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ANIMAL NUTRITION |
* Key Laboratory of Subtropical Agro-ecological Engineering, Institute of Subtropical Agriculture, The Chinese Academy of Sciences, Changsha, Hunan 410125, P. R. China;
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Babcock University, P.M.B. 21244, Ikeja, Lagos, Nigeria;
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Hunan Agricultural University, Changsha, Hunan 410128, P. R. China;
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Hunan Yahua Dairy Industry Co. Ltd., Changsha, Hunan 410005, P. R. China; and
# New Hope Group, Chengdu, Sichuan 610041, P. R. China
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Abstract |
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 Top Abstract INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Key Words: goat • in vitro gas production • rumen fluid • in vitro digestibility
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INTRODUCTION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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; Tan et al., 1995). During recent years, yeast culture and fibrolytic enzymes have been used to improve the nutritive value and utilization efficiency of low-quality roughages. Fibrolytic enzymes and yeast culture supplementation in ruminant diets can increase DMI, production performance, cellulose degradation, and nutrient digestibility (Kung et al., 1997, 2002; Yang et al., 2000; Lesmeister et al., 2004). Addition of cellulolytic enzymes to forage at the time of ensiling can aid in the digestion of cell wall polysaccharides, resulting in greater digestibility and increased intake (Henderson et al., 1982; Nakashima and Ørskov, 1989; Sheperd and Kung, 1996; Colombatto et al., 2003). When an exogenous fibrolytic enzyme was applied to the concentrate portion of diet or sprayed onto the total mixed ration before feeding, the nutrient digestibility in the total digestive tract was increased for early lactating cows (Yang et al., 2000). Similarly, using the exogenous fibrolytic enzyme in high grain diets or in grass forages improved fiber digestion and grain utilization both in situ and in vivo (Feng et al., 1996; Krause et al., 1998). Yeast culture (Saccharomyces cerevisiae) has been extensively used as a dietary supplement in ruminants. The benefits associated with S. cerevisiae include increased DM and NDF digestion (Carro et al., 1992; Plata et al., 1994), increased initial rate of fiber digestion (Williams et al., 1991), improved in situ CP and NDF degradation and microbial efficiency (Olson et al., 1994a,b), and increased DMI and milk production (Williams et al., 1991; Piva et al., 1993; Kung et al., 1997). In vitro studies have also shown that yeast culture favorably altered the mixed ruminal microorganism fermentation and stimulated cellulose digestion by pure cultures of predominant ruminal bacteria (Martin et al., 1989; Callaway and Martin, 1997). There is little information on the additive and interactive effects of fibrolytic enzymes preparation and yeast culture on low-quality roughages. The current experiment was designed to investigate the effect of direct addition of fibrolytic enzymes preparation and yeast culture at different levels on in vitro fermentation characteristics of rice straw, wheat straw, maize stover, and ensiled maize stover using an in vitro gas production technique.
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MATERIALS AND METHODS |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Straw Preparation and Analysis
The experiment was conducted at the experimental farm of the Institute of Subtropical Agriculture in southern China (28°12'N and 113°5'E; altitude 38 m). After wheat (Triticum aestivum var. Taishan 9818) and rice (Oryza sativa var. Xiang 125S/BAR-1) grains were harvested on May 21 and July 25, 2005, respectively, 2 kg each of mature wheat and rice straw were randomly collected and chopped to a length of 1 cm. Mature maize (Zea mays var. Keyu 2) stover (6 kg) was harvested on July 27, 2005, and chopped to a length of 2 cm. Four kilograms of the harvested straw or stover were immediately placed in a plastic bag and then tightly sealed. The plastic bag was opened after 30 d of ensiling. The fresh, mature maize stover silage was analyzed, and DM content was 44.5%, pH was 4.18, and the lactic acid and acetic acid contents were 49 and 17 g/kg of DM, respectively. After straw collection, approximately 500 g of each straw was oven-dried at 60°C and ground through a 1-mm screen (DF-2, Changsha Instrument Factory, Changsha, China) for chemical analysis. Fresh maize stover silage (500 g) was oven-dried at 60°C and ground through a 1-mm screen for chemical analysis. All samples were analyzed for DM, OM, N, NDF, and ADF. The DM content was determined in triplicate by oven-drying at 105°C for 24 h. The OM was determined by ashing at 550°C for 4 h. Total N content was determined according to the Kjeldahl method (AOAC, 1995; method 954.01). The contents of NDF and ADF were determined using the method of Van Soest et al. (1991), but NDF was assayed with the addition of a heat stable amylase (A3306, Sigma-Aldrich, St. Louis, MO) without sodium sulfite. Both NDF and ADF are expressed inclusive of residual ash.
Experimental Design
In vitro incubation experiments were conducted to measure in vitro gas production characteristics of cereal straws. The IVDMD and in vitro OM disappearance (IVOMD) of the 4 types of cereal straw were determined. The concentrated enzyme mixture (Yingheng Biotech Ltd, Guangdong Province, China) contained cellulase and xylanase activities. Enzyme activities were cellulase at 13,000 units of hydroxyethyl cellulase and xylanase at 2,100 IU per gram. (Enzyme activity units were expressed as micromoles of reducing sugars per milligram of enzyme product per minute.) The yeast culture was supplied by Diamond V Mills, Inc. (Cedar Rapids, IA). Supplements were administered in a 4 x 4 factorial arrangement of treatments. Supplements were designed to contain 1 of 4 levels of yeast culture (0, 2.5, 5.0, or 7.5 g/kg of DM of fermented straw) and 1 of 4 levels of fibrolytic enzymes (0, 2.5, 5.0, or 7.5 g/kg of DM of fermented straw) for each straw type. Yeast was mixed with the straw before the commencement of the experiment, whereas fibrolytic enzymes were directly administered in the in vitro incubation solution.
Gas Production, IVDMD, and IVOMD Measurement
In vitro fermentation was carried out in graduated glass syringes (100 mL capacity) following the method described by Lu and Xie (1990). Rumen fluid was collected from 3 ruminally fistulated Chinese Liuyang Black goats (a local breed; BW 14.2 ± 0.5 kg) fed a total mixed ration consisting of maize stover and concentrates (1:1, wt/wt) before the morning feeding. Two samples of the mixed diet were taken for DM determination, and subsamples were composited for chemical analysis (Table 1). Goats were offered equal amounts (up to 5% refusal) of the diet at 0800 and 1600 h daily. An equal volume of rumen fluid obtained from each of the 3 goats was mixed and was strained through 4 layers of cheesecloth into a prewarmed Erlenmeyer flask and taken to the laboratory. All laboratory handling of rumen fluid was carried out under continuous flow of CO2.
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Statistical Analyses
To describe the dynamics of in vitro gas production over time, the following Gompertz function was chosen (Schofield et al., 1994):
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where GP is the cumulative gas production (mL) at time point t, A is the theoretical maximum of gas production (mL), B is the maximum rate of gas production (mL/h), LAG (h) is the lag time, t is the incubation time (h), and e is the Euler constant (base of the natural logarithm). The A, B, and LAG were calculated using the NLREG (Version 5.0) software (Sherrod, 2000).
Data were subjected to statistical analysis using a completely randomized design and the GLM procedure (SAS Inst. Inc., Cary, NC). Parameters analyzed included GP, A, B, LAG, IVDMD, and IVOMD. Two-way ANOVA was used to test the main and interactive effects of fibrolytic enzymes and yeast culture addition on in vitro gas production parameters, IVDMD, and IVOMD. Orthogonal polynomial contrast was used to examine their responses (linear, quadratic, and cubic) to increasing the fibrolytic enzymes and yeast culture supplement.
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RESULTS AND DISCUSSION |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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Chemical composition of rice straw, wheat straw, maize stover, and ensiled maize stover are presented in Table 2. Nitrogen and ash contents in rice straw were numerically greater than in wheat straw and maize stover. Wheat straw and maize stover had greater NDF and ADF content than rice straw. Chemical compositions of these straws were similar to results reported previously (Tan et al., 1995, 1996; Tolera and Sundstøl, 1999). Ensiled mature maize stover had greater N content and lower NDF, ADF content compared with the mature maize stover. This is consistent with previous observations where ensiling decreased DM, NDF, and ADF content, and increased slightly N content for whole maize stover (Filya, 2004).
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In vitro gas production parameters of maize stover, maize stover silage, rice straw, and wheat straw are given in Tables 3 and 4. For maize stover, GP (linear and quadratic, P < 0.01) increased in response to yeast supplementation, A tended to linearly increase (P = 0.06) as yeast supplementation increased, and the greatest value of B (1.75 mL/h) was observed when yeast was supplemented at the level of 5.0 g/kg (quadratic and cubic, P 
0.01), whereas LAG (quadratic, P< 0.01; cubic, P = 0.03) decreased in response to yeast supplementation. General increases in GP (linear, quadratic, and cubic, P < 0.01) and A (linear and cubic, P< 0.01) were noted as yeast supplementation increased for maize stover silage, B reached the greatest value (1.75 mL/h) at yeast supplementation level of 5.0 g/kg (quadratic and cubic, P < 0.01), and LAG tended to linearly decrease (P = 0.09) as yeast supplementation increased. For rice straw, increasing yeast level increased GP (linear and quadratic, P < 0.01) and A (linear and quadratic, P 0.02), but decreased the lag time (linear and quadratic, P < 0.01), and a greater value for B (2.03 mL/h) was observed for yeast supplementation levels of 2.5 and 5.0 g/kg than for the other 2 treatments (linear and quadratic, P < 0.01). For wheat straw, GP and A increased (linear, quadratic, and cubic, P 0.01), but LAG decreased (quadratic and cubic, P < 0.01) as yeast supplementation level increased, and there were no difference (P 
0.28) in B among the 4 treatments.
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0.15). For rice straw, increasing fibrolytic enzymes level increased GP (linear, P < 0.01) and B values (linear, quadratic, and cubic, P 0.04), and LAG increased (linear and cubic, P 0.03) in response to fibrolytic enzymes supplementation; the greatest values of GP (35.6 mL), B (2.07 mL/h), and LAG (0.6 h) were observed when fibrolytic enzymes were supplemented at 7.5, 5.0, and 2.5 g/kg, respectively, and the lowest A value (38.2 mL) was observed at the fibrolytic enzymes supplementation level of 5.0 g/kg (cubic, P < 0.01). For wheat straw, GP (linear and quadratic, P < 0.01), A (linear, quadratic, and cubic, P 0.03), B (linear and quadratic, P < 0.01), and LAG (linear, quadratic, and cubic, P < 0.01) increased in response to the addition of fibrolytic enzymes; the greatest values of GP (30.8 mL), A (42.1 mL), B (1.53 mL/h), and LAG (0.4 h) were observed at the 7.5, 2.5, 5.0, and 2.5 g/kg enzyme supplementation levels, respectively.
Results in the current study show that the rate of gas production can be increased for cereal straws by the addition of yeast culture and fibrolytic enzymes (Tables 3
and 4). This finding is consistent with the results of previous in situ degradation experiments and in vitro digestion, where the rate of cellulose and NDF degradation increased with the addition of yeast culture (Olson et al., 1994a; Sullivan and Martin, 1999). Previous research indicates that treatment with some yeast cultures could increase the number of total and cellulolytic bacteria in the rumen and, at times, cellulose degradation (Dawson et al., 1990; Newbold et al., 1991, 1995). Findings by Newbold et al. (1991, 1995) further suggested that Aspergillus oryzae fermentation extract and S. cerevisiae culture stimulated the rate rather than the extent of degradation by ruminal microorganisms. Sullivan and Martin (1999) found S. cerevisiae culture filtrate stimulated the initial rate of cellulose degradation. This may explain the increase in the rate of gas production by the addition of yeast in the current study. It has been reported that cellulase and other commercial fibrolytic enzymes can increase the cumulative gas production and rates of in vitro fermentation of grass and maize silage (Wallace et al., 2000). Pre-treatment or addition of fibrolytic enzyme has been shown to increase the rate of in situ DM and NDF degradation (Feng et al., 1996). These previous results agree with the current study. In contrast, Yang et al. (2000) and Kung et al. (2002) reported that the gas production rate was not affected by the fibrolytic enzyme supplementation. Nakashima and Ørskov (1989) observed that the rate constant of in situ DM degradation decreased with increasing cellulase supplementation for ensiled barley straw.
The current study demonstrated that yeast supplementation could decrease the lag time of in vitro gas production (Tables 3 and 4). Accordingly, Olson et al. (1994a) reported that the addition of yeast culture numerically decreased the lag time of in situ NDF degradation. Williams et al. (1991) suggested that the stimulation of cellulose degradation by yeast culture is associated with a decreased lag time, which results in increased initial rates of digestion. However, increased NDF disappearance as a result of supplemental yeast culture has not been reported previously (Martin et al., 1989). In the current study, increasing the level of fibrolytic enzymes increased the lag time of gas production for all test materials except for maize stover silage (Tables 3 and 4). This finding is in contrast to the study of Yang et al. (2000), where fibrolytic enzyme treatment reduced the lag time of gas production when the enzyme was applied to the diet of lactating dairy cows. Unfortunately, few relevant experiments have been conducted to study the effects of direct fibrolytic enzymes addition on in vitro gas production parameters of low-quality roughages. Studies have shown that the effects of using fibrolytic enzyme supplements in ruminant diets are not always consistent (Rode et al., 1999; Yang et al., 1999), presumably due to diet composition, type of enzyme used, level of enzyme provided, enzyme stability, and method of application (Yang et al., 2000).
In Vitro DM and OM Disappearance
Effects of yeast and fibrolytic enzymes supplementation on IVDMD and IVOMD of maize stover, maize stover silage, rice straw, and wheat straw are presented in Tables 5 and 6. When yeast was supplemented at the level of 5.0 g/kg, the greatest values of IVDMD occurred for maize stover (36.3%; cubic, P < 0.01), maize stover silage (44.6%; cubic, P < 0.01), and wheat straw (36.4%; linear, quadratic, and cubic, P < 0.01); but IVDMD of rice straw generally decreased (linear, quadratic, and cubic, P 0.01) as yeast supplementation increased. Yeast supplementation increased IVOMD of maize stover (quadratic and cubic, P < 0.01), maize stover silage (quadratic and cubic, P < 0.01), rice straw (linear, quadratic, and cubic, P < 0.01), and wheat straw (linear, quadratic, and cubic, P < 0.01) only at the addition level of 5.0 g/kg, whereas IVOMD values of cereal straws almost decreased at yeast supplementation levels of 2.5 and 7.5 g/kg when compared with no yeast supplementation.
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0.05), rice straw (linear, quadratic, and cubic, P < 0.01), and wheat straw (linear and quadratic, P 0.02) at the levels of 5.0 and 7.5 g/kg; 2.5 g/kg enzymes supplementation treatment had lower IVOMD values for maize stover and its silage and rice straw than those of the 0 g/kg treatment.
With the exception of IVDMD of rice straw, the current results generally agree with previous experiments (Olson et al., 1994a; Sullivan and Martin, 1999; Tan et al., 2004), in which yeast culture supplementation improved IVDMD values of both alfalfa hay and coastal Bermudagrass hay, and increased IVOMD of grass. This effect could be a result of increased protozoa and cellulolytic populations observed with the addition of yeast culture (Harrison et al., 1988; Dawson et al., 1990; Plata et al., 1994). Previous research also demonstrated that application of fibrolytic enzyme directly before incubation is effective in increasing in vitro or in situ DM and NDF disappearance of cool-season grass (Feng et al., 1996). Forwood et al. (1990) reported that treatment of tall fescue with a cellulase solution increased IVDMD, and electron micrographs showed greater cuticular disruption on cellulase-treated leaf blades. Therefore, it can be proposed that the addition or treatment of fibrolytic enzyme preparation enhances ruminal microbial colonization, and consequently reduces particle size of fiber, and increases DM digestion (Feng et al., 1996).
Interactive Effects of Yeast and Fibrolytic Enzymes on In Vitro Fermentation
This study was also conducted to explore the combination of yeast and fibrolytic enzymes that might improve in vitro gas production fermentation of low-quality cereal straws. There were significant interactions between yeast culture and fibrolytic enzymes on in vitro gas production parameters (GP, A, B, and LAG, see Tables 3 and 4), and IVDMD and IVOMD (Tables 5 and 6) for the 4 types of cereal straws. Few studies have been conducted to examine the interactive effects of direct supplementation of fibrolytic enzymes and yeast culture on in vitro fermentation characteristics of ruminant feedstuffs or diets. In the current study, the interactions between yeast culture and fibrolytic enzymes for in vitro gas production parameters and for in vitro DM and OM disappearances suggest that it is important to identify appropriate supplemental levels of both yeast and fibrolytic enzymes for each straw type. In vitro gas production parameters usually reflect the characteristics of the fermentation process, whereas in vitro DM and OM disappearances can be directly used to elucidate the utilization efficiency of cereal straws. Interactive effects between yeast and fibrolytic enzymes for in vitro DM and OM disappearances suggest that the appropriate supplemental levels of yeast and fibrolytic enzymes were 5.0 and 7.5 g/kg of DM of fermented straws, respectively. When yeast culture and fibrolytic enzymes were supplemented at these levels, the greatest values of IVDMD and IVOMD occurred (Tables 5 and 6) in maize stover (42.3 and 42.5%), maize stover silage (48.6 and 48.8%), rice straw (54.5 and 56.8%), and wheat straw (41.3 and 39.5%).
Additionally, some studies conducted to test the effects of fibrolytic enzyme or yeast culture supplementation have indicated that in vivo digestibility data are consistent with the in vitro disappearance data (Olson et al., 1994a,b; Feng et al., 1996). Further study is needed to investigate the effects of direct supplementation of fibrolytic enzymes and yeast on in situ degradation or in vivo digestibility and performance in ruminants. Moreover, some of the lag time values in this study were negative (Tables 3 and 4). Krishnamoorthy et al. (1991) also observed negative values of lag time for in vitro gas curves of oat, rye, and hay standards incubated for 96 h. They assumed that the negative lag time values are a consequence of rapid gas production in the early stages of fermentation, which may have been caused by the characteristics of the fermentable substrate.
In conclusion, the effects of yeast and fibrolytic enzymes supplementation, and their interactions on in vitro gas production, and in vitro DM and OM disappearance observed in the current study demonstrated that the addition of yeast and fibrolytic enzymes may improve in vitro gas production fermentation of low-quality roughages.
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Footnotes |
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2 Corresponding author: zltan{at}isa.ac.cn
Received for publication July 10, 2007. Accepted for publication January 10, 2008.
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LITERATURE CITED |
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TopAbstractINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONLITERATURE CITED |
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