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Effects of Different Fiber to Starch Ratios on Growth Performance,Nutrients Digestibility, and Ruminal Microbiota in Holstein BullsZenglin Wang 

Hebei Agricultural UniversityHui Yan 

Hebei Agriculture Univeristy https://orcid.org/0000-0002-9004-0481Yizhao Shen 

Hebei Agricultural UniversityYan Li 

Hebei Agricultural UniversityMeimei Wang 

Hebei Agricultural UniversityYufeng Cao 

Hebei Agricultural UniversityXiujiang Zhang 

Animal Husbandry Workstation of BaodingYanxia Gao 

Hebei Agriculture UniveristyJianguo Li 

Hebei Agricultural UniversityYunqi Li 

Hebei Agriculture UniversityQiufeng Li  ( lqf582@126.com )

Hebei Agricultural University

Research

Keywords: NDF:starch ratio, Holstein bulls, growth performance, Prevotella 1

Posted Date: November 9th, 2020

DOI: https://doi.org/10.21203/rs.3.rs-102472/v1

License: This work is licensed under a Creative Commons Attribution 4.0 International License.   Read Full License

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AbstractBackground: The objective of this study was to investigate the effects of dietary neutral detergent �ber (NDF) to starch ratios on growth performance,nutrient digestibility, ruminal fermentation, and microbial community patterns. Thirty-six healthy Holstein bulls at nine months of age (averaged282±37 kg of body weight) were randomly allocated into three treatments: dietary NDF to starch ratio of 1.13, 1.38, and 1.63. Dry matter (DM) intake(DMI), body weight gain, serum biochemical characteristics, hormones, total-tract digestibility, ruminal enzyme activities, ruminal fermentation, andmicrobial communities were analyzed.

Results: Though the DMI and feed e�ciency were not affected, the average daily gain (ADG) increased linearly with a decreasing NDF to starch ratio.DM digestibility, ruminal microbial diversity, ruminal amylase activity, propionate and butyrate concentration, serum glucose, acetyl-CoA carboxylaseincreased linearly with decreasing dietary NDF:starch ratio. The abundance of the predominant ruminal genus, Prevotella 1 (19.00%~31.64%) linearlydecreased, while the proportion of two dominant genera (Ruminococcaceae NK4A214 group/4.14%~6.66% and Rikenellaceae RC9group/5.57%~7.76%) increased with the decreasing NDF:starch ratio. Dry matter intake, digestibility of crude protein, ether extract (EE), NDF and ADF,AST, BUN, TP, NEFA, GH, IGF-1, and insulin were not affected by the dietary NDF:starch ratio.

Conclusions: In conclusion, the dietary NDF:starch ratio of 1.13 bene�ts the weight gain of Holstein bulls. A potential reason might be the regulationof propionate productivity, amylase activity, and physiological metabolism through the change of dominant ruminal microbiota (Prevotella 1,Ruminococcaceae NK4A214 group and Rikenellaceae RC9 group), which might have a bene�cial effect on the lipogenesis and body weight gain ofHolstein bulls during the fattening period.

BackgroundStarch and NDF were the two most crucial carbohydrate and energy sources in the diet of ruminants. The fermentation products of starch and NDFwere the primary energy supply for ruminant animals. Starchy feedstuff is more digestible than NDF when feeding to ruminants, and the apparentdigestibility of starch could be twice as high as NDF [1]. Starchy substances usually supply more digestible energy due to its higher digestibility, and arational proportion of starch addition should be taken into account to achieve optimal growth e�ciency [2]. The content of NDF also affects drymatter intake and nutrient digestibility due to its greater �lling effect [3]. Excessive NDF supply may decrease digestible energy concentrations ofdiets, but if the diet has more starch and less NDF, the homeostasis of the rumen ecosystem might be destroyed and become too acid, resulting inpotential health problems. However, changing the dietary starch content necessitates alters of other carbohydrate substrates as well. A suitable ratioof starch to NDF was critical for nutrient digestibility, rumen microbial fermentation pattern, and physiological metabolism of ruminants, so we focuson the dietary NDF:starch ratio. In the previous studies, dietary NDF:starch ratio was an important evaluating index of carbohydrate composition ofdiets for ruminants. It could affect nutrient digestibility [4], energy utilization [4], milk production [5], and the rumen epithelial cell morphologicalstructure and gene expressions of dairy cows [6]. However, the nutrient requirements of different varieties and growth stages are different, the studyof dietary NDF:starch effect on Holstein bulls is still limited.

The objective of this study was to evaluate whether the NDF:starch ratio should be considered as a nutritional indicator to manipulate the ruminalfermentation pattern and growth performance in dairy Holstein bulls. We hypothesized that dietary NDF:starch ratio might affect rumen fermentationmode by affecting different key microbes in the rumen. Thereupon, the physiological metabolism and feed e�ciency were regulated.

Materials And MethodsAnimals and diets

All the experimental procedures were approved by the Animal Welfare and Ethics Committee of Hebei Agriculture University. This experiment wasconducted at the Hongda Dairy Farm (Baoding, Hebei, China) from February to May 2018.

A total of 36 healthy Holstein bulls (12 head/treatment) at nine months of age (initial body weight of 282 ± 37 kg) were blocked by body weight andassigned to a complete randomized design to 1 of 3 treatments. The bulls were housed in individual stalls with free feed and clear water throughoutthe experimental period. The bulls were fed a total mixed diet ad libitum twice daily at 0700 and 1700 h. The bulls were gradually adapted to theexperimental environment over 7 d before starting the experiment, and the experimental period was 90 d. The energy and protein contents in theexperimental diets were similar, whereas the NDF: starch ratios were different (1.13, 1.38, and 1.63; low-NDF:starch ratio group, medium- NDF:starchratio group and high- NDF:starch ratio group, Table 1). The diets were formulated according to NASEM (2017) to meet the nutrient requirement [7].

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Table 1Ingredients and nutritional composition of diets with NDF to starch ratios of 1.13 (Low), 1.38 (Medium) and 1.63 (High)

Items Diets (NDF/starch)

High Medium Low

Ingredients, % of DM      

Whole corn silage 4.90 22.00 41.20

Millet straw 30.90 18.40 4.30

Chinese wildrye 4.50 4.50 4.50

Corn 15.70 12.85 9.60

Flaked corn 10.00 10.00 10.00

Bran 4.00 4.00 4.00

Beet 10.00 10.00 10.00

Soybsuanean meal 8.35 7.55 6.70

DDGS 8.65 7.70 6.70

Limestone 0.10 0.10 0.10

CaHPO4 0.10 0.10 0.10

NaCl 0.10 0.10 0.10

NaHCO3 0.10 0.10 0.10

Premix a 2.60 2.60 2.60

Total 100.00 100.00 100.00

Nutrients b      

NEmf(MJ/kg) 5.88 5.88 5.88

CP 13.08 13.07 13.09

NDF 40.69 37.12 33.10

ADF 23.56 20.87 17.84

ADL 3.84 2.96 1.97

Starch 25.02 26.99 29.19

Ca 0.61 0.60 0.59

P 0.38 0.38 0.31

NDF/starch 1.63 1.38 1.13

a. Per kg premix compositions: VA3600 IU, VD3 1280 IU, VE 30 IU, D-biotin 5.0 mg, Cu 10.0 mg, Mn 20 mg, Zn 30 mg, Mg 1200 mg, Co 0.15 mg,NaCl 80 mg, NaHCO3200 mg, Limestone 80 mg.

b. NEmf was calculated according to NRC (2000),while the other nutrient levels were measured values.

 

Data collection

During the experimental period, the amount of feed offered and refused was recorded daily. Samples of TMR were collected weekly, and feed refusedwere sampled daily for each bull and pooled weekly by the group. Then samples were dried at 105℃ for four h in a forced-air oven followed byequilibration in a desiccator and weighed for DMI analysis. The initial and �nal body weight (BW) of each bull was measured before the morningfeeding on day 1 and 90 of the experimental period.

Blood sampling and analysis

Approximately 20 ml of blood samples were collected from each bull via the jugular vein using sterile blood collection tubes before morning feedingon the 85th day at the end of the experimental period. Serum samples were centrifuged (1500 g, 15 min) after water bath at 37℃ for 30 min and

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frozen (-20℃) for later analysis. Alanine aminotransferase (ALT), aspartate Aminotransferase (AST), acetyl-CoA carboxylase (ACC), non-esteri�edfatty acids (NEFA) were measured with a commercial ELISA kit (Beijing Huayue Chang Institute of Biological Technology, Beijing, China) using amicroplate reader (DNM-9602, Beijing Pulang New Technology Co., Ltd., China.). Growth hormone (GH), insulin (INS), and insulin-like growth factor 1(IGF-1) were measured using ELISA kits (Beijing Hueyue Chang Institute of Biological Technology, Beijing, China). Glucose was measured with aglucose assay kit (361510, Shanghai Rongsheng Biotechnology Co., Ltd). Urea nitrogen (UN) was measured with a commercial kit (105-000452-00,Shenzhen Mairui bio-medical electronics company). Total protein (TP) was measured with a total protein quantitative assay kit (A045-4-2, NanJingJianCheng Bioengineering Institute).

Ruminal enzyme activity analysis

10 mL rumen �uid sample was centrifuged at 1500 g for 10 min at 4 °C,supernatants were collected and stored at -20 °C. Amylase, Carboxymethylcellulase, and xylanase activities were measured with a commercial kit (MM-195502, MM-195602, MM-195702, Beijing Huayue Chang Institute ofBiological Technology, Beijing, China).

Nutrient digestibility analysis

On the 70th day of the experimental period, samples of dietary TMR were collected, then a representative sub-sample was generated by quarteringmethod and dried at 65 °C to determine the nutrients component. Rectal feces samples were collected for three consecutive days, and a subsamplewas preserved by the addition of 20 mL 10% (w/w) H2SO4 per 100 g fresh feces to prevent N loss for crude protein (CP) determination, then all thesamples were frozen immediately at -20 °C. Total-tract nutrient digestibility was estimated using acid insoluble ash (AIA) as an internal markeraccording to the previous methods (method 942.05)[8]. Feeds and feces samples for digestibility measurement were dried and ground through a 1-mm screen, and ground samples were analyzed for ether extract (EE, AOAC 2007, method 960.39), CP(AOAC 2007, method 990.03), NDF (AOAC 2012,method 2002.4), ADF (AOAC 2012, method 973.18) and DM (AOAC 2007, method 930.15) contents according to O�cial Methods of Analysismethodologies[9, 10].

High-throughput sequencing and analysis of ruminal microbiota

On the 88th day of the experimental period, ten bulls were randomly selected from each group, and rumen �uid samples were collected using the oralstomach tube from each bull. Collected rumen �uid samples were �ltered through four-layer cheesecloth and immediately transferred to liquidnitrogen, and then stored at -80 °C in the laboratory until VFA determination and DNA extraction for high-throughput sequencing analysis. DNA puritywas characterized by the A260/A280 absorbance ratio between 1.77 ~ 1.95. DNA concentration was required between 25 ng/uL ~ 97.7 ng/uL. V4region of 16S rRNA gene for amplicon high-throughput sequencing was ampli�ed with primers 515F/806R (515F-5’-GTGCCAGCMGCCGCGGTAA-3’;806R-5’-GGACTACHVGGGTWTCTAAT-3’) and sequenced by Illumina Hiseq platform. Illumina sequencing adapters and primers were removed usingCutadapt[11], then low-quality ends of reads were trimmed off, and low-quality reads were �ltered out with an average quality score lower than 15 inany 4-bp slide window using Trimmomatic-0.36[12]. Paired forward and reversed clean reads were merged into combined sequences based onoverlap longer than 10-bp and maximum mismatch percentage less than 10% (0.1) using FLASH[13]. Then chimeric sequences were detected andremoved with Usearch61, and sequences were pushed into the QIIME analysis pipeline[14]. Sequences were clustered into OTUs based on a 97%similarity threshold with the ulcust method. Taxonomic functions of representative sequences were annotated based on the SILVA132 database.Singletons were removed to reduce the over-estimation of rare OTU. Bacterial community diversity, including observed species, Chao1, Shannonindex, Simpson index, weighted UniFrac distance, and unweighted UniFrac distance, were calculated using scripts in QIIME. All the raw sequencingdata have been deposited and available in National Genomics Dater Center Sequence Read Archive with SRA number CRA002682(https://bigd.big.ac.cn/ ).

Statistical analysis

Statistical differences between treatments were analyzed with the GLM procedure using the aov formula in R (3.6.2). Yi = µ + xi+ei, where Yi is thedependent parameter for the ith treatment, µ is the overall mean, xi is the �xed effect of the ith treatment, and ei is the residual error. The linearregression was performed with lm(y ~ x) in R. The quadratic regression was performed with lm(y ~ x + I(x^2)) in R. Signi�cance between groups ofbeta diversity was calculated by ANOSIM (analysis of similarity) with the vegan package (2.5) in R software (3.6.2). The igraph package (1.2.5) wasused to draw the network.

The statistical signi�cance was declared at P < 0.05, the rumen microbiota abundances were declared at a signi�cance at P < 0.01. The FDR valuecorrection was s performed using R software. The SEM value was calculated with package psych in R. All data are shown as mean ± SEM.

ResultsEffects of NDF:starch ratio on feed intake and growth performance

The DMI and �nal BW were not affected by treatments during the experimental period. While the ADG increased linearly as the NDF:starch ratiodecreased, the ADG of low-NDF:starch ratio group was signi�cantly higher than the high-NDF:starch ratio group. The feed e�ciency increased as the

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dietary NDF:starch decreased, although the difference among groups was not signi�cant (Table 2).

Table 2Effects of dietary NDF/starch on fattening performance of Holstein bulls

Items Diets (NDF/starch ratio) SEM P-value  

  1.63 1.38 1.13   Treatment Linear Quadratic

Initial weight, kg 282 282 282 6.1 ns ns ns

Final weight, kg 427 433 438 6.4 ns ns ns

ADG, kg/d 1.61b 1.67ab 1.73a 0.018 <0.05 <0.05 <0.05

DMI, kg/d 11.2 11.3 10.7 0.28 ns ns ns

F/G 6.95 6.78 6.16 0.179 ns ns ns

ns: Not signi�cant

 

Effects of NDF:starch ratio on nutrient digestibility, fermentation parameters, and ruminal enzyme activities

No signi�cant difference among treatments was observed in the apparent total tract digestibility of CP, EE, NDF, and ADF. Whereas, the digestibility ofDM increased linearly with decreasing NDF:starch ratio, the DM digestibility of low-NDF:starch ratio group was signi�cantly higher than the high ratiogroup (Table 3, P < 0.05).

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Table 3Rumen fermentation parameters, ruminal enzyme activities and nutrient digestibility

Items Diets (NDF/starch ratio) SEM P-value

1.63 1.38 1.13 Treatment Linear Quadratic

Apparent Nutrient Digestibility

DM 70.28b 72.94a 74.71a 0.597 <0.05 <0.05 <0.05

CP 69.03 69.44 69.93 0.214 ns ns ns

EE 86.22 86.55 87.21 0.477 ns ns ns

NDF 47.78 53.48 49.43 1.904 ns ns ns

ADF 46.43 50.41 46.14 1.995 ns ns ns

Fermentation

pH 6.99 6.86 6.79 0.042 ns ns ns

NH3-N/(mg/dL) 10.62 10.05 11.19 0.35 ns ns ns

MCP/(mg/dL) 10.58 10.83 12.14 0.396 ns ns ns

Acetate/(%) 65.12b 60.82a 56.22a 0.012 < 0.01 < 0.01 < 0.01

Propionate/ (%) 18.89c 22.28b 24.80a 0.008 < 0.01 < 0.01 < 0.01

Butyrate/(%) 10.84b 11.80b 13.74a 0.004 < 0.01 < 0.01 < 0.01

TVFA/(mmol/L) 86.69 89.35 89.49 0.641 ns ns ns

A:P 3.45c 2.74b 2.27a 0.152 < 0.01 < 0.01 < 0.01

Enzyme Activity

Amylase/(U/mL) 103.18b 104.86ab 111.35a 1.442 < 0.05 < 0.05 < 0.05

CMC/(IU/L) 220.28b 210.09a 201.30a 2.649 < 0.01 < 0.01 < 0.01

Xylanase/(U/ml) 73.58 74.9 76.38 0.515 ns < 0.05 ns

DM: dry matter; CP, crude protein; EE, ether extract; NDF, neutral detergent �ber; ADF, acid detergent �ber; NH3-N: ammonia-N; MCP: microbial crudeprotein; TVFA: total volatile fatty acid; A:P: acetate: propionate ratio; CMC: carboxymethyl cellulase.

ns: not signi�cant.

 

Ruminal pH, NH3-N, MCP, and total VFA were not affected by the dietary NDF:starch. However, linear effects in ruminal acetate, propionate, butyrateconcentration, and acetate:propionate (A:P) ratio were observed. Low-NDF:starch ratio diet increased the proportions of propionate and butyrate by31.3% and 26.7% over high-NDF:starch ratio group, respectively; correspondingly, acetate proportion decreased by 15.8% (Table 3).

The ruminal amylase and xylanase activities increased linearly as the dietary NDF:starch ratio decreased; meanwhile, the ruminal carboxymethylcellulase (CMC-ase) activity decreased linearly (Table 3).

Effects of NDF:starch ratio on serum biochemical characteristics and hormones

The serum concentration of ALT, AST, glucose, blood urea nitrogen, total protein, ACC, NEFA, GH, IGF-1, and insulin were detected and presented inTable 4. ALT, GLU, and ACC concentrations increased linearly as NDF:starch ratio decreased, low-NDF:starch ratio diet increased serum GLUconcentration by 10.6% over high-NDF:starch ratio group. The dietary treatment did not affect AST, BUN, TP, NEFA, GH, IGF-1, and insulinconcentrations.

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Table 4Effects of dietary NDF/starch on serum biochemical parameters and hormones of Holstein bulls

Items Diets (NDF/starch ratio) SEM P-value

1.63 1.38 1.13 Treatment Linear Quadratic

ALT/ (ng/mL) 13.47b 13.46b 13.84a 0.07 < 0.05 < 0.05 < 0.05

AST/ (ng/mL) 12.57 12.78 12.93 0.132 ns ns ns

GLU/ (mmol/L) 3.96b 4.25ab 4.38a 0.009 < 0.05 < 0.05 ns

BUN/ (mmol/L) 3.28 3.22 3.24 0.012 ns ns ns

TP/ (g/L) 65.25 65.3 65.43 0.093 ns ns ns

ACC/ (ng/mL) 13.08b 13.82ab 14.09a 0.199 < 0.05 < 0.05 ns

NEFA/ (µmol/L) 485.22 516.03 447.87 1.211 ns ns ns

GH/ (µg/L) 32.58 32.62 32.91 0.213 ns ns ns

IGF-1/ (µg/L) 145.98 146.3 146.57 1.246 ns ns ns

INS/ (mU/L) 19.94 20.13 20.38 0.277 ns ns ns

ALT: Alanine Aminotransferase; AST: Aspartate Aminotransferase; GLU: Glucose; BUN: Blood Urea Nitrogen; TP: Total Protein; ACC: Acetyl-CoACarboxylase; NEFA: non-Esteri�ed Fatty Acids; GH: growth hormone; IGF-1: insulin-like growth factor-1; INS: Insulin.

ns: no signi�cant.

 

Effects of NDF:starch ratio on rumen microbiota diversity

16S rRNA high-throughput sequencing from 30 rumen samples generated approximately 2712943 pairs of raw reads by the sequencing procedure.After �ltering the low-quality reads, 2687771 combined sequences were obtained in total. For all samples, the good’s coverages were more than99.9%. Observed-species and richness estimator (Chao1) was similar among different dietary groups; however, bacterial diversity estimators(Shannon and Simpson) increased linearly as the NDF:starch ratio decreased, and the low NDF:starch ratio group had the highest ruminal bacterialdiversity (Table 5).

Table 5Alpha diversity analysis of rumen bacteria

Items Diets (NDF/starch ratio) SEM P-value  

1.63 1.38 1.13 Treatment Linear Quadratic

Observed-species 1000 1001.8 999.5 0.569 ns ns ns

Shannon 7.867b 8.064a 8.158a 0.034 < 0.001 < 0.001 < 0.001

Simpson 0.981b 0.986a 0.989a 0.001 < 0.001 < 0.001 < 0.001

Chao1 1002.74 1002.64 1002.04 0.58 ns ns ns

Goods-coverage 0.99991 0.99995 0.99991 0.00001 ns ns ns

ns: not signi�cant.

 

Correlations among dietary NDF:starch ratio, predominant microbiota, and fermentation parameters

To further identify the origin of ruminal fermentation pattern change, ruminal microbiota responses on the dietary NDF:starch ratio were studied atthe genus level. 160 genera have been identi�ed in total, among which 25 genera signi�cantly changed at the genus level (Table 6). Prevotella 1,Rikenellaceae RC9 group, Christensenellaceae R-7 group, and Ruminococcaceae NK4A214 group were identi�ed as the four most predominantgenera, with the mean relative abundance of 25.8 ± 7.9%, 6.7 ± 1.4%, 6.0 ± 1.4% and 5.3 ± 1.5%, respectively (Supplementary Table 1). Intriguingly, thedominant genera Prevotella 1, Rikenellaceae RC9 group, and Ruminococcaceae NK4A214 group were signi�cantly affected by the dietary NDF: starchlevel. Relative abundances of Prevotella 1 linearly increased as the dietary NDF:starch ratio increased; whereas, Rikenellaceae RC9 group andRuminococcaceae NK4A214 group decreased linearly as the NDF:starch ratio increased (Table 6).

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Table 6Signi�cantly changed genera of rumen microbiota due to different NDF:starch ratios based on relative abundance

Phylum Family Genera Diets (NDF/starch ratio)a SEM P-value  

1.63 1.38 1.13 Treatments Linear Quadratic

Firmicutes Lachnospiraceae LachnospiraceaeND3007 group

0.410b 0.534b 0.822a 0.041 < 0.001 < 0.001

< 0.001

    LachnospiraceaeXPB1014 group

0.521b 0.613b 0.776a 0.028 < 0.01 < 0.001

    Marvinbryantia 0.060b 0.078b 0.113a 0.006 < 0.01 < 0.001

    Roseburia 0.082c 0.080b 0.114a 0.004 < 0.01 < 0.001

    Acetitomaculum 0.126b 0.142b 0.199a 0.009 < 0.01 < 0.001

  Ruminococcaceae RuminococcaceaeNK4A214 group

4.140b 5.128b 6.663a 0.266 < 0.01 < 0.001

    Eubacteriumcoprostanoligenesgroup

0.850b 1.037b 1.266a 0.044 < 0.01 < 0.001

    Papillibacter 0.363b 0.388b 0.464a 0.013 < 0.01 < 0.001

  Erysipelotrichaceae ErysipelotrichaceaeUCG-004 group

0.439b 0.475b 0.587a 0.018 < 0.01 < 0.001

    ErysipelotrichaceaeUCG-009 group

0.103b 0.122b 0.167a 0.008 < 0.01 < 0.001

    Anaerorhabdusfurcosa group

0.080b 0.106b 0.123a 0.005 < 0.01 < 0.001

    uncultured 0.121b 0.149a 0.187a 0.007 < 0.01 < 0.001

  Veillonellaceae Selenomonas 0.010b 0.018b 0.027a 0.002 < 0.01 < 0.001

Bacteroidetes Rikenellaceae Rikenellaceae RC9group

5.572b 6.629b 7.763a 0.259 < 0.01 < 0.001

  Bacteroidales RF16group

uncultured 1.539b 1.574b 2.310a 0.09 < 0.01 < 0.001

  Bacteroidales BS11gut group

uncultured 0.202b 0.234b 0.361a 0.018 < 0.01 < 0.001

  Bacteroidales BS11gut group

uncultured 0.304b 0.334b 0.570a 0.034 < 0.01 < 0.001

  F082 uncultured 0.010b 0.018b 0.025a 0.002 < 0.01 < 0.001

  Marinilabiliaceae uncultured 0.050b 0.066b 0.076a 0.003 < 0.01 < 0.001

Tenericutes uncultured uncultured 0.606b 0.700ab 0.834a 0.026 < 0.01 < 0.001

Patescibacteria Saccharimonadaceae CandidatusSaccharimonasgroup

0.174b 0.211ab 0.343a 0.02 < 0.01 < 0.001

Proteobacteria uncultured uncultured 0.026b 0.044b 0.065a 0.004 < 0.01 < 0.001

Bacteroidetes Prevotellaceae PrevotellaceaeGa6A1 group

0.085b 0.059ab 0.040a 0.005 < 0.01 < 0.001

< 0.01

    PrevotellaceaeYAB2003 group

0.120b 0.102ab 0.044a 0.009 < 0.01 < 0.01

    Prevotella 1 31.636b 26.752ab 19.002a 1.437 < 0.01 < 0.01

a, relative abundance of different genera. Bold, predominant genera in rumen in this study.

 

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Moreover, correlations among dietary NDF:starch ratio, ruminal microbiota, and fermentation parameters were shown in Fig. 1. At the genus level, 3genera belonged to the family Prevotellaceae were positively associated with dietary NDF:starch ratio, and were all positively associated with acetateto propionate ratio. Speci�cally, the proportion of Prevotella 1 was positively associated with acetate (r > 0.83, q < 0.01), and negatively associatedwith propionate (r < -0.79, q < 0.01) and butyrate (r < -0.77, q < 0.01). The proportion of Prevotellaceae YAB2003 group was positively associated withacetate (r > 0.85, q < 0.01), and Prevotellaceae Ga6A1 group was negatively associated with propionate (r < -0.83, q < 0.01) and butyrate (r < -0.83, q < 0.01). The other 22 genera were all negatively associated with dietary NDF:starch ratio, among which, the abundance of the genus RuminococcaceaeNK4A214 was positively related with amylase activity (r > 0.73, q < 0.01), propionate (r > 0.83, q < 0.01) and butyrate concentrations (r > 0.80, q < 0.01).

DiscussionAn increase in ADG and total weight gain throughout the experimental period as the dietary NDF:starch ratio decreased is consistent with �nding byHu [15]. On the contrary, Suarez found that an excessive high starch diet may reduce feed intake and ADG by decreasing the rumen pH value [16]. Forthe low-NDF:starch ratio group, lower concentration of acetate and higher content of propionate and butyrate were observed, as also indicated byprevious studies that lower starch diet resulted in higher A:P [4, 17]. Oba suggested that propionate has greater hypophagic effects than acetate, DMImight decrease compared to higher ruminal acetate concentration [17] due to the physiological stimulator of insulin secretion by propionate [18].Therefore, in the current research, reduced DMI as NDF:starch ratio decreased has been observed, and the suppression of appetite may be caused bythe increased proportion of propionate that was recognized as a satiety signal in the rumen. Nevertheless, starch was a more digestible carbohydratethan NDF[1, 4], which might result in higher DM digestibility[4]. Increased ruminal amylase and xylanase activities in low-NDF:starch ratio diet, as alsofound by Noziere [19], probably resulted in increased DM digestibility. A higher proportion of starch in the diet could supply su�cient substrates forstarch degradation bacteria resulting in increased enzyme activities. Therefore, the effect of DMI decrease might be counteracted by the increasedDM digestibility, which results in increased ADG. In the current research, the digestibility of NDF in the medium ratio group is higher than the low-NDF:starch ratio group; as found in other studies, lower NDF concentration resulted in lower NDF digestibility[4, 19]. However, the NDF concentrationwas 40.69% in the high-NDF:starch ratio group; excessive substrate might cause the decrease of NDF digestibility for the high ratio group. Hence, anoptimum range of dietary NDF:starch ratio was bene�cial for the growth performance of Holstein bulls in a fattening period, NDF:starch ratio of 1.13was more e�cient and pro�table for Holstein bulls from 9 to 12 months old in the current study.

Lower NDF:starch ratio diet caused increasing of serum insulin and glucose concentrations, McCarthy [20] observed a similar increase for cows fedhigher starch diet, and propionate as an insulin secretagogue [21] has also signi�cantly increased as the NDF:starch ratio decreased in this research(Figure 2). For ruminant animals, 50~75% of glucose is derived from propionate[22], and propionate was the most important precursor forgluconeogenesis to supply energy, which might stimulate glucose and fat metabolism, and increased glucose and insulin could stimulate thesynthesis of hepatic [23, 24] and muscle glycogen [25, 26]. Besides, insulin increasing also caused stimulation of fatty acid synthesis, accompanyingby Acetyl-CoA concentration increased[27], ACC as a critical enzyme that catalyzes the carboxylation of acetyl-CoA to malonyl-CoA in the rate-limitingfatty acid synthesis step increased signi�cantly as the dietary NDF:starch ratio decreased. Hence, low-NDF:starch group may improve the weight gainthrough the change of ruminal fermentation products pattern and the stimulation of glucose metabolism and lipid synthesis for Holstein bulls from 9to 12 months old in the current study.

Ruminal microbiota plays a critical role in fermentation patterns and physiological metabolism in ruminants. In this research, in total, 25 signi�cantlychanged genera as dietary NDF:starch ratio change have been observed (FDR < 0.01). Twenty-�ve genera were all �t to the linear form with dietaryNDF:starch ratio, and intriguingly, 3 out of 25 signi�cantly shift genera that linearly increased according to the increasing NDF:starch ratio allbelonged to the family Prevotellaceae (Prevotella 1, Prevotellaceae Ga6A1 group, and Prevotellaceae YAB2003 group). As the predominant genus incurrent research, Prevotella was also found to be a major genus in the rumen of ruminants in previous researches [28, 29], it was typically found inthe intestinal tract of humans and pigs[30], and it plays critical rules in the rumen microbial metabolism process[31]. Prevotella utilizes various plantpolysaccharides[32] as a major degrader of hemicelluloses[33], and shows the potential for break down complex �bers[34]. Kljak found that therelative abundance of Prevotella spp. increased with the decreasing diet starch concentration[37], as observed in the current research; on the contrary,the abundance of Prevotella was also found to be higher in a lower forage diet[35, 36]. One possible reason might be the interaction effect ofdifferent dietary nutrients other than starch [38]. Species Prevotella ruminicola was supposed to produce signi�cantly more acetate than propionateand butyrate[39], and the proportion increase of three genera in family Prevotellaceae might be responsible for the linearly increased acetate in rumenfermentation products.

On the contrary, lower dietary NDF:starch ratio linearly up-regulate the abundances of 22 different genera, accompanied by apparently increasedruminal propionate concentration. These 22 genera belong to 13 different families, the common feature that caused the coincidental increaseremains elusive. It is worth noting that out of 22 genera, Rikenellaceae RC9 group and Ruminococcaceae NK4A214 group belongs to the top fourdominant genera in the rumen microbiota. These two dominant genera might play important roles in starch degradation and compete for nichesagainst some species of genus Prevotella 1, Prevotellaceae Ga6A1 group, and Prevotellaceae YAB2003 group (Figure 2). As the dominant genus inthe rumen ecosystem, Rikenellaceae RC9 and Ruminococcaceae NK4A214 group play important roles in the rumen biochemical metabolic process.The relative abundance of Rikenellaceae RC9 was affected by dietary starch concentration[38], and Rikenellaceae RC9 could produce propionate,acetate as fermentation products[40], that it might play important roles in lipid metabolism[41]. Diet with higher concentrate levels harbored higherproportions of Ruminococcaceae NK4A214 group [29], which was consistent with the �ndings in current research. Ruminococcaceae has been

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associated with the utilization of resistant starch in ruminants and humans[42, 43], and the amylase activity was observed to be positivelyassociated with the abundance of Ruminococcaceae NK4A214 group (Figure 2, Supplementary Table 1). Hence, the higher abundances of thedominant genera Ruminococcaceae NK4A214 group and Rikenellaceae RC9 might be responsible for the increasing amylase activity and improvedlipid metabolism.

Conclusion>A lower dietary NDF:starch ratio (1.13, 1.38, and 1.63) has a positive impact on the ADG of Holstein bulls. The regulation might be majorly caused bythe ruminal microbiota shift of the dominant genus (Prevotella 1, Rikenellaceae RC9 group, and Ruminococcaceae NK4A214 group), then theamylase activity, propionate concentration, and lipid metabolism were positively affected, which further bene�ting the ADG of Holstein bulls. Even so,this �nding and mechanism need to be further veri�ed by future researches. Based on our results, the NDF:starch ratio of 1.13 can be adequate forHolstein bulls from 9 to 12 months old referring to body weight gain and feed e�ciency.

AbbreviationsNDF: Neutral detergent �ber; ADF, acid detergent �ber; A:P, ratio of acetate to propionate; CP: Crude protein; DM: dry matter; VFA: Volatile fatty acid; EE,ether extract; ALT: Alanine Aminotransferase; AST: Aspartate Aminotransferase; GLU: Glucose; BUN: Blood Urea Nitrogen; TP: Total Protein; ACC:Acetyl-CoA Carboxylase; NEFA: non-Esteri�ed Fatty Acids; GH: growth hormone; IGF-1: insulin-like growth factor-1; INS: Insulin; CMC-ase:carboxymethyl cellulase.

DeclarationsEthics approval

All the experimental procedures were approved by the Animal Welfare and Ethics Committee of Hebei Agriculture University(2020).

Consent for publication

Not applicable.

Availability of data and materials

The data analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded by the National Modern Agro-industry Technology Research System (CARS-37), Hebei Modern Agro-industry TechnologyResearch System beef cattle innovation team e�cient breeding post construction project (HBCT2018130202) and the Hebei Provincial Department ofEducation Youth Fund (QN2019134).

Authors’ contributions

QFL designed experiments, ZLW carried out experiments and analyzed data, HY analyzed the sequencing data and wrote the manuscript, YZS, YFCand JGL helped to revise the manuscript, MMW and XJZ helped on sample collection, YL, YXG, and YQL assisted with data analysis.

Acknowledgements

We thank Professor Ji Shoukun in Hebei Agriculture University for supplying help in statistics, and the workers in Hongda Dairy Farm for assist inexperiments.

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Figures

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Figure 1

Correlations among dietary NDF:starch ratio, predominant ruminal microbiota, and rumen fermentation parameters. Red bold-lines, positivecorrelations between dietary NDF:starch ratio and ruminal microbiota; green bold-lines, negative correlations between dietary NDF:starch ratio andruminal microbiota; red thin-lines, positive correlations between ruminal microbiota and fermentation parameters; green thin-lines, negativecorrelations between ruminal microbiota and fermentation parameters. Black dot, dietary NDF:starch ratio; gold dot, genera of phylum Bacteroidetes;red dots, genera of phylum Firmicutes; grey dots, Other phyla; green dots, ruminal fermentation parameters. A:P: acetate: propionate ratio. ×10, radiuswas 10 times smaller than the other dots.

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Figure 1

Correlations among dietary NDF:starch ratio, predominant ruminal microbiota, and rumen fermentation parameters. Red bold-lines, positivecorrelations between dietary NDF:starch ratio and ruminal microbiota; green bold-lines, negative correlations between dietary NDF:starch ratio andruminal microbiota; red thin-lines, positive correlations between ruminal microbiota and fermentation parameters; green thin-lines, negativecorrelations between ruminal microbiota and fermentation parameters. Black dot, dietary NDF:starch ratio; gold dot, genera of phylum Bacteroidetes;red dots, genera of phylum Firmicutes; grey dots, Other phyla; green dots, ruminal fermentation parameters. A:P: acetate: propionate ratio. ×10, radiuswas 10 times smaller than the other dots.

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Figure 2

Consolidation of NDF:starch effects from the rumen microbiota, fermentation, products, and biochemical recruitment. Pink: product enriched or effectincreased signi�cantly. Green, the concentration decreased signi�cantly. Grey, not measured.

Figure 2

Consolidation of NDF:starch effects from the rumen microbiota, fermentation, products, and biochemical recruitment. Pink: product enriched or effectincreased signi�cantly. Green, the concentration decreased signi�cantly. Grey, not measured.