated with the absorption rate
of nutrients absorbed by animals and the quality of feed, which was
very important for the growth and development of ruminants (Zhang
etal., 2022). Relevant research has demonstrated that fermenting feed
could increase the utilization rate of feed (Kawamoto etal., 2009) and
the incorporation of white-rot fungi and enzymatic bacteria in diets
could improve feed quality and digestibility (So etal., 2020; Clark
etal., 2022). In this study, the incorporation of 30% FVMR in diets of
Guizhou black male goats could increase the apparent digestibility of
DM, CP, and NDF. e reason may bethat the nutritional properties
and special composition of the feed raw materials (bacteria residue)
themselves may directly increase the apparent digestibility (Li etal.,
2015; Meng et al., 2016). Moreover, we have adopted dierent
treatment methods, adding white-rot fungi, mixed starter culture and
enzyme preparation to the FVMR feed, which will also greatly
improve the apparent digestibility of the feed. When animals ingest
feed to reach the energy requirements, DMI intake of feed will
bereduced, and because the moisture content of FVFM feed aer
fermentation was higher than that of the control group, it may also
be the reason for reducing DMI intake of feed. Ultimately, the
FCR decreases.
4.2 Eects of rumen fermentation
e uctuation of rumen pH is the most intuitive indicator
reecting the state of rumen fermentation and internal environment
stability, which normal range is 6.0 ~ 7.0 (Guo etal., 2022). Although
the rumen pH value of the FVMR in the FTMR diet was higher than
that of the control group, both groups were within the normal range.
erefore, it could beconsidered that feeding Guizhou black male
goats with FVFM in the FTMR would not cause ruminal acidosis.
Acetic acid and butyric acid can beconverted into each other in
the rumen (Wang etal., 2020). 28% of acetic acid is not absorbed by
the rumen in the form of acetic acid, but acetic acid could react with
acetyl-CoA or butyryl-CoA transferase to produce butyric acid,
which is then absorbed by rumen microorganisms (Kristensen,
2001; Hackmann and Firkins, 2015). In this study, weconcluded that
the acetic acid content of Guizhou black male goats fed 30% FVFM
in the FTMR was signicantly lower than that of the control group,
while the contents of propionic acid and butyric acid tended to
increase. It might bebecause FVFM in the FTMR could improve the
activity of acetyl-CoA and butyryl CoA, and accelerate the
conversion between acetic acid and butyric acid (Henderson etal.,
2015). However, goats may promote microbial metabolism through
an energy dissipation process that continuously converts acetic acid
to butyric acid. Additionally, FVFM in the FTMR might also change
the rumen fermentation mode from acetic acid to propionic acid
(Chen etal., 2021), this model needs to befurther conrmed under
the conditions of this study. Finally, the content of acetic acid and
the ratio of acetic acid to propionic acid is reduced. In addition,
butyric acid could usually participate in the development of rumen
papilla by stimulating the metabolism of rumen epithelial cells
(Poudel etal., 2019) and mutton sheep mainly use propionic acid
produced from sugar and starch fermentation to produce glucose
through gluconeogenesis to provide more energy for the body
(Astawa etal., 2011; Gunun etal., 2018; Zhang etal., 2022). us,
while propionic acid and butyric acid were conducive to improving
FIGURE3
Eects of dierent diets on the phylum-level and genus-level colony abundance of rumen microorganisms in Guizhou black male goats. (A) Relative
abundance of phylum horizontal species. (B) Relative abundance of genus horizontal species.

Long et al. 10.3389/fmicb.2024.1347853
Frontiers in Microbiology 09 frontiersin.org
animal growth, which could explain the higher growth performance
obtained in this study.
4.3 Eects of serum biochemical indicators
Serum biochemical indicators are aected by factors such as
animal species, species, age, sex, dietary nutritional structure, and
seasonal climate changes, which could reect the body’s health, body
nutrition level, and metabolic state (Piccione etal., 2010). As one of
the peptide hormones, GH could participate in the regulation of
animal reproductive function and promote muscle development, but
to a certain extent, it could also promote the dierentiation,
proliferation, and migration of some cancer cells (Imbesi etal., 2014).
is study found that feeding 30%, FVMR FTMR can signicantly
increase goats’ GH value and better promote goats’ growth and
development. IgA, IgG, and IgM produced in the body’s rst immune
response are important immunoglobulins of the body and are the
main antibodies of the body’s second immune response to pathogens
(Çölkesen etal., 2022). is study found that 30% FVMR in FTMR
diets increased the levels of IgA and IgM in goat serum, which
indicates that feeding 30% FVMR in FTMR diets improved the
immune capacity of goats. e IL-6 anti-inammatory factor secreted
by 2 cells can produce a large amount of immunoglobulin by
stimulating B cells, which have anti-infective, inhibit, and kill tumor
cells (Calleja-Agius and Brincat, 2008; Wu etal., 2011). However,
wehave failed to identify the specic reasons and mechanisms of
IL-6in this study was lower than those in the control group, which
needs further study. AST and ALT have the function of catalyzing the
conversion of amino acids into keto acids and are considered to
becrucial transaminase enzymes in various biological processes (Yan g
etal., 2022). Under normal circumstances, the activity of ALT and
AST in the body’s serum is low. If the liver tissue cells are damaged,
lesions occur, and the function is impaired, the body’s AST and ALT
will enter the blood to increase the activity of ALT and AST in the
serum (Akrami etal., 2015; Tan etal., 2016). In this study, it was found
that feeding 30% FVMR in FTMR diets decreased the content of ALT
and AST. It shows that feeding 30% FVMR in FTMR diets could
TABLE7 Eects of dierent diets on the relative abundance of ruminal bacterial communities at the genus level (average relative abundance ≥0.5% for
at least one treatment) of Guizhou black male goats (%).
Phylum Genus Group SEM p-value
I II
Bacteroidota Prevotella 20.72 17.44 2.15 0.459
Muribaculaceae 12.24 11.27 0.66 0.473
F082 10.19 11.22 1.20 0.674
Rikenellaceae_RC9_gut_group 7.65 10.56 0.90 0.107
Bacteroidales_RF16_group 2.86 3.66 0.39 0.311
Prevotellaceae_UCG-003 2.27 2.04 0.28 0.685
Prevotellaceae_UCG-001 1.14 1.70 0.16 0.088
Bacteroidales_BS11_gut_group 2.34a0.20b0.49 0.026
Prevotellaceae_NK3B31_group 0.61 0.72 0.06 0.342
Alistipes 0.52 0.57 0.04 0.590
Firmicutes Lachnospiraceae_NK4A136_group 3.60 3.92 0.19 0.409
Clostridia_UCG-014 1.71 1.61 0.14 0.738
Ruminococcaceae_NK4A214_group 0.86 1.28 0.22 0.349
Ruminococcaceae_UCG_002 1.72 2.20 0.30 0.443
Lachnospiraceae_ND3007_group 0.47b1.64a0.20 <0.01
Eubacterium_coprostanoligenes_group 0.97 1.08 0.11 0.602
Lachnospiraceae_UCG-004 0.99 0.78 0.24 0.678
Ruminococcus 0.80 0.83 0.11 0.912
Ruminococcaceae_UCG-005 0.94 0.59 0.23 0.471
Lachnospiraceae_UCG-010 0.57 0.49 0.07 0.568
Anaeroplasma 0.51 0.53 0.14 0.957
Christensenellaceae_R-7_group 0.60a0.30b0.07 0.024
Proteobacteria Proteu s 8.91 11.85 0.96 0.129
Desulfovibrio 0.58a0.32b0.07 0.049
Other uncultured 1.62 2.42 0.21 0.056
Other 7.91 3.03 1.60 0.130
p < 0.05, signicant; p < 0.01, extremely signicant. SEM, standard error of the mean. Dierent superscript letters indicate signicant dierences.
I, Traditional FTMR without FVMR diets; II, 30% FVMR in FTMR diets.

Long et al. 10.3389/fmicb.2024.1347853
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improve the immune ability of Guizhou black male goats and maintain
liver health.
4.4 Eects of rumen microflora
Rumen microorganisms comprise three major groups of protozoa,
bacteria, and fungi. e unique existence of these microorganism
groups has created a powerful digestive system for ruminants
(Biscarini etal., 2018). Regulating the rumen microbial ecosystem to
improve rumen fermentation in ruminants, improving animal
productivity, and economic benets have always been among the
leading research hotspot in animal nutrition research (Patra and
Saxena, 2011). e change in rumen microbial diversity has a key
relationship with the diet structure. By adjusting the diet structure, the
rumen microbial diversity can be improved, and the health of
ruminants can beguaranteed (Henderson etal., 2015). At the phylum
level, the proportion of Bacteroidetes and Firmicutes accounted for
more than 70% of rumen microorganisms, and they were the
dominant colonies of rumen microorganisms in ruminants (Singh
etal., 2012; Jami etal., 2013; Liu etal., 2017). is study also obtained
consistent results. e sum of Bacteroidetes and Firmicutes colonies
between the two groups accounted for more than 70% of the rumen
microorganisms. Bacteroidetes in the rumen are an essential ora that
promotes the degradation of polysaccharides, proteins, and
carbohydrates in feed in ruminants (Naas etal., 2014); it can degrade
high-molecular organic matter and improve the innate immune
response by enhancing the intestinal mucosal barrier function
(omas etal., 2011; Magrone and Jirillo, 2013). Bacteroidetes in the
rumen are crucial for the synthesis of acetate and propionate as well
as the degradation of non-cellulosic plant-based substances (Söllinger
etal., 2018). is is the main reason that the proportion of acetic acid
and propionic acid in the rumen of the control group is higher than
that of the experimental group II. Firmicutes are related to energy
metabolism, and they carry many genetic codes that can produce a
variety of digestive enzymes to decompose various nutrients; they are
the most critical bacterial group for ruminants to absorb protein and
improve ber utilization (Kaakoush and Nadeem, 2015; Petri etal.,
2019). Proteobacteria are mainly Gram-negative bacteria, including,
Escherichia coli, Salmonella, and other pathogenic bacteria, which can
degrade soluble carbohydrates. When the content is higher than 19%,
indicating that the rumen microbial ecosystem is unstable (Auret
etal., 2017). In this study, the content of Proteobacteria in the two test
groups was lower than 19%, and the rumen microbial ecosystem was
stable. It shows that feeding 30% FVMR in FTMR diets does not aect
the stability of the rumen microbial system of black goats. In addition,
the relative abundance of Bacteroidetes in group II was lower than that
in the control group, but the relative abundance of Firmicutes was
higher than that in the control group. is provides certain evidence
that FVMR can improve the ruminal microbial composition of
Guizhou black male goats.
Generally, the most dominant genus in the rumen of ruminants
is Prevotella (Bowen etal., 2018). Its primary function is to degrade
cellulose, starch, hemicellulose, and protein (Li and Guan, 2017).
Prevotella was positively correlated with the proportion of acetic acid
but negatively correlated with the body’s MCP concentration, gas
production, and the proportion of valerate and butyrate (Zhou etal.,
2022). e reason may bethat why the acetic acid in the group II was
lower than that in the control group, but the butyric acid was higher
than that in the control group. Muribaculaceae belongs to
Bacteroidetes, which is abundant in the intestines of mice. is genus
is a newly identied genus name. ere is still a lack of in-depth
understanding of its specic functions. e changes in its abundance
are mainly related to the host and its dietary conditions (Zhou etal.,
FIGURE4
The microbiomes of the two groups were described using LEfSe and LDA analysis according to ASV dierences. (A) The histogram of the distribution of
LDA values was calculated with a score of LDA scores >2. The length of the bars represents the abundance of dierent species. (B) Example map of
dierent species annotation branches in the figure; dierent colors indicate dierent groups. The yellow nodes represent the species with no
significant dierence between the two groups. The diameter of the node is proportional to the relative abundance. Each layer of nodes represents the
phylum/class/order/family/genus from the inside to the outside and each layer of species. The marked annotations indicate the phylum/class/order/
family/genus from the inside to the outside; the species names represented by English letters in the figure are displayed in the legend on the right.

Long et al. 10.3389/fmicb.2024.1347853
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2020). In this study, because both Muribaculaceae and Prevotella
belong to Bacteroidota, webelieve that the function of Muribaculaceae
may berelated to the degradation of cellulose, starch, and protein, and
further research is needed to prove it. Rikenellaceae_RC9_gut_group
and Ruminococcus are the main bacteria of rumen microorganisms,
which can secrete a large amount of oligosaccharase, cellulase, and
hemicellulase and participate in the decomposition and absorption of
protein and carbohydrates (Dai et al., 2015; Zhu et al., 2021).
erefore, it promoted ber digestion in test group II more than in
control. e relative abundance of Prevotellaceae_UCG-003 was
signicantly positively correlated with the valeric acid and propionic
acid ratio, and there was a promoting eect in the process of
polysaccharide degradation (Flint etal., 2008). is may also lead to
lower propionic and valeric acid in group II in this study than in the
control group. e change of rumen microbes has an important
relationship with the body’s immune regulation, disease resistance,
and animal growth (Wang etal., 2021). Lachnospiraceae_NK4A136_
group and Lachnospiraceae_ND3007_group belong to the
Lachnospiraceae family, and many microorganisms can produce
butyric acid, which can inhibit inammation and enhance the
integrity of the epithelial barrier and are positively correlated with IgG
and IgM (Meehan and Beiko, 2014; Sarmikasoglou and Faciola, 2022).
In addition, Ruminococcus may inhibit inammation and promote
growth in animals through butyrate production (Scaldaferri etal.,
2016). Relevant studies have shown that Fibrobacter and Prevotellaceae
UCG-003 negatively correlate with enhancing immune function
(Wang etal., 2021). In this study, Feeding 30% FVMR in FTMR diets
could improve the rumen microbial composition of Guizhou black
male goats.
5 Conclusion
In the context of this study, weattempted to unravel the eects of
the FVMR diet on the fattening eect and rumen health of Guizhou
black male goats. Our ndings demonstrated that 30% FVMR in
Guizhou black male goats FTMR diets could improve rumen
microbial composition and reduce the content of acetic acid.
Moreover, the apparent digestibility of DM, CP, and NDF is improved,
it also could improve the immunity. Finally, 30% FVMR in FTMR
diets increased ADG and decreased FCR of Guizhou black male goats.
Consequently, the feed cost is 25% lower than that of the control
group, and the average prot per sheep is 17.35 CNY higher than that
of the control group. In conclusion, 30% FVFM in the FTMR to feed
Guizhou black male goats is an eective and promising method to
reduce feeding costs and improve the economic benets of Guizhou
black male goats. Notably, Webelieve that many microorganisms in
the rumen aer feeding FVMR are closely related to the immune
regulation and health of the animal body, and the specic inuencing
mechanisms and functions require further research and verication.
FIGURE5
Correlation analysis of rumen fermentation parameters and nutrient digestibility with microbial abundance. TVFA, Total volatile fatty acids; EED,
Apparent digestibility of crude fat; A/P, Acetic acid/propionic acid; CPD, Apparent digestibility of protein; NDFD, Apparent digestibility of neutral
detergent fiber; DMD, Apparent digestibility of dry matter; ADFD, Apparent digestibility of acid detergent fiber. Red indicates a positive correlation; blue
indicates a negative correlation. *p < 0.05.

Long et al. 10.3389/fmicb.2024.1347853
Frontiers in Microbiology 12 frontiersin.org
Data availability statement
e data presented in the study are deposited in the NCBI
repository, accession number PRJNA1063023.
Ethics statement
All animals were meticulously conducted in accordance with
animal welfare guidelines and were subject to stringent regulatory
oversight by the Experimental Animal Ethics Committee of Guizhou
University in Guizhou, China (EAE-GZU-2021-E024). e study was
conducted in accordance with the local legislation and
institutional requirements.
Author contributions
YL: Writing – original dra, Writing – review & editing. WX:
Data curation, Methodology, Writing – review & editing. YZ: Data
curation, Methodology, Writing – review & editing. CY: Investigation,
Soware, Writing – review & editing. DW: Data curation, Supervision,
Writing – review & editing. YY: Data curation, Supervision, Writing
– review & editing. CS: Data curation, Supervision, Writing – review
& editing. PP: Project administration, Resources, Visualization,
Writing – review & editing. YH: Data curation, Funding acquisition,
Investigation, Project administration, Resources, Supervision, Writing
– review & editing, Writing – original dra.
Funding
e author(s) declare nancial support was received for the
research, authorship, and/or publication of this article. Major Special
Projects of the Guizhou Province Department of Science and
Technology provided funding for this work (Qianke Service Enterprise
[2020] 4009).
Acknowledgments
We first need to thank the reviewers of this journal for their
valuable suggestions. At the same time, we would like to thank the
Guizhou Provincial Institute of Animal Husbandry and Veterinary
Medicine for providing the experimental base, and also thank the
Key Laboratory of Animal Genetics, Breeding and Reproduction
in the Plateau Mountainous Region, Ministry of Education,
Guizhou University for providing the experimental platform and
staff guidance. In addition, the authors express their gratitude to
the personnel of OE Biotech Co., Ltd. (Shanghai, China) for their
valuable assistance in conducting the research. The authors
express their gratitude for the technical support provided by
Wang Tian.
Conflict of interest
e authors declare that the research was conducted in the
absence of any commercial or nancial relationships that could
beconstrued as a potential conict of interest.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
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