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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.49 No.6 pp.217-234
DOI : https://doi.org/10.14397/jals.2015.49.6.217

Effects of Buffer Extraction of Protein Feeds on In Vitro Fermentation Characteristics, Degradation and Methane Production by Rumen Microbes

Seong-Ho Choi1, Guang-Lin Jin1, Wei-Ze Qin2, Sun-Sik Chang3, Joon Jeong4, Man-Kang Song1*
1Department of Animal Science, Chungbuk National University, Cheongju 28644, Korea
2Institue of Animal Science, Yanbian academy of agricultural science, Longjing, Jilin, China
3Hanwoo Experiment Station, National Institute of Animal Science, RDA, PyeongChang, 25340, Korea
4Livestock Research Center, NACF, San 54 Shindoo-Ri, Ansung, 17558, Korea

+ Seong-Ho Choi and Guang-Lin Jin were equally contributed as first author

Corresponding author: Man-Kang Song Tel: +82-43-261-2545 FAX: +82-43-273-2240 mksong@cbnu.ac.kr
November 7, 2015 December 3, 2015 December 6, 2015

Abstract

The present in vitro study was conducted to examine the effect of buffer solubility of eight protein feeds (coconut meal, distillers grain, sesame meal, perilla meal, soy source cake, rape seed meal, soybean meal and lupine) on the fermentation characteristics, degradability of dry matter (DM) and crude protein (CP), and methane (CH4) production by rumen microbes. Buffer extraction increased pH (P<0.05 ~ p<0.001) of the culture solution but tended to lower ammonia- N concentration for all protein feeds. Total volatile fatty acids(VFAs) and each VFAs concentrations in all incubation was decreased by buffer extraction (P<0.01 ~ P<0.001). Also, molar proportion of acetate in 1h (P<0.001), 3h (P<0.01) and 12h (P<0.05) incubations and molar proportion of propionate in 1h (P<0.001), 3h (P<0.01), 6h (P<0.05) and 12h (P<0.05) were decreased by buffer extraction. But molar proportion of butyrate in 1h (P<0.001), 3h (P<0.01) and 6h (P<0.05) were increased by buffer extraction. The in vitro effective degradability of dry matter (P<0.001) and CP (P<0.001) was decreased by buffer extraction. The methane production (P<0.01~P<0.001) in all incubation was decreased by buffer extraction. The results from in the current study might be useful for diet formulation to improve the feed efficiency of the ruminant animals without massive loss of major nutrients.


초록


    Chungbuk National Universityhttp://dx.doi.org/10.13039/501100002461

    Introduction

    There has been a considerable improvement in the ruminants feeds due to the application of research results. If ruminant nutrition is to progress the point at which diets are continually tested in virtually infinite combinations, the details of the fermentation must be considered as Russell and Wallace(1997).

    The quality and quantity of ruminal fermentation products are dependent on the types and activities of microorganisms in the rumen since the ruminal microbial ecosystem is very diverse(Hungate, 1966; Bauchop, 1979; Coleman, 1980; Citron et al., 1987). According to Nocek & Russell(1988), the rate of feedstuff degradation in the rumen was a profound effect on fermentation end products and on animal performance. More satisfactory prediction of rumen degradable protein and rumen undegradable protein supplies are necessary to optimize performance while minimizing losses of nitrogen. The Cornell Net Carbohydrate and Protein System(CNCPS) provide quantitative estimates of fermentation end products and materials that escape ruminal degradation(Fox et al., 2004).

    In ruminants, feedstuffs are fermented in the rumen before hindgut digestion, and this fermentation has confounded the prediction of animal performance from dietary ingredients. The CNCPS has led to balance the carbohydrate and protein in the diet and to maximize the utilization of them by both rumen microorganisms and the ruminants. Although the disappearance of the feed in the rumen is ultimately determined by the relative rates of fermentation and passage(Waldo & Smith, 1972), fermentation rate is an inherent property of the feed. The buffer solubility of the feedstuffs for the ruminant animals might be related the above concept. The CNCPS has been applied to feed industry to improve the utilization efficiency of the ruminant’s diets without massive loss of nutrients. To achieve improved feed efficiency measurement of the buffer solubility of the various feedstuffs could be the beginning step, and might be followed by the estimation of fermentation by rumen microorganisms.

    Ruminal methane(CH4) production is a nutritionally negative process that has been implicated in global warming(Duxbury et al., 1993). Blaxter(1962) reported that cereal grain feeding decreases methane production. The rumen methanogen species differ depending on diet, which can be reduced by modifying dietary composition(Hook et al., 2010).

    Therefore, the present in vitro study was conducted to estimate the buffer solubility that have been used for the ruminant’s diet. The in vitro study was also conducted to examine the fermentation characteristics, degradability and CH4 production before and after extraction of protein feeds with buffer.

    Materials and Methods

    1.Feeds and chemical analyses

    Eight protein feeds(coconut meal, distillers grain, sesame meal, perilla meal, soy sauce cake, rape seed meal, soybean meal and lupine, provided by Livestock Research Center, NACF) were selected and were ground through 1mm screen for proximal analyses according to AOAC(1995). Analysis of neutral detergent fiber(NDF) followed the method of Van Soest et al.(1991). Chemical composition of the protein feeds is shown in Table 1.

    2.Buffer extraction of protein

    The soluble crude protein(CP) of the feeds was determined with borate–phosphate buffer(Licitria et al., 1996), in which 12.20g monosodium phosphate (NaH2PO4·H2O), 8.91g sodium tetraborate(Na2B4O7· 10H2O) and 100ml tertiary butyl alcohol were dissolved in 1,000ml of distilled water to make up borate-phosphate buffer (pH6.7 ~ 6.8). Forty milliliters of borate phosphate buffer and 1ml 10% sodiumazide were added to 1g dry ground(1mm) feed samples in a tube and shaken by shaking incubator for overnight to extract soluble protein in feeds, then was filtered through Whatman #54 filter paper and washed with distilled water 3times. The filtered feeds then were dried in an oven at 60°C for 72h, and 0.5g of buffer extracted protein feeds was analyzed for buffer insoluble protein fraction(IP).

    3.In vitro incubation

    The feeds before or after borate phosphate buffer extraction were used in vitro incubation for the measurement of fermentation characteristics, effective degradability(ED) and CH4 production. Rumen contents were obtained 2h after the morning feeding from two ruminally fistulated Holstein cows (655±20kg) fed 8kg of total diets daily(6kg concentrate and 2kg rye grass, as fed basis), twice(09:00 and 18:00h) per day in an equal volume. The rumen contents collected was blended in equal volume in a Warning blender (Fisher 14-509-1) for 20 seconds to detach the bacteria from the feed particles, and was strained through 12 layers of cheese cloth to minimize the feed particles. Gaseous carbon dioxide(CO2) was flushed into the strained rumen fluid for 20 seconds. Solution for the culture was prepared by mixing 40ml strained rumen fluid with 40ml McDougall`s artificial saliva consisting of 2.0g NaCl, 0.5g (NH4)2SO4, 1.0g K2HPO4, 1.0g KH2PO4, 0.265g CaCl2 ·2H2O and 0.209g MgSO4·7H2O per liter under flushing of N2(Mcdougall, 1948).

    Approximately 1g(DM basis)of each feed before extraction or after extraction with buffer on a dry matter(DM) basis was weighed into a small nylon bag (3 x 3cm, with pore size of 45μm) and was placed in the bottle containing the culture solution. All treatments were done with triplicate. One 0.5g iron bead was also put in the nylon bag to make the bag place in the middle of the culture solution. The bottles were then sealed with aluminum caps, connected with 3-way stopcock, and were incubated anaerobically in a shaking incubator(VS- 8480SR, VISON Science, Korea) at 39°C for up to 24h at the speed of 140rpm. Percent disappearances of DM and CP in the feeds from each incubation time(1, 3, 6, 12 and 24h) were measured after incubation. Disappearance rate was fitted to the equation and ED of DM(EDDM) and CP(EDCP) were calculated according to the equation of Ørskov and McDonald (1979). Fractional outflow rate of 0.05/h was applied for ED calculation.

    4.Measurement and analysis

    The pH of culture solution was measured when incubation was initiated(0h) and the incubation of indicated times (1, 3, 6, 12 and 24h) was terminated. The culture content including 0h incubation was freeze dried to determine the degradability of DM and CP. Ammonia-N concentration in the culture solution was determined by the method of Fawcett and Scott(1960) using spectrophotometer(DU-650). The 0.8ml culture solution was mixed with 0.2ml 25% phosphoric acid and 0.2ml pivalic acid solution(2%, w/v) as the internal standard for the volatile fatty acid(VFA) analysis. The VFA concentration in the culture solution was determined by gas chromatograph(GC, HP5890 series II, Hewlett Packard Co.) equipped with FID. Total gas production was measured from the culture bottles through the 3-way stopcock using a 50ml glass syringe connected to a needle. A gas sample was transferred to a 6ml vacuum tube and analyzed for CH4 by GC(YL6100, Younglin Co., Korea) equipped with TCD. The oven temperature for CH4 was 10 0°C, and temperatures of injector and detector were maintained at 150°C and 200°C, respectively. A 30m fused capillary column(HP- PLOT/Q, 19095P-QO4, 0.53mm i.d. U.S.A.) was used. Ultra high purity Helium gas (He) was used at a flow rate of 30ml/min. Peak was identified and quantified using standard CH4 gas. Mixed gas consisting of H2 (10%), CH4 (30%), N2 (20%) and CO2 (40%) was used as a standard gas to estimate the production of CH4 during the degradation of various protein feeds. Equation was drawn based on peak area obtained from injection of standard gas.

    The equation was follows:

    Yarea = aXvolume + b

    Thus, A(CH1/std) = aV(CH/std) + b

    → V(CH4/0.1ml sample) = (A(CH/0.1ml sample) – b) / a

    → VCH4 = (V(CH4/0.1ml sample) × 10) × Vtotal gas

    Where, A(CH4/std) is peak area of CH4 in standard, V(CH4/std) is amount of CH4 injected, A(CH4/0.1ml sample) is peak area per 0.1ml injection of gas produced from incubation, V(CH4/0.1ml sample) is amount of CH4 in 0.1ml gas produced from incubation. VCH4 is total CH4 production and Vtotal gas is total gas produced from incubation.

    5.Statistical analyses

    All the data were tested for significance using a 2-way ANOVA by GLM procedure of SAS(2002). The statistical model was:

    Y ijk = μ + τ i + β j + τ × β ij + ϵ ijk

    Where, Υijk is the dependent variable, μ is the overall mean, τ i is the treatment effect(i= Unext and Ext), βj is the protein feed effect(j= 8 protein feeds), (τ × β)ij is the interaction effect and ε ijk is the error term. The significance among means for all variables by incubation time was compared by S-N-K’s test(Steel & Torrie, 1980). Difference among means with P<0.05 was accepted as representing statistical differences.

    Results

    1.Content and solubility

    Protein feeds containing more than 30%CP (DM basis) were soybean meal (45.11%), sesame meal (43.37%), rapeseed meal (36.09%) and perilla meal (35.84%), and those containing less than 30% CP were soy source cake (28.16%), distillers grain (23.6%) and palm meal (13.55%) among 8 protein feed sources(Table 1). The proportion of buffer soluble protein in total CP of coconut meal, and distillers grain were 18.0% and 13.9%, respectively, and those of sesame meal, perilla meal and soybean meal were ranged 22.6~29.5% while those of soy sauce cake, rapeseed meal and lupine were ranged 30.7 ~ 48.0% (Table 2).

    2.Fermentation characteristics

    The pH of the culture solution was increased (P<0.05~P<0.001) at all incubations by buffer extraction of the protein feeds(Table 3). The difference (P<0.05~P<0.001) in pH of culture solution among feed sources regardless of extraction was found at 12h and 24h incubations (P<0.001). The mean pH of culture solution was different (P<0.001) among feeds at all incubations. Within the feed source, buffer extraction increased the pH of culture solution at all incubation time in distillers grain and perilla meal while buffer extraction did not influence the pH of coconut meal at 12h, sesame meal at 3h, soy sauce cake at 1h, 3h, and 24h, rapeseed meal and soybean meal at 24h, and lupine at 1h, 3h, 12h and 24h incubations(Table 3).

    Ammonia-N concentration in culture solution was not influenced by buffer extraction (Table 4) but irrespective of extraction, difference was found among feeds (P<0.001). The mean ammonia-N concentration in culture solution was different (P<0.001) among feeds at all incubations. Ammonia-N concentration in culture solution was relatively lower in coconut meal, distillers grain, sesame meal and perilla meal than in the other feeds. Within the feed source, buffer extraction reduced the ammonia-N concentration in sesame meal (P<0.01), rapeseed meal (P<0.05) and lupine (P<0.05) at 6h incubation time(Table 4).

    Total VFA concentration in culture solution was decreased (P<0.01~P<0.001) by buffer extraction (Table 5), and irrespective of extraction, difference (P<0.01) was found among feeds at 1h, 3h and 12h incubations. Within the feed source, buffer extraction reduced the total VFA concentration in coconut meal at 3h (P<0.001) and 6h (P<0.015), sesame meal (P<0.029) and perilla meal (P<0.045) at 24h, soy sauce cake (P<0.034), rapeseed meal (P<0.028) and soybean meal (P<0.025) at 6h, and lupine at 3h (P<0.033) and at 24h (P<0.049) (Table 5).

    Proportion of acetate(C2) in culture solution was decreased by buffer extraction of protein feeds at 1h (P<0.001), 3h (P<0.01) and 12h (P<0.05), (Table 6), and irrespective of extraction, difference (P<0.01) in C2 proportion was found among feeds at 6h and 12h incubations. Within the feed source, buffer extraction reduced the C2 proportion in many protein feeds at 1h (P<0.038~P<0.007) except for perilla meal, rapeseed meal and lupine, and reduced it in soy sauce cake (P<0.009), rapeseed meal (P<0.02) and lupine (P<0.026) at 24h incubations (Table 6).

    Proportion of propionate(C3) in culture solution was decreased (P<0.05~P<0.001) by buffer extraction of protein feeds except for 24h incubation time (Table 7), and irrespective of extraction, difference in C3 proportion was found among feeds at 3h (P<0.01), 6h (P<0.001) and 12h (P<0.01) incubations. Mean C3 proportion in culture solution was different among feeds and buffer extraction at 1h (P<0.009), 3h (P<0.0002) 6h (P<0.0001) and 12h (P<0.001) incubations. Within the feed source, buffer extraction reduced the C3 proportion in coconut meal seed meal (P<0.014) and distillers grain (P<0.005) at 12h, sesame meal at 3h (P<0.006), perilla meal at 6h (P<0.03) and soy sauce cake at 1h (P<0.01) incubation time (Table 7).

    Proportion of butyrate (C4) in culture solution was increased (P<0.05~P<0.001) by buffer extraction of protein feeds up to 6h incubations (Table 8), and irrespective of extraction, difference in C4 proportion was found among feeds at 3h (P<0.01), 6h (P<0.001) and 12h (P<0.01) incubations. The mean C4 proportion in culture solution was different (P<0.003~P<0.0001) among feeds and buffer extraction up to 12h incubations. Within the feed source, buffer extraction increased the C4 proportion in distillers grain at 3h (P<0.005), sesame meal at 1h (P<0.005), soy sauce cake at 1h (P<0.025), soybean meal at 1h (P<0.031) and at 3h (P<0.027), and lupine at 24h (P<0.002) incubation time Table 8).

    3.Effective degradability

    Buffer extraction decreased fraction a (P<0.01), c (P<0.001) and EDDM (P<0.001) of protein feeds, and difference was not observed in the mean value of all parameters such as a, b and c and EDDM (Table 9). Within feed, extraction decreased fraction b of rapeseed meal (P<0.127) and EDDM of coconut meal (P<0.043), distillers grain (P<0.044), soy sauce cake (P<0.049), rapeseed meal (P<0.029) (Table 9). Buffer extraction decreased fraction b (P<0.001), c (P<0.001) and EDCP (P<0.001) of protein feeds, but difference was not observed in the mean value of all the protein feeds. Within feed, extraction decreased fraction b of soy sauce cake (P<0.03, Table 10).

    4.Gas production

    Extraction of protein feeds with borate-phosphate buffer decreased CH4 production at all incubations (P<0.01~P<0.001, Table 11). Difference in CH4 production among feeds was found at all incubations (P<0.01~P<0.001, Table 11). Within the feed source, buffer extraction decreased CH4 production from coconut meal (P<0.046~P<0.005), distillers grain (P<0.014~P<0.002), rapeseed meal (P<0.023~P<0.007), soybean meal (P<0.027~P<0.001) and lupine (P<0.048 ~P<0.004) at all incubations, sesame meal (P<0.047~ P<0.015) up to 12h, perilla meal at 1h (P<0.042) and 6h (P<0.031), soy sauce cake (P<0.048~P<0.012) except for 3h incubations (Table 11).

    The regression (y) between DM degradation and CH4 production in protein feeds before extraction was 0.0006x2 + 0.036x - 2.294 and regression coefficient (R2) was 0.856 (Fig. 1), and the regression between DM degradation and CH4 production after extraction was 0.0001x2 + 0.01x - 0.273 and regression coefficient (R2) was 0.863 (Fig. 2). Thus, the CH4 production (ml) per DM degradation (mg) of protein feeds in vitro after extraction is lower than before extraction.

    Also, the regression between CP degradation and CH4 production in protein feeds before extraction was - 0.001x2 + 0.136x - 1.985 and regression coefficient (R2) was 0.546 (Fig. 3), and the regression between CP degradation and CH4 production after extraction was 0.0002x2 + 0.069x - 0.483 and regression coefficient (R2) was 0.587 (Fig. 4). The results indicated that CH4 production (ml) per CP degradation (mg) of protein feeds in vitro after extraction is lower than before extraction.

    Discussion

    The rate of feedstuff degradation in the rumen can have a profound effect on fermentation end products and on animal performance(Nocek & Russell, 1988). Any economic approach to diet balancing needs to seek the maximal beneficial aspects of ruminal fermentation while minimizing fermentation losses. Much of the feed, which enters the rumen, is fermented, but some feed escapes ruminal degradation. Although the disappearance of the feed is ultimately determined by the relative rates of fermentation and passage(Waldo & Smith, 1972), fermentation rate is an inherent property of the feed. The buffer solubility and classification of protein in the feedstuffs might be closely related the above concept.

    In ruminants, feedstuffs are fermented in the rumen before hindgut digestion, and this fermentation has confounded the prediction of animal performance from dietary ingredients. The CNCPS has led to balance the carbohydrate and protein in the diet and to maximize the utilization of them by both rumen microorganisms and the ruminants. One of the possible ways to achieve the goal is to match the carbohydrate and protein in fermentation rate (synchronization) for the rumen microorganisms, and adequate supply of protein (amino acids and peptides) for the ruminant animals.(Bauchop, 1979; Coleman, 1980). The concept of CNCPS has been applied to feed manufacturing to improve the utilization efficiency of the ruminant’s diets by maximizing the production of microbial protein and has animal production without massive loss of nutrients(Citron et al., 1987). Measurement of the buffer solubility of the protein feeds should be the initial step, and might be followed by the estimation of fermentation by rumen microorganisms. Measurement of CH4 production could also be an indirect way to know the fermentation pattern by rumen microbes.

    The present in vitro studies was conducted to examine the effect of the buffer extraction on the degradability of protein feeds and gas production in order to generate the basic information for the diet formulation which has been lack in Korea.

    Characteristics of microbial fermentation, and rate and extent of protein feeds in the rumen, in general, depend upon the chemical and physical structural of the protein feeds. Generally, the fermentation characteristics, rate and extent of protein feeds and gas production in the rumen are closely correlated each other. Buffer extraction slightly increased pH of the culture solution but tended to lower ammonia-N concentration for all protein feeds. This may due to the fact that soluble carbohydrate and protein, which can be utilized as a substrate at initial fermentation by rumen microbes was extracted. Similar trends were observed in VFA concentration, rate and extent of in vitro degradation, and gas production to those of pH and ammonia-N concentration.

    The extracts with buffer may include mostly soluble carbohydrates and nitrogen, which can be rapidly fermentable. Carbohydrate fermentation in the rumen mostly results in the production of VFA and lactate as well as microbial protein synthesis (Hungate, 1966). The VFA and lactate can be used as energy source in the body, and microbes can be used as a high quality protein. Because methane, however, is produced during the process of carbohydrate, its amount is closely related with the loss of dietary energy. Unlikely to the carbohydrates, final product of protein degradation in the rumen is mostly ammonia when feed protein was degraded, thus the faster the rate of degradation of feed protein, soluble proteins, the more protein will be lost by rumen microbes in the rumen. Condition that above concept is accepted, buffer extracts may affect more protein than carbohydrate of feeds in ruminant nutrition. Rate of fermentation(fermentation characteristics, effective degradability and methane production) of all the protein feedstuffs estimated in the present study was decreased by buffer extraction, and most of all, rate of transfer of feed protein to ammonia-N leads to protein loss.

    The results from in the current study might be useful for diet formulation to improve the feed efficiency of the ruminant animals without massive loss of major nutrients. The CH4 production by the microbial degradation major components(DM and CP) of the feeds could also be applied as indirect method in the estimation of the rate of fermentation in the rumen and degradability.

    Figure

    JALS-49-217_F1.gif

    Relationship between the DM degradation and the CH4 production of protein feeds in vitro before buffer extraction.

    JALS-49-217_F2.gif

    Relationship between the DM degradation and the CH4 production of protein feeds in vitro after buffer extraction.

    JALS-49-217_F3.gif

    Relationship between the CP degradation and the CH4 production of protein feeds in vitro before buffer extraction.

    JALS-49-217_F4.gif

    Relationship between the CP degradation and the CH4 production of protein feeds in vitro after buffer extraction.

    Table

    Chemical composition of protein feeds

    1)CP: crude protein; EE: ether extract; NDF: neutral detergent fiber.

    Solubility and protein fractionation of protein feeds

    1)CP: crude protein, SP: soluble protein, IP: insoluble protein.

    Effect of buffer extraction and protein source of feeds on pH in culture solution by rumen microbes

    1)SEM, Standard error of means;
    2)Pr < F, Probability;
    3)Differences between buffer extraction(BE), feed sources(F) and it’s interaction(BE x F)
    *P < 0.05;
    **P < 0.01;
    ***P < 0.001;
    NS, non-significant.;
    Superscript were present the interaction between buffer extraction and feed sources(BE x F)

    Effect of buffer extraction and protein source of feeds on ammonia-N concentration(mg/100ml) in culture solution by rumen microbes

    1)SEM, Standard error of means;
    2)Pr < F, Probability;
    3)Differences between buffer extraction(BE), feed sources(F) and it’s interaction(BE x F)
    *P < 0.05;
    **P < 0.01;
    ***P < 0.001;
    NS, non-significant.;
    Superscript were present the interaction between buffer extraction and feed sources(BE x F)

    Effect of buffer extraction and protein source of feeds on Total VFA concentration(mmoles/100ml) in culture solution by rumen microbes

    1)SEM, Standard error of means;
    2)Pr < F, Probability;
    3)Differences between buffer extraction(BE), feed sources(F) and it’s interaction(BE x F)
    *P < 0.05;
    **P < 0.01;
    ***P < 0.001;
    NS, non-significant.;
    Superscript were present the interaction between buffer extraction and feed sources(BE x F)

    Effect of buffer extraction and protein source of feeds on molar proportion of acetate(mmoles/ 100mmoles) in culture solution by rumen microbes

    1)SEM, Standard error of means;
    2)Pr < F, Probability;
    3)Differences between buffer extraction(BE), feed sources(F) and it’s interaction(BE x F)
    *P < 0.05;
    **P < 0.01;
    ***P < 0.001;
    NS, non-significant.;
    Superscript were present the interaction between buffer extraction and feed sources(BE x F)

    Effect of buffer extraction and protein source of feeds on molar proportion of propionate(mmoles/ 100mmoles) in culture solution by rumen microbes

    1)SEM, Standard error of means;
    2)Pr < F, Probability;
    3)Differences between buffer extraction(BE), feed sources(F) and it’s interaction(BE x F)
    *P < 0.05;
    **P < 0.01;
    ***P < 0.001;
    NS, non-significant.;
    Superscript were present the interaction between buffer extraction and feed sources(BE x F)

    Effect of buffer extraction and protein source of feeds on molar proportion of butyrate(mmoles/ 100mmoles) in culture solution by rumen microbes

    1)SEM, Standard error of means;
    2)Pr < F, Probability;
    3)Differences between buffer extraction(BE), feed sources(F) and it’s interaction(BE x F)
    *P < 0.05;
    **P < 0.01;
    ***P < 0.001;
    NS, non-significant.;
    Superscript were present the interaction between buffer extraction and feed sources(BE x F)

    Effect of buffer extraction and protein source of feeds on degradation parameters(a, b and c) and effective degradability of dry matter(EDDM) of feeds by rumen microbes in vitro

    1)a, rapidly soluble fraction; b, degradable fraction in the rumen at time infinity; c, degradation rate of b per time
    2)SEM: Standard error of means;
    3)Pr < F, Probability;
    4)Differences between buffer extraction(BE), feed sources(F) and it’s interaction(BE x F)
    *P < 0.05;
    **P < 0.01;
    ***P < 0.001;
    NS, non-significant.;
    Superscript were present the interaction between buffer extraction and feed sources(BE x F)

    Effect of buffer extraction and protein source of feeds on degradation parameters(a, b and c) and effective degradability of crude protein(EDCP) of feeds by rumen microbes in vitro

    1)a, rapidly soluble fraction; b, degradable fraction in the rumen at time infinity; c, degradation rate of b per time
    2)SEM: Standard error of means;
    3)Pr < F, Probability;
    4)Differences between buffer extraction(BE), feed sources(F) and it’s interaction(BE x F)
    *P < 0.05;
    **P < 0.01;
    ***P < 0.001;
    NS, non-significant.;
    Superscript were present the interaction between buffer extraction and feed sources(BE x F)

    Effect of buffer extraction and protein source of feeds on methane(CH4) production(ml) by rumen microbes in vitro

    1)SEM, Standard error of means;
    2)Pr < F, Probability;
    3)Differences between buffer extraction(BE), feed sources(F) and it’s interaction(BE x F)
    *P < 0.05;
    **P < 0.01;
    ***P < 0.001;
    NS, non-significant.;
    Superscript were present the interaction between buffer extraction and feed sources(BE x F)

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