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Effects of Neutral Detergent Soluble Fiber and Sucrose Supplementation on Ruminal Fermentation, Microbial Synthesis, and Populations of Ruminal Cellulolytic Bacteria Using the Rumen Simulation Technique (RUSITEC)
Journal of Integrative Agriculture
August 2013
2013, 12(8): 1471-1480
RESEARCH ARTICLE
Effects of Neutral Detergent Soluble Fiber and Sucrose Supplementation on Ruminal Fermentation, Microbial Synthesis, and Populations of Ruminal Cellulolytic Bacteria Using the Rumen Simulation Technique (RUSITEC) ZHAO Xiang-hui, LIU Chan-juan, LI Chao-yun and YAO Jun-hu College of Animal Science and Technology, Northwest A&F University, Yangling 712100, P.R.China
Abstract We evaluated the effects of neutral detergent soluble fiber (NDSF) and sucrose supplementation on ruminal fermentation, microbial synthesis, and populations of ruminal cellulolytic bacteria using the rumen simulation technique (RUSITEC). The experiment had a 2×2 factorial design with two dosages of sucrose, low (ca. 0.26 g d-1, low-sucrose) and high (ca. 1.01 g d-1, high-sucrose), and two dosages of supplied NDSF, low (1.95 g d-1, low-NDSF) and high (2.70 g d-1, high-NDSF). Interactions between NDSF and sucrose were detected for xylanase activity from solid fraction and apparent disappearance of neutral detergent fiber (NDF) and hemicellulose, with the lowest values observed for high-NDSF and high-sucrose treatment. Supplemental NDSF appeared to increase the molar proportion of acetate and reduce that of butyrate; however, the effects of supplemental sucrose on VFA profiles depended upon NDSF amount. There was a NDSF×sucrose interaction for the production of methane. High-NDSF fermenters had lower ammonia-N production, greater daily N flow of solidassociated microbial pellets and total microorganisms, and greater microbial synthesis efficiency compared with lowNDSF fermenters. Supplementation with NDSF resulted in an increase in 16S rDNA copies of Ruminococcus flavefaciens and a reduction in copies of Ruminococcus albus. Supplementation with sucrose tended to increase the 16S rDNA copies of R. albus from liquid fraction, but did not affect daily total microbial N flow and cellulolytic bacterium populations from solid fraction. These data indicate that the effects of the interaction between NDSF and sugars on ruminal fermentation and fiber digestion should be taken into account in diet formulation. Ruminal fermentation and metabolism of sugars warrant further investigation. Key words: neutral detergent soluble fiber, sucrose, ruminal fermentation, Rusitec
INTRODUCTION Due to their high content of neutral detergent soluble fiber (NDSF; largely pectic substances) and sugars, sugar beet pulp (with or without molasses inclusion) and citrus pulp are widely used in ruminant feeding as a source of energy. Although many studies have determined the effects of the inclusion of sugar beet pulp or
citrus pulp in diets on ruminal fermentation and nutrient metabolism, results remain inconclusive. Taking citrus pulp for example, the digestibility of neutral detergent fiber (NDF) and acid detergent fiber (ADF) increased in certain studies (Bueno et al. 2002) but was unaffected or reduced in others (Brown and Johnson 1991; Ariza et al. 2001). Similarly, inconsistent effects have also been observed on ruminal pH (Pinzon and Wing 1976; Gholizadeh and Naserian 2010), volatile fatty
Received 5 June, 2012 Accepted 22 December, 2012 ZHAO Xiang-hui, Tel: +86-29-87092102, E-mail: [email protected]; Correspondence YAO Jun-hu, Tel: +86-29-87092102, E-mail: [email protected]
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acids (VFA) profiles, and microbial synthesis (Ariza et al. 2001; Barrios-Urdaneta et al. 2003) when dietary cereals were replaced with citrus pulp. The variation in the chemical composition of citrus pulp may be one of the reasons resulting in the discrepancies among studies. Citrus pulp can vary from 25 to 44% NDSF and 12 to 40% sugars on a dry matter basis; however, NDSF and sugars differ in digestion and fermentation characteristics (Hall et al. 2010). Many studies (Broderick et al. 2008; Alamouti et al. 2009) have investigated the effects of dietary NDSF or sugar on ruminal fermentation and feed utilization, but little information is available on their interaction. Huhtanen (1988) observed increased flow of microbial crude protein to the small intestine and higher efficiency of microbial synthesis (EMS) when molasses was supplemented to cattle fed grass silage. However, Hemingway et al. (1986) found that including dried molassed sugar-beet pulp, containing either 200 or 400 g of molasses per kg at 400 g kg-1 of a compound food, did not affect either milk yield or milk components when given to cows receiving grass silage and hay. We speculate the interaction between NDSF and sugars may be a noteworthy factor that influences ruminal fermentation and animal production. Therefore, the objective of the present
study was to evaluate the effects of NDSF, sucrose, and their interaction on ruminal fermentation, microbial synthesis, and populations of ruminal cellulolytic bacteria in Rusitec fermenters.
RESULTS Dietary treatment did not affect pH before feeding, but supplemental sucrose reduced mean pH over 12 h after feeding (P<0.01; Table 1). Numerical or significant interactions between NDSF and sucrose were detected for apparent disappearance of dry matter (DM) (P=0.08), organic matter (OM) (P=0.04), NDF (P=0.01), and hemicellulose (P=0.09), which were reduced by supplemental NDSF only in high-sucrose diets. Apparent disappearance of ADF was not affected by dietary treatment and averaged 16.3% across treatments. Dietary treatment did not affect daily production of total VFA, which averaged 32.1 mmol d-1 (Table 2). Although interactions between NDSF and sucrose were detected for the molar proportions of butyrate (P=0.05), on the whole, high-NDSF diets increased the molar proportion of acetate and reduced that of
Table 1 Effects of experimental treatments on pH, apparent disappearance of diet in the Rusitec fermenters (n=4) Item
Low sucrose Low NDSF
pH before feeding pH1) (0 to 12 h) Apparent disappearance (%) DM OM NDF ADF Hemicellulose 2)
High sucrose
High NDSF
6.87 6.86 a
6.85 6.82 ab
49.2 48.0 21.8 ab 14.2 31.1
50.4 49.3 22.4 ab 17.5 31.8
Low NDSF
High NDSF
6.86 6.80 b
6.79 6.79 b
53.2 52.0 26.9 a 16.8 39.4
49.9 47.8 18.2 b 16.8 27.8
SEM
P NDSF
Sucrose
0.025 0.013
0.16 0.14
0.10 <0.01
NDSF×Sucrose 0.34 0.34
1.10 1.11 1.42 1.23 3.16
0.38 0.22 0.02 0.23 0.12
0.14 0.31 0.74 0.44 0.51
0.08 0.04 0.01 0.22 0.09
Treatment values were determined at 0, 4, 8, and 12 h after feeding. Hemicellulose=NDF-ADF Different letters within a row differ at P=0.05 (Tukey’s test). The same as below.
1) 2)
Table 2 Effects of experimental treatments on daily production of methane and VFA in the Rusitec fermenters (n=4) Item Total VFA (mmol d-1) Individual (mol 100 mol -1) Acetate Propionate Butyrate Acetate:Propionate Methane (mmol d-1)
Low sucrose
High sucrose
Low NDSF
High NDSF
Low NDSF
High NDSF
30.8
31.6
33.6
32.5
52.9 b 35.6 11.5 b 1.49 b 4.4 b
54.9 a 35.1 10.1 b 1.57 a 6.7 a
50.5 c 34.4 15.1 a 1.47 b 5.9 ab
54.4 a 34.8 10.8 b 1.56 a 5.3 ab
SEM
P NDSF
Sucrose
1.05
0.86
0.12
NDSF×Sucrose 0.38
0.39 0.33 0.49 0.020 0.23
<0.01 0.89 <0.01 <0.01 0.08
0.03 0.05 <0.01 0.95 0.61
0.18 0.19 0.05 0.86 <0.01
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Effects of Neutral Detergent Soluble Fiber and Sucrose Supplementation on Ruminal Fermentation, Microbial Synthesis
butyrate compared with low-NDSF diets; however, supplemental sucrose increased the molar proportion of butyrate and reduced that of acetate only under lowNDSF conditions. The ratio between acetate and propionate was higher (P<0.01) for fermenters fed highNDSF diets than those fed low-NDSF diets. A NDSF×sucrose interaction was observed for the production of methane (P<0.01) where supplementation with NDSF only increased the production of methane in low-sucrose diets. Supplementation with NDSF and sucrose both reduced the net production of ammonia-N (P<0.01 and P=0.03, respectively) (Table 3). High-NDSF fermenters had greater daily flow of total NAN (P=0.01), total microbial N (P=0.01), and SAM (P<0.01) and greater efficiency of microbial synthesis (P=0.02), expressed as g microbial N kg-1 OM fermented compared with low-NDSF fermenters. Supplementation with sucrose numerically increased the daily flow of LAM (P=0.12), but did not affect daily total microbial N flow. Fermenters fed high-sucrose diets had greater 16S rDNA copy numbers of R. albus (P=0.01) in liquid fraction than those fed low-sucrose diets (Table 4). HighNDSF diets resulted in an increase in the 16S rDNA copy numbers of R. flavefaciens (P<0.02) and a re-
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duction in copies of R. albus (P<0.01) in both liquid and solid fraction compared with low-NDSF diets. A NDSF×sucrose interaction trend was detected for carboxymethylcellulase activity in fermenter liquid (P=0.07), where supplementation with sucrose increased carboxymethylcellulase activity in low-NDSF diets but did not appear to affect it in high-NDSF diets (Table 5). Xylanase activity in fermenter liquid tended to increase with supplemental sucrose (P=0.06). Supplementation with NDSF increased the carboxymethylcellulase activity from solid fraction (P=0.04). There was a NDSF×sucrose interaction for xylanase from solid phase, with the lowest value observed in high-sucrose and high-NDSF treatment (P=0.04). Supplementation with NDSF or sucrose increased or tended to increase the xylose concentration from solid fraction (P=0.05 and P=0.07, respectively).
DISCUSSION Few data are available on the effects of supplemental NDSF or pectin on ruminal nutrients digestibility and although many studies have investigated the effect of adding sucrose to diets on the digestibility of NDF, re-
Table 3 Effects of experimental treatments on daily production of ammonia-N and NAN, daily N flow of liquid-associated (LAM) and solid-associated microorganisms (SAM), and efficiency of microbial synthesis (EMS) in the Rusitec fermenters (n=4) Item
Low sucrose Low NDSF
80.2 Ammonia-N (mg d-1) 255.6 Total NAN flow (mg d-1) Microbial N flow (mg d-1) Total microorganisms 117.2 LAM 63.4 SAM 53.8 EMS (mg microbial N g-1 OM fermented) 19.2
High sucrose
SEM
P
High NDSF
Low NDSF
High NDSF
a b
52.5 b 285.0 a
67.1 a 270.7 ab
43.7 b 291.0 a
4.09 6.45
<0.01 0.01
0.03 0.14
0.61 0.50
b
136.6 a 61.6 74.9 a 20.6 a
115.8 b 70.9 45.1 c 16.6 b
134.9 a 65.2 69.7 ab 19.6 ab
3.84 3.14 4.32 0.73
0.01 0.28 <0.01 0.02
0.70 0.12 0.15 0.04
0.98 0.57 0.69 0.33
bc ab
NDSF
Sucrose
NDSF×Sucrose
Table 4 Effects of experimental treatments on 16S rDNA gene copy numbers of three predominant ruminal cellulolytic bacteria from liquid fraction and solid fraction in the Rusitec fermenters (n=4) Item
Low sucrose Low NDSF
High NDSF
High sucrose Low NDSF
Liquid fraction, log10 of 16S rDNA gene copy numbers per mL fermenter liquid F. succinogenes 6.5 6.0 6.4 R. albus 4.2 3.8 4.9 R. flavefaciens 4.1 5.0 4.5 Solid fraction, log10 of 16S rDNA gene copy numbers per g undigested feed1) F. succinogenes 8.9 8.8 8.9 R. albus 7.5 6.7 7.4 R. flavefaciens 6.7 b 7.7 a 6.9 ab 1)
High NDSF
SEM
P NDSF
Sucrose
NDSF×Sucrose
6.2 4.3 5.1
0.14 0.13 0.21
0.12 <0.01 0.02
0.51 0.01 0.11
0.21 0.47 0.36
9.3 6.8 7.6 a
0.20 0.17 0.16
0.37 <0.01 <0.01
0.25 0.55 0.18
0.74 0.76 0.29
Dry matter basis.
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Table 5 Effects of experimental treatments on carboxymethylcellulase activity, xylanase activity, and xylose concentration from liquid fraction and solid fraction in the Rusitec fermenters (n=4)1) Item Liquid fraction Carboxymethylcellulase Xylanase Xylose (μg mL-1) Solid fraction Carboxymethylcellulase Xylanase Xylose2)
Low sucrose
High sucrose
Low NDSF
High NDSF
Low NDSF
High NDSF
0.09 b 3.82 5.89
0.17 ab 3.98 6.32
0.25 a 6.65 14.30
0.18 ab 4.74 10.30
0.27 2.36 ab 130.3 b
0.52 2.68 a 187.0 ab
0.28 2.57 a 161.5 b
0.38 1.82 b 346.5 a
P SEM
NDSF
Sucrose
NDSF×Sucrose
0.024 0.700 2.210
0.65 0.25 0.78
0.05 0.06 0.07
0.07 0.45 0.47
0.038 0.123 45.15
0.04 0.55 0.05
0.26 0.12 0.07
0.25 0.04 0.18
Carboxymethylcellulase and xylanase activities are as nanomoles or micromoles of glucose or xylose released from the corresponding substrates by 1 mL of ruminal fluid, for liquid fraction, or 1 g of undigested feed (DM basis), for solid fraction, in 1 min at 39°C and pH 6.5. 2) Microgramme of xylose determined from enzyme solution of 1 g undigested feed (Pan et al. 2003). 1)
sults remain inconclusive. Some studies have found that the digestibility of NDF is not affected by the addition of sucrose in diets (Owens et al. 2008), while others have shown there to be a decrease in digestibility (Khalili and Huhtanen 1991). In the present study, apparent disappearance of DM, OM, and NDF were all affected by the treatments and each had or tended to have a NDSF×sucrose interaction, with the lowest values observed in the high-sucrose and high-NDSF treatment. A reduction in xylanase activity and consequently in hemicellulose digestibility resulting from the high-sucrose and high-NDSF diet contributed to the reduced apparent disappearance of other nutrients. Hemicelluloses can be hydrolyzed to xylose and xylose-oligosaccharides, together with glucose and arabinose, by hemicellulolytic or xylanolytic ruminal b a c t e r i a i n c l u d i n g B u t y r i v i b r i o f i b r i s o l v e n s, F. succinogenes, R. albus, R. flavefaciens, and Prevotella ruminicola (Walker and Hopgood 1961; Cotta and Zeltwanger 1995), but F. succinogenes and R. flavefaciens do not appear to utilize effectively the breakdown products for growth (Coen and Dehority 1970; Odenyo et al. 1991). In the present study, the highest xylose concentration was observed in the highsucrose and high-NDSF treatment, which maybe resulted in depressed xylanase activity. Some researches (Rajaram and Varma 1990; Jommuengbout et al. 2009) have reported that production and activity of xylanase can be inhibited by the high concentration of end products of xylan hydrolysis, such as xylose, xylobiose, and xylotriose. Dehority (1967) demonstrated that soluble oligosaccharides from hemicellulose degradation accumulated in fermentations with the non-utilizing strain. Besides R. albus, B. fibrisolvens,
P. ruminicola, and Selenomonas ruminantium are also able to utilize the oligosaccharide mixture as a growth substrate (Cotta 1993). Therefore, the reduced R. albus with increased NDSF supplementation observed in the present study should not result in the accumulation of soluble oligosaccharides. However, some researchers have found that when mixed sugars were provided, R. albus (Thurston et al. 1994), B. fibrisolvens (Strobel and Dawson 1993), and S. ruminantium (Strobel 1993) preferentially utilized sucrose and glucose over pentoses (xylose and arabinose), which may explain the accumulation of pentoses for high-sucrose treatments in the present study, and especially for the high-sucrose and high-NDSF treatment, in which R. albus copies were reduced by supplemental NDSF and, as a result, activity of xylanase and apparent disappearance of hemicellulose were reduced. Improved xylanase activity in fermenter liquid with increased sucrose supplementation was consistent with correspondingly increased copies of R. albus and R. flavefaciens. Total 16S rDNA copy numbers of three representative fibrolytic bacterial species in both liquid and solid phases did not differ among treatments in the present study (data not shown), which is consistent with previous studies using sugar beet pulp as energy supplements for cattle fed forage diets (Fluharty and Dehority 1995). However, the 16S rDNA copy numbers of R. albus were reduced by supplemental NDSF in the present study, which is unexpected and remains unexplained. The copies of R. flavefaciens increased with increased NDSF, which may be due to the reduced R. albus copies. Odenyo et al. (1994) found that R. albus 8 produced proteinaceous factors that inhibited the growth of R. flavefaciens FD-1. Supple-
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Effects of Neutral Detergent Soluble Fiber and Sucrose Supplementation on Ruminal Fermentation, Microbial Synthesis
mentation with sucrose only tended to increase the copies of R. albus in liquid phase in the present study, which may be due to the preference of this species for sucrose (Thurston et al. 1994). With similar infusion of artificial saliva, production of fermentation acid is a major determinant of ruminal pH. In the present study, variation in the mean pH over 12 h after feeding was numerically consistent with the change in total VFA production. Supplemental sucrose increased the molar proportion of butyrate in low-NDSF diets, which is consistent with previous studies (Chamberlain et al. 1993). Pectin primarily ferments to acetate (Marounek and Dušková 1999). Supplementation with NDSF produced a greater molar proportion of acetate and the acetate:propionate ratio in the present study, which is consistent with results obtained in previous studies (Strobel and Russell 1986). Higher production of methane was observed for the high-NDSF diet than for the low-NDSF diet under low-sucrose conditions, which may be due to increased pectin digestion involving the removal of methoxyl groups to form methanol, with subsequent conversion to methane (Waghorn et al. 2006). Supplementation with sucrose tended to increase methane production in lowNDSF diets, which is in agreement with Hindrichsen et al. (2004), who found that butyrate, a major product from sugar fermentation, was positively correlated with methane production. Under high-NDSF diets, however, supplemental sucrose reduced the production of methane, which may relate to the reduced hemicellulose digestion. Moe and Tyrrell (1979) reported that the amount of digestible hemicellulose was positively correlated with methane production. As expected, supplemental NDSF reduced the daily production of ammonia-N and increased microbial synthesis, particularly increased SAM synthesis. However, supplementation with sucrose only slightly increased the daily flow of LAM and did not affect total microbial N flow in the present study. In contrast, Chamberlain et al. (1993) observed an increase in the daily output of purine derivatives in urine and the calculated amount of microbial N entering the small intestine with sugar supplements in sheep given silage-based diets. Similar results were also reported by Oh et al. (1999). Several previous studies have, however, reported similar observations to the current study. Owens
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et al. (2008) found that sucrose (90 g kg-1 DM) had no effect on microbial N flow when supplementing a grass silage diet. Similarly, Migwi et al. (2011) reported that microbial N production did not increase when 112.5 g of sucrose was added to a urea-treated low quality roughage diet. In addition, no increase in microbial growth was observed in some in vitro (Vallimont et al. 2004) and in vivo (Broderick et al. 2008) studies when starch was replaced by sucrose. The reasons for the lack of improvement in microbial growth after the addition of sucrose to diets have been elucidated by Penner et al. (2009). Considering no monosaccharide transport across ruminal epithelial cells and a similar passage rate in the present study, bacteria maybe convert soluble dietary sugars to glycogen as a short-term storage of energy that can be utilized later. Hall and Weimer (2007) demonstrated that adding 65, 130, or 195 mg of sucrose to 130 mg of isolated NDF resulted in increased glycogen concentration at 0 and 4 h but found that glycogen concentration decreased thereafter. In addition, this speculation is partly supported by the observation that supplementation with sucrose in highNDSF diets did not result in a significant increase in the production of total VFA in the present study. However, ruminal fermentation and metabolism of sugars warrant further investigation.
CONCLUSION In conclusion, a NDSF×sucrose interaction was detected for the apparent disappearance of NDF and a high sucrose and high-NDSF content diet depressed xylanase activity and consequently digestion of hemicellulose. Supplemental NDSF appeared to increase the molar proportion of acetate and reduce that of butyrate; however, the effects of supplemental sucrose on the VFA profile depended upon NDSF amount. Supplementation with NDSF changed the population of ruminal cellulolytic bacteria (i.e., R. albus and R. flavefaciens) and improved microbial synthesis, but few effects on those were observed when sucrose was added to diets. These data would indicate that the effects of interaction between NDSF and sugars on ruminal fermentation and fiber digestion should be taken into account in diet formulation. Ruminal fermentation and metabolism of sugars warrant further investigation.
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MATERIALS AND METHODS Apparatus, animals, and diet This study was carried out using rumen simulation (Rusitec, Sanshin Co. Ltd., Tokyo, Japan) as described by Kajikawa et al. (2003). The fermentation equipment included eight fermenters with an effective volume of 800 mL each. The general incubation period was as described by Kajikawa et al. (2003). The inoculum used in the fermenters was obtained from four ruminally fistulated goats (with 40 kg mean body weight) fed two equal meals at 08:00 and 20:00 daily containing alfalfa hay and concentrate (40:60, DM basis). Rumen content was collected through the ruminal fistula before the morning feeding and strained through two layers of surgical gauze to separate the liquid and solid fractions. Squeezed solid inoculum (70 g wet weight) was enclosed in a nylon bag (14 cm×7 cm with 50 μm pore size). On the first day, 400 mL of liquid inoculum was dispensed to each fermenter under CO2 flux after mixing with an equal volume of McDougal’s buffer (McDougall 1948), and two bags were placed in the fermenter, one with feed and the other with solid inoculum. After 24 h, the bag with the solid inoculum was withdrawn and a new bag with feed was supplied. On subsequent days the bag containing the feed which has been incubated for 2 d was replaced by a new feed bag. Therefore, each fermenter always had two bags, one of which was removed each day allowing feed to
be incubated for 48 h. The experiment had a 2×2 factorial design with two dosages of sucrose (Sigma 84100, Sigma-Aldrich, Shanghai, China), low (ca. 0.26 g d-1, low-sucrose) and high (ca. 1.01 g d -1 , high-sucrose), two dosages of supplied NDSF, low (1.95 g d-1, low-NDSF) and high (2.70 g d-1, high-NDSF). All diets were formulated to contain similar amounts of CP, NDF, and starch (Table 6). The differences among treatments only resulted from amounts of sugar and NDSF. Alfalfa hay and concentrate were ground through 4 and 2 mm sieves, respectively. A continuous infusion of artificial saliva at a rate of approximately 600 mL d-1 was maintained in each fermenter.
Experimental procedure and sampling Experimental treatments were randomly assigned to one of eight fermenters. This experiment was conducted in two independent 15-d incubation periods, with 7 d for stabilization and 8 d for sample collection. On days 8, 9, and 10, the pH of the fluid from each fermenter was determined immediately before exchanging the feed bags, and the following samples were collected. The gas produced was collected in Tedlar bags to determine gas production and concentrations of methane. Liquid effluent was collected in effluent-collection bottles containing a solution of H2SO4 (20%; v/v) to maintain pH values below 2. 1 mL of effluent was preserved by adding 1 mL of deproteinising solution (100 g L-1 metaphosphoric acid and 0.6 g L-1 crotonic acid) to determine VFA. 5 mL of
Table 6 Daily quantity of the diets supplied to the individual fermenters (DM basis) Item Diet ingredient supply (g d -1 ) Alfalfa hay Ground corn Wheat bran Soybean meal Pelleted beet pulp Sucrose Limestone Mineral-vitamin premix1) Nutrients supply (g d-1 ) DM OM CP Starch NDF ADF NDSF2) TESC3) Sum of NFC4)
Low sucrose
High sucrose
Low NDSF
High NDSF
Low NDSF
High NDSF
6.00 2.42 4.02 0.70 0.09 0.28 0.14 0.09
6.00 3.48 0.35 1.32 2.77 0.24 0.00 0.09
6.00 2.42 4.02 0.70 0.09 1.03 0.14 0.09
6.00 3.48 0.35 1.32 2.77 0.99 0.00 0.09
13.74 12.73 2.48 2.55 4.42 2.67 1.95 0.45 4.95
14.25 13.41 2.48 2.55 4.45 3.04 2.70 0.45 5.70
14.49 13.48 2.48 2.55 4.42 2.67 1.95 1.20 5.70
15.00 14.16 2.48 2.55 4.45 3.04 2.70 1.20 6.45
Vitamin-mineral mix contained per kilogram of DM: 450 mg of nicotinic acid, 600 mg of Mn, 950 mg of Zn, 430 mg of Fe, 650 mg of Cu, 30 mg of Se, 45 mg of I, 20 mg of Co, 800 mg of vitamin E, 45 000 IU of vitamin D, and 120 000 IU of vitamin A. 2) NDSF, neutral detergent-soluble fiber. 3) TESC, total 80% ethanol-soluble carbohydrates. 4) Sum of NFC=TESC+Starch+NDSF. 1)
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Effects of Neutral Detergent Soluble Fiber and Sucrose Supplementation on Ruminal Fermentation, Microbial Synthesis
effluent were preserved to determine ammonia-N concentration. The samples were frozen at -40°C until analysis. One feed bag from each vessel was collected, washed once with 100 mL of artificial saliva, washed in the cold rinse cycle (10 min) of a washing machine, dried at 60°C, and stored to determine DM disappearance. The residues were also analysed for OM, NDF, and ADF. On day 11, 4 mL of each fermenter fluid were collected at 0, 4, 8, and 12 h after replacing the feed bag, and the pH was measured immediately. On day 12 and 13, 5 mL of saturated HgCl2 were added to the effluent-collection bottles, which were held in an ice-bath to impede microbial growth. The effluent on day 13 and 14 was collected, mixed and homogenized in a blender. One sample (300 mL) was frozen and lyophilized for determination of NAN and total purines (adenine and guanine) and their metabolites (xanthine and hypoxanthine). Approximately 400 mL of effluent was collected for isolation of liquid-associated microbial pellets (LAM). The contents of the nylon bags removed on day 13 and 14 were collected and mixed to determine the solid-associated microbial pellets (SAM). Approximately 20% of solids content was frozen and lyophilized for determination of DM, NAN, and purines concentration. The residual contents were used to isolate the SAM according to Ranilla and Carro (2003). The bacterial samples from LAM and SAM were lyophilized, ground using a mortar and pestle, and analyzed for N and total purines concentration. On day 15, 4 mL of fermenter fluid as liquid fraction and one feed bag containing undigested feed as solid fraction from each fermenter were collected after replacing the feed bag and were frozen at -80°C for DNA extraction and determination of carboxymethylcellulase and xylanase according to Pan et al. (2003). The enzyme solution obtained from liquid and solid fraction according to Pan et al. (2003) was also used for determination of xylose concentration according to Brückner (1955).
Analytical procedures Dry matter, ash and N were determined according to AOAC methods (AOAC 1990). Neutral detergent fiber and ADF analyses were carried out according to van Soest et al. (1991). Heat-stable α-amylase (Sigma A3306, SigmaAldrich, Shanghai, China) was used for NDF determination, with NDF corrected for CP content but not corrected for ash content. Total 80% ethanol-soluble carbohydrates (TESC) and NDSF were determined using the procedures described by Hall et al. (1999). Total starch content was determined using a modified protocol of the Megazyme Total Starch Assay Kit (Megazyme International Ireland Ltd., Wicklow, Ireland). Ammonia-N in samples was analyzed according to Weatherburn (1967). To determine total and individual VFA, acidified samples were centrifuged at 11 000×g for 10 min, and the supernatant fraction was fil-
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tered through a 0·45 μm filter. The VFA concentrations in the filtered samples were determined by HPLC (model L2000; Hitachi High-Technologies Corporation, Tokyo, Japan) with a reversed-phase Agilent TC-C18 column (4.6 mm×250 mm; 5 μm, Agilent Technologies, Santa Clara, CA) (Akalin et al. 2002). Crotonic acid was used as an internal standard. The concentration of methane was analysed by GC (model 663-30; Hitachi High-Technologies Corporation, Tokyo, Japan) equipped with a flame ionization detector. Total purines in the NAN fraction of the digesta and bacterial pellets were quantified by HPLC (Reynal and Broderick 2009). The volume of total gas produced was measured by the displacement of water as previously described (Soliva and Hess 2007). Carboxymethylcellulase and xylanase activities were determined following the procedures of Pan et al. (2003) using carboxymethylcellulose sodium salt (Sigma C4888, Sigma-Aldrich, Shanghai, China) and beechwood xylan (Sigma X4252, Sigma-Aldrich, Shanghai, China) as substrates, respectively, with a modification that incubation reaction is terminated by adding 3.0 mL of alkaline 3,5dinitrosalicylic acid reagent and heating at 100°C for 5 min. The absorbance is read at 540 nm using a spectrophotometer (U-3900, Hitachi, Japan). Enzymatic activities were expressed as nanomoles or micromoles of glucose or xylose released from the corresponding substrates by 1 mL of ruminal fluid, for liquid fraction, or 1 g of undigested feed (DM basis), for solid fracrion, in 1 min at 39°C and pH 6.5. For microbial determination, total genomic DNA was extracted and purified from fermenter liquid (220 μL) and undigested feed (220 μg) samples using the QIAamp DNA Stool Mini Kit (Qiagen China (Shanghai) Co., Ltd., Shanghai, China). The quality and quantity of these DNA samples were also determined by agarose gel electrophoresis and spectrophotometry. The bacterial species determined were Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens as representatives of fibrolytic (cellulolytic and hemicellulolytic) species. The PCR primers used and DNA fragments in the conservative regions amplified by conventional PCR were as described by Koike and Kobayashi (2001). The PCR products were cloned into the pMD18-T vector according to the procedures of the PCR Cloning Kit (TaKaRa Biotechnology Co., Dalian, China) and transformed into competent Escherichia coli cells. The plasmid DNA carrying the PCR products was extracted from transformed Escherichia coli by the E.Z.N. A. Plasmid Miniprep Kit (Omega Bio-Tek, Inc., GA, USA). The plasmids containing the correct insert were screened out by PCR amplification with respective primer sets and were sequenced by the Shanghai Invitrogen DNA Sequencing Service. The concentration of the plasmid was determined with a spectrometer (NanoDrop 1000; Thermo Fisher Scientific, Inc., Wilmington, Delaware, USA). The copy number of each standard plasmid was calculated using the molecular weight of nucleic acid and the length (base pair)
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of the cloned standard plasmid. Real-time PCR was performed using a Bio-Rad iQ5 Multicolour Real-Time PCR Detection System (Bio-Rad Laboratories Inc., CA, USA), with fluorescence detection of SYBRGreen dye. The PCR conditions were as follows: one cycle of 95°C for 30 s for initial denaturation, and 40 cycles of 95°C for 5 s, 55°C for 30 s for annealing, and 72°C for 30 s for extension. The plasmid DNA was diluted to obtain a 10-fold dilution series and amplified by real-time PCR. Different copy numbers and cycle threshold (Ct) values were used to construct species-specific calibration curves. These curves were used for the calculation of species-specific DNA copy numbers. Assays for all the experimental samples were performed in triplicate. The gene copy number was linearised by log10 before processing for data analysis.
Calculations and statistical analyses The proportion of digesta NAN (liquid or solid) of microbial origin were estimated in each fermenter by dividing the ratio total purines/N of the NAN portion of digesta by the ratio total purines/N in the corresponding microbial pellets (LAB or SAB). Daily microbial N production (mg d-1; LAM or SAM) in each fermenter was calculated by multiplying the proportion of NAN of microbial origin by the amount of NAN in the corresponding digesta (liquid or solid). Total daily microbial production was calculated as the sum of the flows of LAM and SAM. Data were analysed by the PROC MIXED procedure (SAS Inst. Inc., Cary, NC) according to a randomized complete block design (blocking by incubation). The mixed model used for each dependent variable was: Yijk=μ+Pi (i=1, 2)+Nj (j=low NDSF, high NDSF)+Sk (k=low sucrose, high sucrose)+NS jk+εijk, where μ is the common mean; P i, the incubation period (block); N j, the NDSF treatment; S k , the sucrose treatment; NS jk, the interaction between NDSF and sucrose; ε ijk, the residual error. The model included N j, S k, and NS jk as fixed effects and P i as a random effect. Ruminal pH data from four sampling time were analyzed using the PROC MIXED procedure for repeated measures. Sampling time was considered as the repeated factor. The model was: Yijkm=μ+Pi+Nj+Sk+NSjk+Hm+NHjm+SHkm+NSHjkm + ε ijkm . The covariance structures tested were variance components, compound symmetric, unstructured, and autoregressive of order one. The most desirable covariance structure was determined according to the lowest Akaike Information Criterion (AIC) and least squares means for treatments are reported. Significance was declared at P=0.05, and trends were discussed at P=0.10. When a significant effect of treatment was detected, differences among means were tested using Tukey’s multiple comparison test.
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Acknowledgements This research was supported by the National Key Technologies R&D Program of China (2012BAD12B02), the Program of International S&T Cooperation of China (2010DFB34230), and the Scientific & Technological Innovation Project of Shaanxi, China (2011KTCQ02-02).
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dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science, 74, 3583-3597. Thurston B, Dawson K, Strobel H. 1994. Pentose utilization by the ruminal bacterium Ruminococcus albus. Applied and Environmental Microbiology, 60, 1087-1092. Vallimont J, Bargo F, Cassidy T, Luchini N, Broderick G, Varga G. 2004. Effects of replacing dietary starch with sucrose on ruminal fermentation and nitrogen metabolism in continuous culture. Journal of Dairy Science, 87, 4221-4229. Waghorn G, Woodward S, Tavendale M, Clark D. 2006. Inconsistencies in rumen methane production - effects of forage composition and animal genotype. International Congress Series, 1293, 115-118. Walker D, Hopgood M. 1961. The hydrolysis of wheaten hay hemicellulose by a purified enzyme from rumen microorganisms. Australian Journal of Agricultural Research, 12, 651-660. Weatherburn M. 1967. Phenol-hypochlorite reaction for determination of ammonia. Analytical Chemistry, 39, 971-974. (Managing editor ZHANG Juan)
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2013, 12(8): 1471-1480
RESEARCH ARTICLE
Effects of Neutral Detergent Soluble Fiber and Sucrose Supplementation on Ruminal Fermentation, Microbial Synthesis, and Populations of Ruminal Cellulolytic Bacteria Using the Rumen Simulation Technique (RUSITEC) ZHAO Xiang-hui, LIU Chan-juan, LI Chao-yun and YAO Jun-hu College of Animal Science and Technology, Northwest A&F University, Yangling 712100, P.R.China
Abstract We evaluated the effects of neutral detergent soluble fiber (NDSF) and sucrose supplementation on ruminal fermentation, microbial synthesis, and populations of ruminal cellulolytic bacteria using the rumen simulation technique (RUSITEC). The experiment had a 2×2 factorial design with two dosages of sucrose, low (ca. 0.26 g d-1, low-sucrose) and high (ca. 1.01 g d-1, high-sucrose), and two dosages of supplied NDSF, low (1.95 g d-1, low-NDSF) and high (2.70 g d-1, high-NDSF). Interactions between NDSF and sucrose were detected for xylanase activity from solid fraction and apparent disappearance of neutral detergent fiber (NDF) and hemicellulose, with the lowest values observed for high-NDSF and high-sucrose treatment. Supplemental NDSF appeared to increase the molar proportion of acetate and reduce that of butyrate; however, the effects of supplemental sucrose on VFA profiles depended upon NDSF amount. There was a NDSF×sucrose interaction for the production of methane. High-NDSF fermenters had lower ammonia-N production, greater daily N flow of solidassociated microbial pellets and total microorganisms, and greater microbial synthesis efficiency compared with lowNDSF fermenters. Supplementation with NDSF resulted in an increase in 16S rDNA copies of Ruminococcus flavefaciens and a reduction in copies of Ruminococcus albus. Supplementation with sucrose tended to increase the 16S rDNA copies of R. albus from liquid fraction, but did not affect daily total microbial N flow and cellulolytic bacterium populations from solid fraction. These data indicate that the effects of the interaction between NDSF and sugars on ruminal fermentation and fiber digestion should be taken into account in diet formulation. Ruminal fermentation and metabolism of sugars warrant further investigation. Key words: neutral detergent soluble fiber, sucrose, ruminal fermentation, Rusitec
INTRODUCTION Due to their high content of neutral detergent soluble fiber (NDSF; largely pectic substances) and sugars, sugar beet pulp (with or without molasses inclusion) and citrus pulp are widely used in ruminant feeding as a source of energy. Although many studies have determined the effects of the inclusion of sugar beet pulp or
citrus pulp in diets on ruminal fermentation and nutrient metabolism, results remain inconclusive. Taking citrus pulp for example, the digestibility of neutral detergent fiber (NDF) and acid detergent fiber (ADF) increased in certain studies (Bueno et al. 2002) but was unaffected or reduced in others (Brown and Johnson 1991; Ariza et al. 2001). Similarly, inconsistent effects have also been observed on ruminal pH (Pinzon and Wing 1976; Gholizadeh and Naserian 2010), volatile fatty
Received 5 June, 2012 Accepted 22 December, 2012 ZHAO Xiang-hui, Tel: +86-29-87092102, E-mail: [email protected]; Correspondence YAO Jun-hu, Tel: +86-29-87092102, E-mail: [email protected]
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acids (VFA) profiles, and microbial synthesis (Ariza et al. 2001; Barrios-Urdaneta et al. 2003) when dietary cereals were replaced with citrus pulp. The variation in the chemical composition of citrus pulp may be one of the reasons resulting in the discrepancies among studies. Citrus pulp can vary from 25 to 44% NDSF and 12 to 40% sugars on a dry matter basis; however, NDSF and sugars differ in digestion and fermentation characteristics (Hall et al. 2010). Many studies (Broderick et al. 2008; Alamouti et al. 2009) have investigated the effects of dietary NDSF or sugar on ruminal fermentation and feed utilization, but little information is available on their interaction. Huhtanen (1988) observed increased flow of microbial crude protein to the small intestine and higher efficiency of microbial synthesis (EMS) when molasses was supplemented to cattle fed grass silage. However, Hemingway et al. (1986) found that including dried molassed sugar-beet pulp, containing either 200 or 400 g of molasses per kg at 400 g kg-1 of a compound food, did not affect either milk yield or milk components when given to cows receiving grass silage and hay. We speculate the interaction between NDSF and sugars may be a noteworthy factor that influences ruminal fermentation and animal production. Therefore, the objective of the present
study was to evaluate the effects of NDSF, sucrose, and their interaction on ruminal fermentation, microbial synthesis, and populations of ruminal cellulolytic bacteria in Rusitec fermenters.
RESULTS Dietary treatment did not affect pH before feeding, but supplemental sucrose reduced mean pH over 12 h after feeding (P<0.01; Table 1). Numerical or significant interactions between NDSF and sucrose were detected for apparent disappearance of dry matter (DM) (P=0.08), organic matter (OM) (P=0.04), NDF (P=0.01), and hemicellulose (P=0.09), which were reduced by supplemental NDSF only in high-sucrose diets. Apparent disappearance of ADF was not affected by dietary treatment and averaged 16.3% across treatments. Dietary treatment did not affect daily production of total VFA, which averaged 32.1 mmol d-1 (Table 2). Although interactions between NDSF and sucrose were detected for the molar proportions of butyrate (P=0.05), on the whole, high-NDSF diets increased the molar proportion of acetate and reduced that of
Table 1 Effects of experimental treatments on pH, apparent disappearance of diet in the Rusitec fermenters (n=4) Item
Low sucrose Low NDSF
pH before feeding pH1) (0 to 12 h) Apparent disappearance (%) DM OM NDF ADF Hemicellulose 2)
High sucrose
High NDSF
6.87 6.86 a
6.85 6.82 ab
49.2 48.0 21.8 ab 14.2 31.1
50.4 49.3 22.4 ab 17.5 31.8
Low NDSF
High NDSF
6.86 6.80 b
6.79 6.79 b
53.2 52.0 26.9 a 16.8 39.4
49.9 47.8 18.2 b 16.8 27.8
SEM
P NDSF
Sucrose
0.025 0.013
0.16 0.14
0.10 <0.01
NDSF×Sucrose 0.34 0.34
1.10 1.11 1.42 1.23 3.16
0.38 0.22 0.02 0.23 0.12
0.14 0.31 0.74 0.44 0.51
0.08 0.04 0.01 0.22 0.09
Treatment values were determined at 0, 4, 8, and 12 h after feeding. Hemicellulose=NDF-ADF Different letters within a row differ at P=0.05 (Tukey’s test). The same as below.
1) 2)
Table 2 Effects of experimental treatments on daily production of methane and VFA in the Rusitec fermenters (n=4) Item Total VFA (mmol d-1) Individual (mol 100 mol -1) Acetate Propionate Butyrate Acetate:Propionate Methane (mmol d-1)
Low sucrose
High sucrose
Low NDSF
High NDSF
Low NDSF
High NDSF
30.8
31.6
33.6
32.5
52.9 b 35.6 11.5 b 1.49 b 4.4 b
54.9 a 35.1 10.1 b 1.57 a 6.7 a
50.5 c 34.4 15.1 a 1.47 b 5.9 ab
54.4 a 34.8 10.8 b 1.56 a 5.3 ab
SEM
P NDSF
Sucrose
1.05
0.86
0.12
NDSF×Sucrose 0.38
0.39 0.33 0.49 0.020 0.23
<0.01 0.89 <0.01 <0.01 0.08
0.03 0.05 <0.01 0.95 0.61
0.18 0.19 0.05 0.86 <0.01
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Effects of Neutral Detergent Soluble Fiber and Sucrose Supplementation on Ruminal Fermentation, Microbial Synthesis
butyrate compared with low-NDSF diets; however, supplemental sucrose increased the molar proportion of butyrate and reduced that of acetate only under lowNDSF conditions. The ratio between acetate and propionate was higher (P<0.01) for fermenters fed highNDSF diets than those fed low-NDSF diets. A NDSF×sucrose interaction was observed for the production of methane (P<0.01) where supplementation with NDSF only increased the production of methane in low-sucrose diets. Supplementation with NDSF and sucrose both reduced the net production of ammonia-N (P<0.01 and P=0.03, respectively) (Table 3). High-NDSF fermenters had greater daily flow of total NAN (P=0.01), total microbial N (P=0.01), and SAM (P<0.01) and greater efficiency of microbial synthesis (P=0.02), expressed as g microbial N kg-1 OM fermented compared with low-NDSF fermenters. Supplementation with sucrose numerically increased the daily flow of LAM (P=0.12), but did not affect daily total microbial N flow. Fermenters fed high-sucrose diets had greater 16S rDNA copy numbers of R. albus (P=0.01) in liquid fraction than those fed low-sucrose diets (Table 4). HighNDSF diets resulted in an increase in the 16S rDNA copy numbers of R. flavefaciens (P<0.02) and a re-
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duction in copies of R. albus (P<0.01) in both liquid and solid fraction compared with low-NDSF diets. A NDSF×sucrose interaction trend was detected for carboxymethylcellulase activity in fermenter liquid (P=0.07), where supplementation with sucrose increased carboxymethylcellulase activity in low-NDSF diets but did not appear to affect it in high-NDSF diets (Table 5). Xylanase activity in fermenter liquid tended to increase with supplemental sucrose (P=0.06). Supplementation with NDSF increased the carboxymethylcellulase activity from solid fraction (P=0.04). There was a NDSF×sucrose interaction for xylanase from solid phase, with the lowest value observed in high-sucrose and high-NDSF treatment (P=0.04). Supplementation with NDSF or sucrose increased or tended to increase the xylose concentration from solid fraction (P=0.05 and P=0.07, respectively).
DISCUSSION Few data are available on the effects of supplemental NDSF or pectin on ruminal nutrients digestibility and although many studies have investigated the effect of adding sucrose to diets on the digestibility of NDF, re-
Table 3 Effects of experimental treatments on daily production of ammonia-N and NAN, daily N flow of liquid-associated (LAM) and solid-associated microorganisms (SAM), and efficiency of microbial synthesis (EMS) in the Rusitec fermenters (n=4) Item
Low sucrose Low NDSF
80.2 Ammonia-N (mg d-1) 255.6 Total NAN flow (mg d-1) Microbial N flow (mg d-1) Total microorganisms 117.2 LAM 63.4 SAM 53.8 EMS (mg microbial N g-1 OM fermented) 19.2
High sucrose
SEM
P
High NDSF
Low NDSF
High NDSF
a b
52.5 b 285.0 a
67.1 a 270.7 ab
43.7 b 291.0 a
4.09 6.45
<0.01 0.01
0.03 0.14
0.61 0.50
b
136.6 a 61.6 74.9 a 20.6 a
115.8 b 70.9 45.1 c 16.6 b
134.9 a 65.2 69.7 ab 19.6 ab
3.84 3.14 4.32 0.73
0.01 0.28 <0.01 0.02
0.70 0.12 0.15 0.04
0.98 0.57 0.69 0.33
bc ab
NDSF
Sucrose
NDSF×Sucrose
Table 4 Effects of experimental treatments on 16S rDNA gene copy numbers of three predominant ruminal cellulolytic bacteria from liquid fraction and solid fraction in the Rusitec fermenters (n=4) Item
Low sucrose Low NDSF
High NDSF
High sucrose Low NDSF
Liquid fraction, log10 of 16S rDNA gene copy numbers per mL fermenter liquid F. succinogenes 6.5 6.0 6.4 R. albus 4.2 3.8 4.9 R. flavefaciens 4.1 5.0 4.5 Solid fraction, log10 of 16S rDNA gene copy numbers per g undigested feed1) F. succinogenes 8.9 8.8 8.9 R. albus 7.5 6.7 7.4 R. flavefaciens 6.7 b 7.7 a 6.9 ab 1)
High NDSF
SEM
P NDSF
Sucrose
NDSF×Sucrose
6.2 4.3 5.1
0.14 0.13 0.21
0.12 <0.01 0.02
0.51 0.01 0.11
0.21 0.47 0.36
9.3 6.8 7.6 a
0.20 0.17 0.16
0.37 <0.01 <0.01
0.25 0.55 0.18
0.74 0.76 0.29
Dry matter basis.
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Table 5 Effects of experimental treatments on carboxymethylcellulase activity, xylanase activity, and xylose concentration from liquid fraction and solid fraction in the Rusitec fermenters (n=4)1) Item Liquid fraction Carboxymethylcellulase Xylanase Xylose (μg mL-1) Solid fraction Carboxymethylcellulase Xylanase Xylose2)
Low sucrose
High sucrose
Low NDSF
High NDSF
Low NDSF
High NDSF
0.09 b 3.82 5.89
0.17 ab 3.98 6.32
0.25 a 6.65 14.30
0.18 ab 4.74 10.30
0.27 2.36 ab 130.3 b
0.52 2.68 a 187.0 ab
0.28 2.57 a 161.5 b
0.38 1.82 b 346.5 a
P SEM
NDSF
Sucrose
NDSF×Sucrose
0.024 0.700 2.210
0.65 0.25 0.78
0.05 0.06 0.07
0.07 0.45 0.47
0.038 0.123 45.15
0.04 0.55 0.05
0.26 0.12 0.07
0.25 0.04 0.18
Carboxymethylcellulase and xylanase activities are as nanomoles or micromoles of glucose or xylose released from the corresponding substrates by 1 mL of ruminal fluid, for liquid fraction, or 1 g of undigested feed (DM basis), for solid fraction, in 1 min at 39°C and pH 6.5. 2) Microgramme of xylose determined from enzyme solution of 1 g undigested feed (Pan et al. 2003). 1)
sults remain inconclusive. Some studies have found that the digestibility of NDF is not affected by the addition of sucrose in diets (Owens et al. 2008), while others have shown there to be a decrease in digestibility (Khalili and Huhtanen 1991). In the present study, apparent disappearance of DM, OM, and NDF were all affected by the treatments and each had or tended to have a NDSF×sucrose interaction, with the lowest values observed in the high-sucrose and high-NDSF treatment. A reduction in xylanase activity and consequently in hemicellulose digestibility resulting from the high-sucrose and high-NDSF diet contributed to the reduced apparent disappearance of other nutrients. Hemicelluloses can be hydrolyzed to xylose and xylose-oligosaccharides, together with glucose and arabinose, by hemicellulolytic or xylanolytic ruminal b a c t e r i a i n c l u d i n g B u t y r i v i b r i o f i b r i s o l v e n s, F. succinogenes, R. albus, R. flavefaciens, and Prevotella ruminicola (Walker and Hopgood 1961; Cotta and Zeltwanger 1995), but F. succinogenes and R. flavefaciens do not appear to utilize effectively the breakdown products for growth (Coen and Dehority 1970; Odenyo et al. 1991). In the present study, the highest xylose concentration was observed in the highsucrose and high-NDSF treatment, which maybe resulted in depressed xylanase activity. Some researches (Rajaram and Varma 1990; Jommuengbout et al. 2009) have reported that production and activity of xylanase can be inhibited by the high concentration of end products of xylan hydrolysis, such as xylose, xylobiose, and xylotriose. Dehority (1967) demonstrated that soluble oligosaccharides from hemicellulose degradation accumulated in fermentations with the non-utilizing strain. Besides R. albus, B. fibrisolvens,
P. ruminicola, and Selenomonas ruminantium are also able to utilize the oligosaccharide mixture as a growth substrate (Cotta 1993). Therefore, the reduced R. albus with increased NDSF supplementation observed in the present study should not result in the accumulation of soluble oligosaccharides. However, some researchers have found that when mixed sugars were provided, R. albus (Thurston et al. 1994), B. fibrisolvens (Strobel and Dawson 1993), and S. ruminantium (Strobel 1993) preferentially utilized sucrose and glucose over pentoses (xylose and arabinose), which may explain the accumulation of pentoses for high-sucrose treatments in the present study, and especially for the high-sucrose and high-NDSF treatment, in which R. albus copies were reduced by supplemental NDSF and, as a result, activity of xylanase and apparent disappearance of hemicellulose were reduced. Improved xylanase activity in fermenter liquid with increased sucrose supplementation was consistent with correspondingly increased copies of R. albus and R. flavefaciens. Total 16S rDNA copy numbers of three representative fibrolytic bacterial species in both liquid and solid phases did not differ among treatments in the present study (data not shown), which is consistent with previous studies using sugar beet pulp as energy supplements for cattle fed forage diets (Fluharty and Dehority 1995). However, the 16S rDNA copy numbers of R. albus were reduced by supplemental NDSF in the present study, which is unexpected and remains unexplained. The copies of R. flavefaciens increased with increased NDSF, which may be due to the reduced R. albus copies. Odenyo et al. (1994) found that R. albus 8 produced proteinaceous factors that inhibited the growth of R. flavefaciens FD-1. Supple-
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Effects of Neutral Detergent Soluble Fiber and Sucrose Supplementation on Ruminal Fermentation, Microbial Synthesis
mentation with sucrose only tended to increase the copies of R. albus in liquid phase in the present study, which may be due to the preference of this species for sucrose (Thurston et al. 1994). With similar infusion of artificial saliva, production of fermentation acid is a major determinant of ruminal pH. In the present study, variation in the mean pH over 12 h after feeding was numerically consistent with the change in total VFA production. Supplemental sucrose increased the molar proportion of butyrate in low-NDSF diets, which is consistent with previous studies (Chamberlain et al. 1993). Pectin primarily ferments to acetate (Marounek and Dušková 1999). Supplementation with NDSF produced a greater molar proportion of acetate and the acetate:propionate ratio in the present study, which is consistent with results obtained in previous studies (Strobel and Russell 1986). Higher production of methane was observed for the high-NDSF diet than for the low-NDSF diet under low-sucrose conditions, which may be due to increased pectin digestion involving the removal of methoxyl groups to form methanol, with subsequent conversion to methane (Waghorn et al. 2006). Supplementation with sucrose tended to increase methane production in lowNDSF diets, which is in agreement with Hindrichsen et al. (2004), who found that butyrate, a major product from sugar fermentation, was positively correlated with methane production. Under high-NDSF diets, however, supplemental sucrose reduced the production of methane, which may relate to the reduced hemicellulose digestion. Moe and Tyrrell (1979) reported that the amount of digestible hemicellulose was positively correlated with methane production. As expected, supplemental NDSF reduced the daily production of ammonia-N and increased microbial synthesis, particularly increased SAM synthesis. However, supplementation with sucrose only slightly increased the daily flow of LAM and did not affect total microbial N flow in the present study. In contrast, Chamberlain et al. (1993) observed an increase in the daily output of purine derivatives in urine and the calculated amount of microbial N entering the small intestine with sugar supplements in sheep given silage-based diets. Similar results were also reported by Oh et al. (1999). Several previous studies have, however, reported similar observations to the current study. Owens
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et al. (2008) found that sucrose (90 g kg-1 DM) had no effect on microbial N flow when supplementing a grass silage diet. Similarly, Migwi et al. (2011) reported that microbial N production did not increase when 112.5 g of sucrose was added to a urea-treated low quality roughage diet. In addition, no increase in microbial growth was observed in some in vitro (Vallimont et al. 2004) and in vivo (Broderick et al. 2008) studies when starch was replaced by sucrose. The reasons for the lack of improvement in microbial growth after the addition of sucrose to diets have been elucidated by Penner et al. (2009). Considering no monosaccharide transport across ruminal epithelial cells and a similar passage rate in the present study, bacteria maybe convert soluble dietary sugars to glycogen as a short-term storage of energy that can be utilized later. Hall and Weimer (2007) demonstrated that adding 65, 130, or 195 mg of sucrose to 130 mg of isolated NDF resulted in increased glycogen concentration at 0 and 4 h but found that glycogen concentration decreased thereafter. In addition, this speculation is partly supported by the observation that supplementation with sucrose in highNDSF diets did not result in a significant increase in the production of total VFA in the present study. However, ruminal fermentation and metabolism of sugars warrant further investigation.
CONCLUSION In conclusion, a NDSF×sucrose interaction was detected for the apparent disappearance of NDF and a high sucrose and high-NDSF content diet depressed xylanase activity and consequently digestion of hemicellulose. Supplemental NDSF appeared to increase the molar proportion of acetate and reduce that of butyrate; however, the effects of supplemental sucrose on the VFA profile depended upon NDSF amount. Supplementation with NDSF changed the population of ruminal cellulolytic bacteria (i.e., R. albus and R. flavefaciens) and improved microbial synthesis, but few effects on those were observed when sucrose was added to diets. These data would indicate that the effects of interaction between NDSF and sugars on ruminal fermentation and fiber digestion should be taken into account in diet formulation. Ruminal fermentation and metabolism of sugars warrant further investigation.
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MATERIALS AND METHODS Apparatus, animals, and diet This study was carried out using rumen simulation (Rusitec, Sanshin Co. Ltd., Tokyo, Japan) as described by Kajikawa et al. (2003). The fermentation equipment included eight fermenters with an effective volume of 800 mL each. The general incubation period was as described by Kajikawa et al. (2003). The inoculum used in the fermenters was obtained from four ruminally fistulated goats (with 40 kg mean body weight) fed two equal meals at 08:00 and 20:00 daily containing alfalfa hay and concentrate (40:60, DM basis). Rumen content was collected through the ruminal fistula before the morning feeding and strained through two layers of surgical gauze to separate the liquid and solid fractions. Squeezed solid inoculum (70 g wet weight) was enclosed in a nylon bag (14 cm×7 cm with 50 μm pore size). On the first day, 400 mL of liquid inoculum was dispensed to each fermenter under CO2 flux after mixing with an equal volume of McDougal’s buffer (McDougall 1948), and two bags were placed in the fermenter, one with feed and the other with solid inoculum. After 24 h, the bag with the solid inoculum was withdrawn and a new bag with feed was supplied. On subsequent days the bag containing the feed which has been incubated for 2 d was replaced by a new feed bag. Therefore, each fermenter always had two bags, one of which was removed each day allowing feed to
be incubated for 48 h. The experiment had a 2×2 factorial design with two dosages of sucrose (Sigma 84100, Sigma-Aldrich, Shanghai, China), low (ca. 0.26 g d-1, low-sucrose) and high (ca. 1.01 g d -1 , high-sucrose), two dosages of supplied NDSF, low (1.95 g d-1, low-NDSF) and high (2.70 g d-1, high-NDSF). All diets were formulated to contain similar amounts of CP, NDF, and starch (Table 6). The differences among treatments only resulted from amounts of sugar and NDSF. Alfalfa hay and concentrate were ground through 4 and 2 mm sieves, respectively. A continuous infusion of artificial saliva at a rate of approximately 600 mL d-1 was maintained in each fermenter.
Experimental procedure and sampling Experimental treatments were randomly assigned to one of eight fermenters. This experiment was conducted in two independent 15-d incubation periods, with 7 d for stabilization and 8 d for sample collection. On days 8, 9, and 10, the pH of the fluid from each fermenter was determined immediately before exchanging the feed bags, and the following samples were collected. The gas produced was collected in Tedlar bags to determine gas production and concentrations of methane. Liquid effluent was collected in effluent-collection bottles containing a solution of H2SO4 (20%; v/v) to maintain pH values below 2. 1 mL of effluent was preserved by adding 1 mL of deproteinising solution (100 g L-1 metaphosphoric acid and 0.6 g L-1 crotonic acid) to determine VFA. 5 mL of
Table 6 Daily quantity of the diets supplied to the individual fermenters (DM basis) Item Diet ingredient supply (g d -1 ) Alfalfa hay Ground corn Wheat bran Soybean meal Pelleted beet pulp Sucrose Limestone Mineral-vitamin premix1) Nutrients supply (g d-1 ) DM OM CP Starch NDF ADF NDSF2) TESC3) Sum of NFC4)
Low sucrose
High sucrose
Low NDSF
High NDSF
Low NDSF
High NDSF
6.00 2.42 4.02 0.70 0.09 0.28 0.14 0.09
6.00 3.48 0.35 1.32 2.77 0.24 0.00 0.09
6.00 2.42 4.02 0.70 0.09 1.03 0.14 0.09
6.00 3.48 0.35 1.32 2.77 0.99 0.00 0.09
13.74 12.73 2.48 2.55 4.42 2.67 1.95 0.45 4.95
14.25 13.41 2.48 2.55 4.45 3.04 2.70 0.45 5.70
14.49 13.48 2.48 2.55 4.42 2.67 1.95 1.20 5.70
15.00 14.16 2.48 2.55 4.45 3.04 2.70 1.20 6.45
Vitamin-mineral mix contained per kilogram of DM: 450 mg of nicotinic acid, 600 mg of Mn, 950 mg of Zn, 430 mg of Fe, 650 mg of Cu, 30 mg of Se, 45 mg of I, 20 mg of Co, 800 mg of vitamin E, 45 000 IU of vitamin D, and 120 000 IU of vitamin A. 2) NDSF, neutral detergent-soluble fiber. 3) TESC, total 80% ethanol-soluble carbohydrates. 4) Sum of NFC=TESC+Starch+NDSF. 1)
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Effects of Neutral Detergent Soluble Fiber and Sucrose Supplementation on Ruminal Fermentation, Microbial Synthesis
effluent were preserved to determine ammonia-N concentration. The samples were frozen at -40°C until analysis. One feed bag from each vessel was collected, washed once with 100 mL of artificial saliva, washed in the cold rinse cycle (10 min) of a washing machine, dried at 60°C, and stored to determine DM disappearance. The residues were also analysed for OM, NDF, and ADF. On day 11, 4 mL of each fermenter fluid were collected at 0, 4, 8, and 12 h after replacing the feed bag, and the pH was measured immediately. On day 12 and 13, 5 mL of saturated HgCl2 were added to the effluent-collection bottles, which were held in an ice-bath to impede microbial growth. The effluent on day 13 and 14 was collected, mixed and homogenized in a blender. One sample (300 mL) was frozen and lyophilized for determination of NAN and total purines (adenine and guanine) and their metabolites (xanthine and hypoxanthine). Approximately 400 mL of effluent was collected for isolation of liquid-associated microbial pellets (LAM). The contents of the nylon bags removed on day 13 and 14 were collected and mixed to determine the solid-associated microbial pellets (SAM). Approximately 20% of solids content was frozen and lyophilized for determination of DM, NAN, and purines concentration. The residual contents were used to isolate the SAM according to Ranilla and Carro (2003). The bacterial samples from LAM and SAM were lyophilized, ground using a mortar and pestle, and analyzed for N and total purines concentration. On day 15, 4 mL of fermenter fluid as liquid fraction and one feed bag containing undigested feed as solid fraction from each fermenter were collected after replacing the feed bag and were frozen at -80°C for DNA extraction and determination of carboxymethylcellulase and xylanase according to Pan et al. (2003). The enzyme solution obtained from liquid and solid fraction according to Pan et al. (2003) was also used for determination of xylose concentration according to Brückner (1955).
Analytical procedures Dry matter, ash and N were determined according to AOAC methods (AOAC 1990). Neutral detergent fiber and ADF analyses were carried out according to van Soest et al. (1991). Heat-stable α-amylase (Sigma A3306, SigmaAldrich, Shanghai, China) was used for NDF determination, with NDF corrected for CP content but not corrected for ash content. Total 80% ethanol-soluble carbohydrates (TESC) and NDSF were determined using the procedures described by Hall et al. (1999). Total starch content was determined using a modified protocol of the Megazyme Total Starch Assay Kit (Megazyme International Ireland Ltd., Wicklow, Ireland). Ammonia-N in samples was analyzed according to Weatherburn (1967). To determine total and individual VFA, acidified samples were centrifuged at 11 000×g for 10 min, and the supernatant fraction was fil-
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tered through a 0·45 μm filter. The VFA concentrations in the filtered samples were determined by HPLC (model L2000; Hitachi High-Technologies Corporation, Tokyo, Japan) with a reversed-phase Agilent TC-C18 column (4.6 mm×250 mm; 5 μm, Agilent Technologies, Santa Clara, CA) (Akalin et al. 2002). Crotonic acid was used as an internal standard. The concentration of methane was analysed by GC (model 663-30; Hitachi High-Technologies Corporation, Tokyo, Japan) equipped with a flame ionization detector. Total purines in the NAN fraction of the digesta and bacterial pellets were quantified by HPLC (Reynal and Broderick 2009). The volume of total gas produced was measured by the displacement of water as previously described (Soliva and Hess 2007). Carboxymethylcellulase and xylanase activities were determined following the procedures of Pan et al. (2003) using carboxymethylcellulose sodium salt (Sigma C4888, Sigma-Aldrich, Shanghai, China) and beechwood xylan (Sigma X4252, Sigma-Aldrich, Shanghai, China) as substrates, respectively, with a modification that incubation reaction is terminated by adding 3.0 mL of alkaline 3,5dinitrosalicylic acid reagent and heating at 100°C for 5 min. The absorbance is read at 540 nm using a spectrophotometer (U-3900, Hitachi, Japan). Enzymatic activities were expressed as nanomoles or micromoles of glucose or xylose released from the corresponding substrates by 1 mL of ruminal fluid, for liquid fraction, or 1 g of undigested feed (DM basis), for solid fracrion, in 1 min at 39°C and pH 6.5. For microbial determination, total genomic DNA was extracted and purified from fermenter liquid (220 μL) and undigested feed (220 μg) samples using the QIAamp DNA Stool Mini Kit (Qiagen China (Shanghai) Co., Ltd., Shanghai, China). The quality and quantity of these DNA samples were also determined by agarose gel electrophoresis and spectrophotometry. The bacterial species determined were Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens as representatives of fibrolytic (cellulolytic and hemicellulolytic) species. The PCR primers used and DNA fragments in the conservative regions amplified by conventional PCR were as described by Koike and Kobayashi (2001). The PCR products were cloned into the pMD18-T vector according to the procedures of the PCR Cloning Kit (TaKaRa Biotechnology Co., Dalian, China) and transformed into competent Escherichia coli cells. The plasmid DNA carrying the PCR products was extracted from transformed Escherichia coli by the E.Z.N. A. Plasmid Miniprep Kit (Omega Bio-Tek, Inc., GA, USA). The plasmids containing the correct insert were screened out by PCR amplification with respective primer sets and were sequenced by the Shanghai Invitrogen DNA Sequencing Service. The concentration of the plasmid was determined with a spectrometer (NanoDrop 1000; Thermo Fisher Scientific, Inc., Wilmington, Delaware, USA). The copy number of each standard plasmid was calculated using the molecular weight of nucleic acid and the length (base pair)
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of the cloned standard plasmid. Real-time PCR was performed using a Bio-Rad iQ5 Multicolour Real-Time PCR Detection System (Bio-Rad Laboratories Inc., CA, USA), with fluorescence detection of SYBRGreen dye. The PCR conditions were as follows: one cycle of 95°C for 30 s for initial denaturation, and 40 cycles of 95°C for 5 s, 55°C for 30 s for annealing, and 72°C for 30 s for extension. The plasmid DNA was diluted to obtain a 10-fold dilution series and amplified by real-time PCR. Different copy numbers and cycle threshold (Ct) values were used to construct species-specific calibration curves. These curves were used for the calculation of species-specific DNA copy numbers. Assays for all the experimental samples were performed in triplicate. The gene copy number was linearised by log10 before processing for data analysis.
Calculations and statistical analyses The proportion of digesta NAN (liquid or solid) of microbial origin were estimated in each fermenter by dividing the ratio total purines/N of the NAN portion of digesta by the ratio total purines/N in the corresponding microbial pellets (LAB or SAB). Daily microbial N production (mg d-1; LAM or SAM) in each fermenter was calculated by multiplying the proportion of NAN of microbial origin by the amount of NAN in the corresponding digesta (liquid or solid). Total daily microbial production was calculated as the sum of the flows of LAM and SAM. Data were analysed by the PROC MIXED procedure (SAS Inst. Inc., Cary, NC) according to a randomized complete block design (blocking by incubation). The mixed model used for each dependent variable was: Yijk=μ+Pi (i=1, 2)+Nj (j=low NDSF, high NDSF)+Sk (k=low sucrose, high sucrose)+NS jk+εijk, where μ is the common mean; P i, the incubation period (block); N j, the NDSF treatment; S k , the sucrose treatment; NS jk, the interaction between NDSF and sucrose; ε ijk, the residual error. The model included N j, S k, and NS jk as fixed effects and P i as a random effect. Ruminal pH data from four sampling time were analyzed using the PROC MIXED procedure for repeated measures. Sampling time was considered as the repeated factor. The model was: Yijkm=μ+Pi+Nj+Sk+NSjk+Hm+NHjm+SHkm+NSHjkm + ε ijkm . The covariance structures tested were variance components, compound symmetric, unstructured, and autoregressive of order one. The most desirable covariance structure was determined according to the lowest Akaike Information Criterion (AIC) and least squares means for treatments are reported. Significance was declared at P=0.05, and trends were discussed at P=0.10. When a significant effect of treatment was detected, differences among means were tested using Tukey’s multiple comparison test.
ZHAO Xiang-hui et al.
Acknowledgements This research was supported by the National Key Technologies R&D Program of China (2012BAD12B02), the Program of International S&T Cooperation of China (2010DFB34230), and the Scientific & Technological Innovation Project of Shaanxi, China (2011KTCQ02-02).
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