Effects of total mixed ration with various silage on growth performance, serum parameters, ruminal fermentation, and bacteria community profile in beef cattle

Abstract The possibilities of using high‐quality forages in incorporation with total mixed ration (TMR) during the fattening period of beef cattle have been investigated. A total of 30 Simmental bulls (438.94 ± 11.45 kg) were selected and randomly divided into two groups, TMR with single corn silage (SS) and TMR with various silage (MS). The whole experiment consisted of 15 days preparation period and 69 days experimental period. Rumen fluid and blood samples were taken from six beef cattle per treatment at the end of the experiment. The results showed that the average daily gain of the MS group (1.56 kg/day) was higher than (p < .05) the SS group (1.30 kg/day), and a decrease of feed conversion ratio in the MS (10.83) group was observed compared with SS group (12.36) (p < .05). The concentration of total volatile fatty acids for MS group was greater than (p < .05) the SS group. The activities of total antioxidant capacity and superoxide dismutase from MS group were also higher than the SS group, but lower urea nitrogen was found in the MS group from serum (p < .05). In addition, the abundances of the Prevotella‐1 and Verrucomicrobia were higher in the MS group than the SS group (p < .05). An increase in the flavonoid biosynthesis was detected in the MS group compared with the SS group by Kyoto Encyclopaedia of Genes and Genomes analysis. The present findings suggest that it is economical and healthy to substitute high‐quality forage +low level of concentrate for a relatively low proportion forage +high level of concentrate in a finishing diet of beef cattle, which was a feasible and healthy strategy in the intensive feeding system.


| INTRODUC TI ON
In recent years, by the promotion of the government, the plantation of forages (alfalfa, oats, corn, and sweet sorghum) has been widely implemented leading to the production of high-quality forage in China, bringing in rich forage resources to herbivorous animals.
Additionally, many studies have proved that forage ensiled has a better nutritional value and longer storage time due to the fermentation of lactic acid bacteria (Ke et al., 2018;Zhao et al., 2019).
The development of animal husbandry has focused on expanding

Abstract
The possibilities of using high-quality forages in incorporation with total mixed ration (TMR) during the fattening period of beef cattle have been investigated. A total of 30 Simmental bulls (438.94 ± 11.45 kg) were selected and randomly divided into two groups, TMR with single corn silage (SS) and TMR with various silage (MS). The whole experiment consisted of 15 days preparation period and 69 days experimental period. Rumen fluid and blood samples were taken from six beef cattle per treatment at the end of the experiment. The results showed that the average daily gain of the MS group (1.56 kg/day) was higher than (p < .05) the SS group (1.30 kg/day), and a decrease of feed conversion ratio in the MS (10.83) group was observed compared with SS group (12.36) (p < .05). The concentration of total volatile fatty acids for MS group was greater than (p < .05) the SS group. The activities of total antioxidant capacity and superoxide dismutase from MS group were also higher than the SS group, but lower urea nitrogen was found in the MS group from serum (p < .05). In addition, the abundances of the Prevotella-1 and Verrucomicrobia were higher in the MS group than the SS group (p < .05). An increase in the flavonoid biosynthesis was detected in the MS group compared with the SS group by Kyoto Encyclopaedia of Genes and Genomes analysis. The present findings suggest that it is economical and healthy to substitute high-quality forage +low level of concentrate for a relatively low proportion forage +high level of concentrate in a finishing diet of beef cattle, which was a feasible and healthy strategy in the intensive feeding system.

K E Y W O R D S
beef cattle, growth performance, microbial community, ruminal fermentation, silage the use of forage resources, reducing feeds cost, and increasing economic benefits. As a result, the high-quality forages instead of concentrate is becoming increasingly popular in herbivore diets, decreasing the disadvantages of many metabolic diseases and high feed costs related to high concentrate diets in ruminant production (Alstrup et al., 2016;Marques et al., 2016;Schwaiger et al., 2013;Touno et al., 2013). The previous result found that the feeding of relatively high-quality roughage could improve animal intake (Keady et al., 2007).
Furthermore, the traditional feeding mode (single silage or straw forage added concentrate) was used in most beef cattle farming, which limited the efficient use of forage resources (Keady et al., 2007). Thus, it is very necessary to study efficient and highquality feeding strategies for beef cattle. Interestingly, some studies have revealed that compared to alfalfa hay alone as a starter feed for postweaning calves, alfalfa and oat hay mixed feeding can improve dietary nitrogen efficiency and growth performance by impacting rumen fermentation, which was a better feeding strategy (Contreras-Govea et al., 2011;Zou et al., 2018). In the context of these studies and extrapolation of in vitro study data (unpublished; Zhang, Li, Zhou, Wang), it is hypothesized that total mixed ration (TMR) with various forage silage had a positive combination effect on rumen bacteria, rumen fermentation, and growth performance of fattening beef cattle compared with a TMR with single corn silage.
To verify this hypothesis, the present study investigated the effects on rumen fermentation characteristics, rumen bacteria profiles, serum parameters and growth performance of beef cattle fed TMR with alfalfa silage, oat silage, corn silage, and TMR with corn silage.

| Experimental area
The experimental site (North latitude 35 ′07′34″, east longitude 104 ″59′23″, altitude 1,899 m) belongs to the typical place of the semiarid and hilly area of the Loess Plateau, with an average annual temperature from 5.7 to 7.7°C, frost-free period only 122-160 days, with an average annual rainfall from 300 to 400 mm. The growing season for forage is short, only 120-180 day of the year, and droughts are common. More than 3 million acres of artificial forage were planted as alfalfa, corn, oat, and sorghum (Sorghum bicolor L.) et al. The annual yield of silage forage was 2.69 million tons (Table 1).

| Animals, diets, and experimental design
The ingredient and nutrient levels of two types of diets are shown in Table 2. Thirty Simmental bulls (438.94 ± 11.45 kg) with similar age (12 months) were used in a single factor design. Animals were assigned randomly within-pair to one of two treatments (15 animals per group). The corn silage (SS)-based diets were composed of corn silage (40%), alfalfa hay (8%), and 52% concentrate supplement. The various silage (MS)-based diets were composed of corn silage (27%), oat silage (15%), alfalfa silage (10%), wheat straw (10%), and 38% concentrate supplement. The TMR was formulated according to NRC (2000) recommendations for beef cattle. All animals of each group were fed in a 100-m 2 house, each animal lived in a separate shed, and can only touch its diets, and fed twice daily (0700 and 1700 hr) considering 5%-10% feed refusals and water was supplied ad libitum.
There was 15 days adaptation period and the experimental period lasted 69 days. The daily feed intake was recorded, and the average intake during the experiment period was calculated. Meanwhile, the animals were weighed using the Electronic scale (SCS) at the beginning and end of the experiment period, and calculated the average daily gain (ADG) and feed conversion rate (FCR).

| Diets and rumen samples collection
The TMR diets were sampled once a week and stored at −20°C until analysis of nutrient components of diets.
Rumen fluids were taken from six beef cattle per treatment using an esophageal catheter (Colibo A1164K) at feeding before the end of the experiment reference to Zhou et al. (2012), and then were filtered by four layers using a medical gauze. The pH values were measured immediately using a portable pH meter (pHS-3C). Then, a part of the ruminal fluid was stored at −80°C for DNA extraction, the other parts of extracts were centrifuged at 10,000 g for 15 min at 4°C, the supernatants approximately 10 ml were collected and stored at −20°C for subsequent analysis of the concentrations of volatile fatty acids (VFA) profiles and ammonia nitrogen (NH 3 -N). The nutrient levels of experimental diets were analyzed following AOAC (1995) method. The VFA concentration of rumen fluid was measured with a gas chromatograph as described by Li et al. (2017).

Item
The NH 3 -N concentration of rumen fluid was determined by phenolsodium hypochlorite colorimetry (Zhou et al., 2015).

| Collection of blood samples and determination of indicators
Ten ml blood samples were collected from six beef cattle per treatment using the jugular vein at feeding before the end of the experiment and centrifuged at 3,500 g for 10 min at 4°C to separate the serum, then was kept frozen at −20°C for subsequent analysis. Serum

| DNA extracting and sequencing analysis
The rumen fluid DNA was extracted from 300 ml of the sample, following DNA extraction kit (EZNA DNA Stool Mini) method.
Each sample was extracted three times for microbial DNA, which was tested for quality and concentration, and then repeatedly mixed for 16S rRNA sequencing analysis. Bacterial amplicons were sequenced with the Illumina MiSeq platform at BMK Cloud (www.biocl oud.net). Briefly, the (V3-V4) region of the bacterial 16S rRNA was amplified. The amplification primers: Forward primer (5′-ACTCCTACGGGAGGCAGCA-3′) and Reverse primer (5′-GGACTACHVGGGTWTCTAAT-3′). A 10 μl amplification system (including 0.5 μl of full-type gold enzyme, and 2 μl of stock solution, 1 μl of each primer, 2 μl of DNA template, and 3.5 μl of ddH 2 O 2 ) was used to measure sample quality. Amplification procedure: PCR amplification procedure includes initial denaturation at 98°C for 2 min, then 25 cycles at 98°C for 30 s, 50°C for 30 s, and 72°C for 1 min, finally last extension at 72°C for 5 min. PCR products were electrophoresed on a 1.8% (w/v) agarose gel. This band was separated and purified using a gel extraction kit (TAKARA).
FLASH v1.2.7 software was used to stitch the reads of each sample to obtain raw tags, and Trimmomatic v0.33 software was used to filter the stitched raw tags to obtain clean tags. UCHIME v4.2 software (version 10.0, http://drive5.com/upars e/) was used to obtain effective tags. Using UCLUST (Edgar, 2010)

| Data analysis
The rumen bacteria sequence data were analyzed by R language, p < .05 as the criterion for significance. The data of rumen fermentation parameters and growth performance were analyzed using the general linear model of SPSS 19.0 software (SPSS Inc.). The general linear model of statistical analysis is as follows: Y ij : jth observation of the ith treatment, μ: overall average, τ i : fixed effect of the ith treatment, and e ij : random effect. T-test was used to analyze the differences between the treatments, p < .05 was considered statistically significant, and the trend was 0.05 ≤ p < .1.

| Growth performance
The animal performance measured in 30 beef cattle is illustrated in

| Serum biochemical and antioxidant parameters
The different treatments did not affect serum concentrations of MDA, GSH-Px, β-HB, T-CHO, and TP ( Figure 1). However, the concentrations of T-AOC and SOD of MS group were higher than the SS group (p < .05), while the UN concentration was lower in the MS group (p < .05).

| Rumen fermentation parameters
The results of the rumen fermentation parameters are illustrated in

| Diversity of ruminal bacterial communities
A total of 958,947 reads were obtained from the 12 samples, and a total of 651,435 Clean tags were generated after the two-end reads were spliced and filtered, with an average of 54,286 Clean tags obtained from each sample. It was identified that 1,902 OTUs in total in all samples were based on 97% similarity. A total of 18 phyla, 32 classes, 44 orders, 62 families, and 131 genera were identified in the two groups in cattle rumen. Rarefaction curve reflected sufficient sequencing volume (Figure 2).
There were no significant differences in the OTUs, ACE, and Chao1 indices in the MS and SS groups (p > .05). But, the Shannon index of the MS group was higher than the SS group (p < .05), and the Simpson index of the MS group tended to be higher than the SS group (p = .10) ( Table 5).
At the phylum level ( Redundancy (RDA) analysis showed that two axes explained 27.83% of the differentiation of the microbial community ( Figure 8).
The arrow length of the environmental factors represents their effect degree on the bacterial genus, and the obtuse angle between environmental factors and bacterial genus indicated that there was a negative correlation between the two groups. The effects of environmental factors on the bacterial genus ranked from high to low were acetate, propionate, TVFA, NH 3 -N, butyric acid, and pH. There was a positive correlation among the following: acetate concentration with Ruminococcaceae_NK4A214_group, Prevotella_1; and propionate and TVFA concentrations with Ruminococcaceae_UCG_014 and Rikenellaceae_RC9_gut_group, moreover, the NH 3 -N concentration with Rikenellaceae_RC9_gut_group.

| DISCUSS IONS
As previous studies reported that combination of high-quality forage can produce a positive combination effect and improve rumen fermentation properties and further elevate the production performance of livestock (Zhang et al., 2015(Zhang et al., , 2019. In the present study, the diet of the MS group improved the ADFI (5.30%), ADG (16.67%), and reduced FCR (12.76%) of fattening beef cattle compared to the diet of the SS group. The dry matter intake was higher in the MS group compared with the SS group, which could explain the increase of ADG in the MS group compared with the SS group, and also could be explained with the increase in VFA concentration in the MS group, resulting in the increase of metabolizable energy of the diet (Langille et al., 2017).
Blood parameters are an indicator related to pathological diagnosis that reflects the health status of animals exposed to antinutritional or harmful substances (Manoeuvrier et al., 2017). In the present study, serum T-AOC, SOD, and UN concentrations differed between the two groups, indicating that the antioxidant capacity and protein metabolism of the organism were affected by the roughage ratio and composition. Oxidative stress puts ruminants in sub-healthy status, thus it is crucial to improve the antioxidant activity of the animal organism. Generally, serum UN is used for diagnosing kidney damage (Cozzi et al., 2011). In the present study, all serum parameters were within normal ranges (Solanas et al., 2010); thus, the difference of UN was mainly due to dietary nutrient composition in the experimental diet, while the proportion of roughage ratio increased in the MS group, the soybean meal and corn amount decreased to balance the dietary crude protein level among diets.
However, soybean meal had good protein digestibility (Langille et al., 2017), a decrease might occur in serum UN with a decline in soybean meal proportion.
It was found that the ruminal pH and concentration of NH 3 -N were not significantly different between the SS and the MS groups, which might be related to rumen environment stability (Duffield et al., 2004;Ellis et al., 2009). Moreover, it also observed that the NH 3 -N concentration was averagely 14.73 mg/dl (14.88 mg/dl in the SS group vs. 14.57 mg/dl in the MS group). Mcdonald et al. (1995) recommended that the optimum NH 3 -N concentration range from 8.5 to 30 mg/dl, and if the NH 3 -N concentration was below the minimum threshold, microbial production and productivity would be inhibited. In this study, the rumen NH 3 -N concentration of the cattle was within the optimal range.
The VFA is a major carbon source for rumen microbial proliferation (Spears et al., 2004), and provides 60%-80% digestible energy for ruminant (Galyean, 2014). And the composition ratios were TA B L E 5 Species richness and alpha-diversity indexes of the bacterial community (at 97% similarity level)  (Bergman, 1990). Previous studies showed that higher VFA concentration with equivalent to more efficient energy utilization for animal growth and production (Ozadali et al., 1996).
Interestingly, in the present study, the TMR with various silage improved the VFA concentration, implying that the combination of a high-quality forage diet could improve rumen digestibility and energy conversion by a higher intake of dry matter, and then increase the VFA concentration (Zhou et al., 2012).
The present study data of the higher Shannon index indicated that TMR with multitudinous high-quality forage would increase the diversity of rumen microbiota. Furthermore, the abundances of Bacteroidetes and Firmicutes were the highest in the rumen microorganism, which was similar to previous reports in bovine (Bo Trabi  (Lopes et al., 2015;Perumbakkam et al., 2011), describing the Bacteroidetes and Firmicutes were dominant phyla in the rumen microbiome. Previous studies had reported that the content of dietary fiber was proportional to the change in Bacteroidetes/ Firmicutes ratio (Parnell & Reimer, 2012;Trompette et al., 2014). Our present results also showed a higher ratio of Bacteroidetes/Firmicutes in the MS group than that the SS group which was relative to higher NDF intake (38.89% vs. 32.77%). Moreover, it was found that a higher Verrucomicrobia in the MS group compared with the SS group.
Verrucomicrobia plays a major role in immune tolerance of the gastrointestinal tract for mammals, and the decrease of species abundance is closely related to the decline of host immunity (Derrien et al., 2011).
Therefore, our results indicated that the beef cattle fed TMR with various forage silage could contribute to better health, which corresponds to the results of the previous serum antioxidant parameters. However, the long-term effects should be further studied.
The genus Prevotella, a dominant genus of rumen microorganisms, is involved in the degradation of all dietary components in the rumen (Alzahal et al., 2017;Henderson et al., 2015;Schären et al., 2018). Consistently, the abundance of Prevotella_1 was greater in the MS group (18.5%) than the SS group (16.9%), indicating that the beef cattle fed TMR with various forage silage was beneficial to the degradation of dietary components in the rumen.
Ruminococcaceae, is a kind of cellulose-degrading bacteria, its main products are acetic acid and formic acid (Wood, 1988 The KEGG analysis indicated that the flavonoid biosynthesis was higher in the MS group compared with the SS group. The activity of enzymes catalyzed the O 2 -production could be inhibited by flavonoids, activated antioxidant enzymes, and thus reduced the production of free radicals (Bao et al., 2016). As a polyphenol, flavonoids endow high antioxidant activity (Kamiloglu et al., 2013). This study detected beef cattle-fed MS group diets may be beneficial to the synthesis of flavonoids, and may improve the antioxidant function of the animal organism. The RDA analysis revealed that there is a positive relationship between Prevotella_1 and acetate concentration. Similarly, Henderson et al. (2015) observed that Prevotella_1 was positively correlated with acetate and pH value. In agreement with our results, Biddle et al. (2013) found Ruminococcaceae_RC9_ gut_group had a positive correlation with NH 3 -N concentration.

| CON CLUS IONS
In summary, the beef cattle fed TMR with a variety of silage increased the concentration of TVFAs and individual acids, improved rumen fermentation, average daily weight gain, and feed effectiveness, moreover, improved serum T-AOC and SOD activities. The species involved in the degradation of dietary components (Prevotella_1) and immune tolerance of the gastrointestinal tract (Verrucomicrobia) were higher in diet with a variety of silage compared with the single silage. Therefore, the present findings suggest that it is economical and healthy to substitute various silage forage +low level of concentrate for relative single silage forage +high level of concentrate in a finishing diet of beef cattle. Furthermore, the effect of various silage supplementation on production performance and the livestock products quality should be continuously studied for long-term feeding.

ACK N OWLED G M ENTS
We appreciate the support from the National Key R&D Program of China (No. 2017YFE0104300) and Dingxi City Science and Technology Plan (071100032). We gratefully acknowledged helps from Prof. An Jizhong, Mr. Wang Weizhong, Ms. Ma Peilin, and all staff of the Jia Tianxia beef cattle farm in Dingxi City, Gansu Province. We also want to thank Ishaq Muhammad for guidance on English correction.

CO N FLI C T O F I NTE R E S T
We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

E TH I C S S TATEM ENT
All the animal experimental procedures adhered to the Guidelines for the Care and Use of Laboratory Animals (Ministry of Science and Technology of the People's Republic of China) and were approved by the Ethical Committee for Animal Experiments, Lan Zhou University.

DATA AVA I L A B I L I T Y S TAT E M E N T
All authors confirm that the data supporting the findings of this study are available within the article.