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Effects of short-term fasting on in vivo rumen microbiota and in vitro rumen fermentation characteristics.

INTRODUCTION

In vitro feed evaluation using small tubes and ruminal fluid has been extensively used to examine ruminant diets in the academia and industry [1]. Such techniques are based on the ruminal fluid obtained from live animals equipped with permanent rumen cannulae and require several steps, including the removal of feed particles via muslin, cheesecloth, or centrifugation. Microbial activity is assumed to reach a peak after feeding; accordingly, numerous studies have examined the rumen contents collected post feeding [2-4]. However, some studies have noted that the ruminal fluid might mask the true effect of feed or feed additives if these effects are large. Therefore, the rumen contents are collected prior to feeding (i.e., before morning feeding) [5,6]. However, nutrient perturbation, even for short intervals, influences the rumen microbiota and microbial activity in the rumen [7]. In many countries where cannulated animals are seldom available owing to their maintenance costs, it is not uncommon for scientists to obtain the rumen contents only at an abattoir where the animals are fasted without feed and water for up to a day before they are slaughtered [8]. Therefore, one might speculate that the rumen contents obtained from animals after such short-term changes in status (i.e., fasting for a day) could affect the feed evaluation process. Unfortunately, little information is known about how the microbiome responds to short-term fasting. Fasting could cause microorganisms in the rumen to encounter decreased nutrients and habitat resources as well as increased competition for food. Therefore, fasting may lead to changes in the microbiota and activity in the rumen. The aim of the present study was to evaluate the potential effects of short-term starvation on rumen microbiota using in vivo molecular culture-independent methods as well as the effects on in vitro fermentation characteristics using the ruminal fluid obtained from animals that were fasted at least for 24 h.

MATERIALS AND METHODS

This study was approved by the Institutional Animal Care and Use Committee at the Chung-Ang University, Seoul, Korea (No. 2013-0047).

Animals and experimental design

The representative rumen contents were obtained from three cannulated Holstein steers (793 [+ or -] 8 kg) 2 h after morning feeding (control). Then, fasting was induced by withdrawing both feed and water for 24 h, and the rumen contents were obtained (fasting) from the same steers. The steers were offered typical commercial concentrates and rice straw at a ratio of 40:60. The rumen contents were filtrated through four layers of muslin, immediately sampled (50 mL), and snap-frozen for microbial analysis. Approximately 1 L of filtrated ruminal fluids was stored in individual Thermos bottles and transported to the laboratory for in vitro experiments.

Isolation and purification of DNA

For molecular microbial analyses, DNA was isolated from the ruminal fluid samples that were collected before (control) and after fasting (fasting) from steers using a previously described method [9]. Briefly, genomic DNA was extracted by bead-beating using a Mini Bead-beater (BioSpec Products, Bartlesville, OK, USA) for 4 min at full speed in the presence of 0.7 g of zirconium beads (0.1 mm in diameter), 282 [micro]L of Buffer A (0.2 M NaCl, 0.2 M Tris, and 0.02 M ethylenediaminetetraacetic acid [EDTA]; pH 8), 26.8 [micro]L of Buffer PM (QIAquick 96 PCR Purification Kit, Qiagen, Valencia, CA, USA), 200 [micro]L of 20% sodium dodecyl sulfate, and 550 [micro]L of a phenol-chloroformisoamyl alcohol mixture (25:24:1 by volume, pH 8). After centrifugation (16,000xg for 20 min at 4[degrees]C), the supernatant was thoroughly mixed with 650 pL of Buffer PB (Qiagen, USA), and the DNA sample was purified using the Qiagen PCR Purification Kit according to the manufacturer's protocol.

Denaturing gradient gel electrophoresis analysis

To perform denaturing gradient gel electrophoresis (DGGE) analysis, 16S rRNA gene fragments of the V3 region were amplified using the primers GC-clamp-341f (5'-TCC TAC GGG AGG CAG CAG-5') and 518r (5'-ATT ACC GCC GCT GCT GG-3') as described previously [10,11]. Polymerase chain reaction (PCR) was performed using a TaKaRa Bio instrument (PCR Thermal Cycler, Otsu, Japan) in a final volume of 25 [micro]L with EmeraldAmp (GT PCR Master Mix, TaKaRa Bio, Japan), 1 [micro]L of each primer (GC-clamp 341f and GC-clamp 354r), 2 U of Taq polymerase (Ex Taq, TaKaRa Bio, Japan), and 1 [micro]L of template. After the initial denaturation at 94[degrees]C for 5 min, amplification consisted of 30 cycles of denaturation (at 94[degrees]C for 30 s), annealing (at 55[degrees]C for 30 s), extension (at 72[degrees]C for 30 s), and final extension step (at 72[degrees]C for 7 min). The PCR product was checked using 2% agarose gel electrophoresis and visualized using a Gel Doc System (Bio-Rad, Hercules, CA, USA). PCR products were concentrated and purified using the QIAquick PCR Purification Kit (Qiagen, USA). The DGGE was conducted using the D-Code System (Bio-Rad, USA) with 8% (w/v) polyacrylamide gels containing a 40% to 65% denaturant gradient, 1 mm thick, in 1xTris acetate-EDTA buffer. Equal amounts of purified PCR products were loaded on the gel, and electrophoresis was performed at 25 V for 15 min and then 70 V for 16 h and 30 min at 60[degrees]C. The gel was stained in 250 mL of running buffer containing ethidium bromide (50 pg/mL) for 15 min. The stained gels were photographed under UV light using the Gel Doc XR documentation system (BioRad, USA). The normalization and analysis of gel profiles were conducted using the XLSTAT program (Addinsoft, New York, NY, USA).

Quantitative polymerase chain reaction

Populations of Fibrobacter succinogenes, Ruminococcus albus, Streptococcus bovis, Prevotella ruminicola, Prevotella albensis, Eubacterium ruminantium, Anaerovibrio lipolytica, Ruminococcus flavefaciens, methanogenic archaea, general protozoa, and general fungi were analyzed using a previously described quantitative PCR method [12-16]. Forty nanograms of extracted DNA was mixed with primers for the 16S rDNA region of target bacteria or the ITS region of eukaryotic microbes and amplified using SYBR Premix Ex Taq (TaKaRa Bio, China) and the LightCycler 480 Real-Time PCR System (Roche, Mannheim, Germany). The PCR mixtures were pre-incubated at 95[degrees]C for 5 min, denatured at 95[degrees]C for 10 s, annealed at 58[degrees]C for 10 s, and extended at 70[degrees]C for 10 s. After 45 cycles of amplification, a melting point test was performed. The annealing temperatures for individual primers varied depending on the primer sequences. The PCR amplicon was inserted into the TOP10 competent cell (Invitrogen, Carlsbad, CA, USA). After DNA extraction, serially diluted DNAs were amplified to create a standard curve for the absolute quantification of individual microorganisms.

In vitro batch culture experiment

To investigate the effects of ruminal fluid on fermentation characteristics, two sets of in vitro experiments with the ruminal fluids obtained from steers were conducted. The substrates for the in vitro analysis were similar to the diets offered to the experimental steers, which received a mixture of 40% commercial concentrates and 60% rice straw. The concentrate and rice straw (Tables 1, 2) were oven-dried at 60[degrees]C for 3 days, milled to pass through a 1-mm sieve, and analyzed for chemical composition using the appropriate AOAC [17] and Van Soest methods [18].

Incubation conditions

The medium [19] was dispensed into serum bottles under an aerobic conditions [20]. It was then infused with [O.sub.2]-free C[O.sub.2] gas, and, simultaneously, strained rumen fluid was added as a microbial suspension (5%, v/v) using a syringe. The serum bottles were crimped with butyl rubber stoppers with aluminum seals and then incubated at 39[degrees]C for 0, 2, 4, 8, 12, and 24 h in a shaking water bath at 100 rpm. The experiments were performed in triplicate and conducted separately using the ruminal fluids obtained from each of three control and fasting animal. From each bottle, the gas volume was measured using a pressure detector (model PSGH-28PCCA, DECO Co., Seoul, Korea). Gas samples were collected in syringes with 3-way stopcocks from fermented gas-tight serum bottles, and C[O.sub.2] and C[H.sub.4] concentrations were estimated by gas chromatography (7890B GC, Agilent Technologies, Santa Clara, CA, USA). At the end of each incubation period, the supernatants were collected for pH determination and stored at -20[degrees]C for analyses of N[H.sub.3]-N [21], volatile fatty acids (VFAs) [22], and microbial protein synthesis [23]. Dry matter digestibility was also determined by filtering residues in a filter crucible, drying at 100[degrees]C, and weighing the resulting samples.

Statistical analyses

The rumen microbial fermentation characteristics, including pH, gas production, N[H.sub.3]-N, microbial protein, VFA, acetate: propionate ratio, and C[H.sub.4] at 0, 2, 4, 8, 12, and 24 h, and the quantity of the rumen microbial DNA extracted from the rumen contents were analyzed statistically using the LSMEANS statement of the MIXED procedure in the SAS program package [24]. Statistical differences were determined at p<0.05.

RESULTS

During fasting, steers did not show any abnormal symptoms until feed and water were reintroduced at the end of the study. The DGGE method was used to examine the differences in bacterial community between samples obtained before and after fasting (Figure 1). Although little variation was observed in fasting samples, we statistically compared the DGGE profiles of bacterial communities in control steers with those of each fasting steer. The variation and differences in community composition were analyzed by a clustering analysis of the DGGE gel profiles. Figure 1 shows that the DGGE profiles formed clusters representing each steer. The ruminal fluids of the control and fasted steers within treatments clustered together (steer A: 88.5% and steer B: 88.5%), except for steer C (67.6%), and a variation was observed among steers, which suggests that the rumen bacterial community changes slightly with short-term starvation (i.e., 24 h fasting). However, further analysis of the rumen microbiota showed marginal variation in the number of specific microorganisms (Table 3). The number of total bacteria (estimated as log copies) determined by qPCR was significantly (p<0.05) higher in the rumen contents of the control steers than in those of fasted steers. These differences reflect the differences between control and fasted steers in several specific rumen microorganisms, including Anaerovibrio lipolytica (p<0.05), Eubacterium ruminantium (p<0.05), Prevotella albensis (p<0.05), Prevotella ruminicola (p<0.05), and Ruminobacter amylophilus (p<0.05). However, the numbers of other major microorganisms in the rumen, such as protozoa, methanogenic archaea, and anaerobic fungi, did not differ between control and fasted animals (Table 3). Interestingly, substantial differences were found in the in vitro fermentation patterns when the rumen inoculum originated from control (no fasting) and fasted steers (Table 4). Although dry matter digestibility between control and fasted steers over the incubation period did not differ, total gas production, C[H.sub.4], C[O.sub.2], and VFA were higher (p<0.05) in the ruminal fluids of control animals than in the fluids from fasted animals throughout the incubation period.

DISCUSSION

The rumen microbiota changes according to various factors, including diet, time after feeding, ruminant species, and physiological status of the animal, among others [25]. Our results suggest that when animals are fasted for a short period of time (i.e., 24 h), the rumen bacterial community changes slightly, but the number of total bacteria and specific populations differs between the rumen contents obtained before and after fasting. A previous study has shown that fasting impacts the gut microbiomes of tilapia, toads, geckos, quail, and mice [26]. According to the previous study, fasting induces changes in the microbiome in various host species and gut regions; however, microbial diversity increases with fasting in the colons of fish, toads, and mice. Presumably, this is explained by the limited nutrient supply in response to fasting, including water changes [27]. In addition, nutrient and water limitations may result in alterations in the relative abundance of ruminal microbes. This is interesting because large portions of ruminal microbes are solid-associated bacteria comprising 70% of the total bacteria in the rumen [7]. Therefore, when steers fast, even for just a short period (i.e., 24 h), limited feed particles in the rumen are likely to reduce the number of solid-associated bacteria. Indeed, the qPCR results in our study suggest that Prevotella and Ruminobacter decreased during fasting, whereas major cell wall-degrading bacteria (R. albus, R. flavefaciens, and F. succinogenes) did not change. Presumably, this may be attributed to slower degradation and longer retention of fiber in the rumen; cellulose-degrading bacteria could maintain their niches for longer than other bacteria. According to compartment theory, rumen microbes are classified by their distance from the feed particles or rumen epithelial tissue [7].

The planktonic bacterium Prevotella and weakly particle attached bacterium Ruminobacter are classified as compartment 1 and 2, respectively. The viability of those bacteria are more sensitive to the existence of available substrate than the other compartments which are tightly bind to substrate or rumen epithelial tissue. The decreased amounts of accessible substrates in the rumen caused decrease in the populations of planktonic and weakly particle attached bacteria during short-term fasting.

The objective of the present study was to examine the effect of fasting on rumen microbiota. Many laboratories worldwide use ruminal fluids as the microbial inoculum for in vitro fermentation studies, and differences in the rumen content properties may explain the differences in study outcomes. This concern is particularly important when comprehensive approaches, such as meta-analyses, are used for in vitro gas production and/or digestibility studies. Indeed, a recent meta-analysis of in vitro techniques indicated issues with these techniques for measuring gas and methane production and suggested "greater harmonization of analytical procedure" to improve our understanding of the results [28,29]. A previous study by Johnson [1] indicated the importance of the rumen inoculum from animals offered different forages when examining forage digestion in vitro, and more recently, excellent reviews have highlighted the critical requirements for in vitro studies [3]. According to Payne et al [4], gas production profiles are less variable at 4 or 8 h post-feeding than those either just before or 2 h post-feeding. This is in contrast with the findings of Menke and Steingass [6].

Our results suggest that the use of the ruminal fluid from fasting animals should be interpreted with caution. Although such findings do not preclude the use of the ruminal fluid from a slaughterhouse, additional care may be imperative, especially when comparisons are attempted among in vitro analyses. Extensive variation among animals was observed, and hence the use of single animals for in vitro or even in situ techniques may be suboptimal.

CONFLICT OF INTEREST

We certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

https://doi.org/10.5713/ajas.18.0489

ACKNOWLEDGMENTS

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through the Agri-Bio industry Technology Development Program, funded by the Ministry for Agriculture, Food and Rural Affairs (MAFRA) (118073-03). We are also grateful to the EasyBio Co. Ltd. for their support for this study.

REFERENCES

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[15.] Koike S, Kobayashi Y Development and use of competitive PCR assays for the rumen cellulolytic bacteria: Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens. FEMS Microbiol Lett 2001;204:361-6.

[16.] Shin EC, Choi BR, Lim WJ, et al. Phylogenetic analysis of archaea in three fractions of cow rumen based on the 16S rDNA sequence. Anaerobe 2004;10:313-9.

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Jong Nam Kim (1,2), Jaeyong Song (3), Eun Joong Kim (3), Jongsoo Chang (4), Chang-Hyun Kim (5), Seongwon Seo (1), Moon Baek Chang (6), and Gui-Seck Bae (6) *

* Corresponding Author: Gui-Seck Bae Tel: +82-31-670-3029, Fax: +82-31-675-7481, E-mail: gsbae@cau.ac.kr

(1) Deparment of Animal Biosystem Sciences, Chungnam National University, Daejeon 34134, Korea

(2) Department of Food Science & Nutrition, Dongseo University, Busan 47011, Korea

(3) Department of Animal Science, Kyungpook National University, Sangju 37224, Korea

(4) Department of Agricultural Science, Korea National Open University, Seoul 03087, Korea

(5) Department of Animal Life and Environmental Science, Hankyung National University, Anseong 17579, Korea

(6) Department of Animal Science and Technology, Chung-Ang University, Anseong 17546, Korea

ORCID

Jong Nam Kim https://orcid.org/0000-0002-8034-7156

Jaeyong Song https://orcid.org/0000-0002-8613-5605

Eun Joong Kim https://orcid.org/0000-0002-5962-6994

Jongsoo Chang https://orcid.org/0000-0003-4118-120X

Chang-Hyun Kim https://orcid.org/0000-0001-6325-9755

Seongwon Seo https://orcid.org/0000-0002-4131-0545

Moon Baek Chang https://orcid.org/0000-0001-7872-5421

Gui-Seck Bae https://orcid.org/0000-0003-4006-1871

Submitted Jul 1, 2018; Revised Aug 11, 2018; Accepted Sept 10, 2018

Caption: Figure 1. Effects of fasting on rumen microbiota based on the banding profile of denaturing gradient gel electrophoresis (A, B, and C indicate specific animals).
Table 1. Ingredients of the concentrates (% of dry matter) and
composition (% of dry matter) of concentrate and rice straw

Items                                  Concentrate   Rice straw

Ingredients
  Ground corn                             3.02
  Wheat                                   2.10
  Soybean meal                            2.40
  Rice bran                               1.00
  Tapioca                                 17.8
  Sesame oil meal                         1.40
  Palm kernel meal                        41.08
  DDGS (1)                                22.0
  Molasses                                5.00
  Condensed molasses soluble              1.00
  Salt                                    0.30
  Limestone                               2.00
  CaC[O.sub.3]                             0.7
  Minerals and vitamins mixture (2)        0.7
Chemical composition
  Dry matter                              88.27        87.83
  Crude protein                           14.50         3.25
  Ether extract                           6.77          1.54
  Crude fiber                             12.68        28.40
  Undegradable protein                    7.36           --
  Ash                                     7.49         15.68
  Nitrogen-free extract                   47.10        38.74
  Non-fiber carbohydrate                  19.83         1.66
  Acid detergent fiber                    23.08        44.13
  Neutral detergent fiber                 39.69        65.70
  Total digestible nutrients              71.02        38.29

(1) DDGS, dried distillers grain with solubles (USA).

(2) Minerals and vitamins mixture, vitamin A 28,000 IU; vitamin D3
4,000 IU; vitamin E 80 IU; Mn 80 ppm; Zn 100 ppm; Fe 70 ppm;
Cu 50 ppm; Co 0.5 ppm; I 2.0 ppm; Se 1.0 ppm.

Table 2. Rumen fermentation parameters of experimental animals
before and after fasting

Items                                  Control (1)    Fasting (1)

pH                                       5.88 (B)      6.27 (A)
N[H.sub.3]-N (mg/L)                     14.50 (B)      4.53 (A)
Total volatile fatty acids (mmol/L)     77.50 (B)      39.44 (A)

Items                                   SEM     p-value

pH                                     0.0263   0.0005
N[H.sub.3]-N (mg/L)                    0.5807   0.0003
Total volatile fatty acids (mmol/L)    2.8125   0.0007

SEM, standard error of the mean.

(1) Control, inoculum (2 h after feeding); Fasting,
inoculum (fasting for 24 h).

(A,B) Means in a row with different letters differ
significantly (p < 0.05).

Table 3. The effects of fasting on the rumen microbiota based on
real-time polymerase chain reaction

Microorganisms               Control (1)   Fasting (1)

                                    Log copies/ng

Fibrobacter succinogenes        8.03          8.05
Ruminococcus albus              5.85          5.48
Ruminococcus flavefaciens       3.83          3.72
Anaerovibrio lipolytica       3.32 (A)      3.02 (B)
Eubacterium ruminantium       4.24 (A)      4.05 (B)
Prevotella albensis           6.08 (A)      5.89 (B)
Prevotella ruminicola         6.01 (A)      5.80 (B)
Ruminobacter amylophilus      5.83 (A)      5.20 (B)
Streptococcus bovis             3.66          3.36
Treponema bryantii              6.61          3.57
Total bacteria               11.52 (A)     11.31 (B)
Protozoa                        4.73          4.75
Methanogen                      3.97          4.08
Fungi                           2.28           2.5

Microorganisms                SEM    p-value

Fibrobacter succinogenes     0.045     ns
Ruminococcus albus           0.012     ns
Ruminococcus flavefaciens    0.078     ns
Anaerovibrio lipolytica      0.061    0.026
Eubacterium ruminantium      0.041    0.030
Prevotella albensis          0.031    0.012
Prevotella ruminicola        0.045    0.027
Ruminobacter amylophilus     0.117    0.020
Streptococcus bovis          0.134     ns
Treponema bryantii           0.047     ns
Total bacteria               0.006    0.002
Protozoa                     0.104     ns
Methanogen                   0.082     ns
Fungi                        0.126     ns

SEM, standard error of the mean; ns, not significant.

(1) Control, inoculum (2 h after feeding); Fasting,
inoculum (fasting for 24 h).

(A,B) Means in a row with different letters differ
significantly (p < 0.05).

Table 4. Rumen fermentation characteristics determined using the
rumen inoculum of non-fasted (control) and fasted (fasting) steers
in vitro

                                             Incubation time (h)

Items                                          0            2

pH value
  Control (1)                              7.18 (B)     7.11 (B)
  Fasting (1)                              7.31 (A)     7.20 (A)
  SEM                                        0.016        0.015
  p-value                                    0.0001       0.0004
Gas production (mL)
  Control (1)                                  --       79.0 (A)
  Fasting (1)                                  --       14.4 (B)
  SEM                                          --          9.82
  p-value                                      --         0.0003
Dry matter digestibility (%)
  Control (1)                                 22.2         20.5
  Fasting (1)                                 22.2         20.3
  SEM                                         1.19         2.23
  p-value                                    0.9658       0.9312
N[H.sub.3]-N concentration (mg/100 mL)
  Control (1)                              0.95 (B)     1.41 (B)
  Fasting (1)                              1.52 (A)     2.27 (A)
  SEM                                         0.07         0.27
  p-value                                    0.0001       0.0419
Microbial protein synthesis (mg/100 mL)
  Control (1)                              86.9 (A)         85
  Fasting (1)                              62.0 (B)        83.3
  SEM                                         1.38         1.94
  p-value                                    0.0001       0.5632
Total VFA concentration (mmol)
  Control (1)                              11.18 (A)    13.87 (A)
  Fasting (1)                              5.77 (B)     7.12 (B)
  SEM                                        1.651        0.695
  p-value                                    0.0401       0.0001
Acetate/propionate ratio
  Control (1)                                 3.39      2.48 (B)
  Fasting (1)                                 4.55      4.28 (A)
  SEM                                         0.4          0.07
  p-value                                    0.0579       0.001
C[H.sub.4] production (mL)
  Control (1)                                 0.14      2.36 (A)
  Fasting (1)                                 0.1       0.44 (B)
  SEM                                         0.29         0.3
  p-value                                    0.3172       0.0004
C[O.sub.2] production (mL)
  Control (1)                              37.7 (B)     75.5 (B)
  Fasting (1)                              71.7 (A)     86.8 (A)
  SEM                                         6.79         1.91
  p-value                                    0.0038       0.001

                                             Incubation time (h)

Items                                          4            6

pH value
  Control (1)                                 7.03      7.06 (A)
  Fasting (1)                                 7.03      6.98 (B)
  SEM                                        0.024        0.014
  p-value                                    0.8975       0.0013
Gas production (mL)
  Control (1)                              125.3 (B)    141.2 (B)
  Fasting (1)                              63.2 (A)     108.8 (A)
  SEM                                        12.06         4.9
  p-value                                    0.0022       0.0003
Dry matter digestibility (%)
  Control (1)                                 28.3         28.7
  Fasting (1)                                 28.7         30.3
  SEM                                         1.75         0.82
  p-value                                    0.0819       0.1837
N[H.sub.3]-N concentration (mg/100 mL)
  Control (1)                                 1.58         1.2
  Fasting (1)                                 1.5          1.11
  SEM                                         0.26         0.12
  p-value                                    0.8226       0.6081
Microbial protein synthesis (mg/100 mL)
  Control (1)                              78.7 (B)        75.3
  Fasting (1)                              92.0 (A)         81
  SEM                                         1.63         2.17
  p-value                                    0.0001       0.0797
Total VFA concentration (mmol)
  Control (1)                              18.01 (A)    23.52 (A)
  Fasting (1)                              10.36 (B)    13.69 (B)
  SEM                                        0.746        0.926
  p-value                                    0.0001       0.0001
Acetate/propionate ratio
  Control (1)                              1.77 (B)     1.53 (B)
  Fasting (1)                              3.80 (A)     3.13 (A)
  SEM                                         0.1          0.07
  p-value                                    0.0001       0.0001
C[H.sub.4] production (mL)
  Control (1)                              8.17 (A)     6.99 (A)
  Fasting (1)                              1.69 (B)     3.24 (B)
  SEM                                         1.07         0.41
  p-value                                    0.0006       0.0001
C[O.sub.2] production (mL)
  Control (1)                                 86.5         70.7
  Fasting (1)                                  77          73.9
  SEM                                         7.51         7.72
  p-value                                    0.3722       0.7659

                                              Incubation time (h)

Items                                          8            12

pH value
  Control (1)                                 6.97         6.88
  Fasting (1)                                 6.96         6.89
  SEM                                        0.012        0.009
  p-value                                    0.5215       0.1151
Gas production (mL)
  Control (1)                              161.0 (A)    237.4 (A)
  Fasting (1)                              130.3 (B)    202.5 (B)
  SEM                                         4.86         5.09
  p-value                                    0.0004       0.0002
Dry matter digestibility (%)
  Control (1)                                 36.5         40.1
  Fasting (1)                                  33          41.9
  SEM                                         1.91         1.16
  p-value                                    0.2144       0.2714
N[H.sub.3]-N concentration (mg/100 mL)
  Control (1)                                 1.09         1.55
  Fasting (1)                                 0.93         0.99
  SEM                                         0.13         0.28
  p-value                                    0.4166       0.1804
Microbial protein synthesis (mg/100 mL)
  Control (1)                                 76.3      85.9 (A)
  Fasting (1)                                 77.8      77.7 (B)
  SEM                                         1.7          1.66
  p-value                                    0.5455       0.0032
Total VFA concentration (mmol)
  Control (1)                              28.58 (A)    40.69 (A)
  Fasting (1)                              18.43 (B)    35.52 (B)
  SEM                                        1.081        1.164
  p-value                                    0.0001       0.0063
Acetate/propionate ratio
  Control (1)                              1.46 (B)     1.46 (B)
  Fasting (1)                              2.57 (A)     2.18 (A)
  SEM                                         0.38         0.63
  p-value                                    0.0001       0.0001
C[H.sub.4] production (mL)
  Control (1)                              9.74 (A)     12.12 (A)
  Fasting (1)                              3.43 (B)     4.07 (B)
  SEM                                         0.48         1.15
  p-value                                    0.0001       0.0001
C[O.sub.2] production (mL)
  Control (1)                              85.9 (A)     75.3 (A)
  Fasting (1)                              28.7 (B)     42.8 (B)
  SEM                                         5.03         9.52
  p-value                                    0.0001       0.0336

                                           Incubation time (h)

Items                                              24

pH value
  Control (1)                                     6.87
  Fasting (1)                                     6.85
  SEM                                             0.008
  p-value                                        0.1607
Gas production (mL)
  Control (1)                                  269.6 (A)
  Fasting (1)                                  231.2 (B)
  SEM                                              4.5
  p-value                                        0.0001
Dry matter digestibility (%)
  Control (1)                                     46.3
  Fasting (1)                                     44.2
  SEM                                             1.25
  p-value                                        0.2465
N[H.sub.3]-N concentration (mg/100 mL)
  Control (1)                                   2.85 (A)
  Fasting (1)                                   1.34 (B)
  SEM                                             0.46
  p-value                                        0.0339
Microbial protein synthesis (mg/100 mL)
  Control (1)                                  100.6 (A)
  Fasting (1)                                   90.8 (B)
  SEM                                             2.47
  p-value                                        0.0129
Total VFA concentration (mmol)
  Control (1)                                  50.16 (A)
  Fasting (1)                                  41.97 (B)
  SEM                                             1.049
  p-value                                         0.001
Acetate/propionate ratio
  Control (1)                                   1.40 (B)
  Fasting (1)                                   2.24 (A)
  SEM                                             0.06
  p-value                                        0.0001
C[H.sub.4] production (mL)
  Control (1)                                  15.52 (A)
  Fasting (1)                                   8.30 (B)
  SEM                                             0.63
  p-value                                        0.0001
C[O.sub.2] production (mL)
  Control (1)                                   78.2 (B)
  Fasting (1)                                   84.3 (A)
  SEM                                             1.75
  p-value                                        0.0312

SEM, standard error of the mean; VFA, volatile fatty acids.

(1) Control, inoculum (2 h after feeding); Fasting, inoculum
(fasting for 24 h).

(A,B) Means in a column with different letters differ
significantly (p < 0.05).
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Author:Kim, Jong Nam; Song, Jaeyong; Kim, Eun Joong; Chang, Jongsoo; Kim, Chang-Hyun; Seo, Seongwon; Chang,
Publication:Asian - Australasian Journal of Animal Sciences
Geographic Code:9SOUT
Date:Jun 1, 2019
Words:5138
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