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Effect of Total Mixed Ration Composition on Fermentation and Efficiency of Ruminal Microbial Crude Protein Synthesis In Vitro

Institut für Ernährungswissenschaften, Martin-Luther-Universität Halle-Wittenberg, 06099 Halle (Saale), Germany

¹ Corresponding author: markus.rodehutscord{at}landw.uni-halle.de

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The goal of this study was to identify dietary factors that affect fermentation and efficiency of microbial crude protein (CP_M) synthesis in the rumen in vitro. We used 16 total mixed, dairy cow rations with known digestibilities that varied in ingredient composition and nutrient content. Each ration was incubated in a Rusitec (n = 3) for 15 d, and fermentation of different fractions was assessed. Observed extents of fermentation in 24 h were 35 to 47% for organic matter, 25 to 60% for crude protein, 3 to 28% for neutral detergent fiber, and 31 to 45% for gross energy. Organic matter fermentation depended on the content of crude protein and neutral detergent fiber in the ration. We studied net synthesis of CP_M using an ¹⁵N dilution technique and found that 7 d of continuous ¹⁵N application are needed to achieve an ¹⁵N enrichment plateau in the N of isolated microbes in this type of study. The efficiency of CP_M synthesis was 141 to 286 g/kg of fermented organic matter or 4.9 to 11.1 g/MJ of metabolizable energy, and these ranges agree with those found in the literature. Multiple regressions to predict the efficiency of CP_M synthesis by diet data showed that crude protein was the only dietary chemical fraction that had a significant effect. Fat content and the inclusion rate of corn silage in the ration also tended to improve efficiency. We suggest that microbial need for preformed amino acids may explain the crude protein effect. A large part of the variation in efficiency of microbial activity still remains unexplained.

Key Words: Rusitec • fermentation • efficiency • microbial yield

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
The amount of microbial CP (CP_M) synthesized in the rumen plays a major role in providing the cow with amino acids for milk synthesis. Because fermentable energy limits CP_M synthesis (Hoover and Stokes, 1991) and protein supply at the duodenum may limit milk protein synthesis (Clark, 1975; Wright et al., 1998), the prediction of the efficiency of CP_M synthesis is of value for feeding high-yielding dairy cows. If dietary factors were known to alter CP_M synthesis in the rumen, they could be used to influence or to predict microbial yield.

The mean of 69 observations reviewed by Stern and Hoover (1979) indicates that 169 g of CP_M/kg of apparently digested OM is synthesized with a range from 63 to 307 g. The efficiency of CP_M synthesis, determined with cannulated cows, is about 10.1 g of CP_M/MJ of ME (SD = 1.5 g) and 156 g of CP_M/kg of digestible OM (SD = 24 g), respectively, based on a recent review (Gesellschaft für Ernährungsphysiologie, 2001). In vitro investigations have shown similar results (Stokes et al., 1991; Bach et al., 1999).

Reasons for this wide range may originate from the methods of determination of CP_M (Stern and Hoover, 1979), the supply of energy and nitrogen (Veira et al., 1980; Kang-Meznarich and Broderick, 1981; Madsen and Hvelplund, 1988) and its synchronization (Herrera-Saldana et al., 1990; Aldrich et al., 1993; Sinclair et al., 1993), the availability of N (Siddons et al., 1985; Kajikawa et al., 2002), the supply of P (Petri et al., 1988), the feedstuffs (Hristov and Broderick, 1996; Sutton et al., 2000; Younge et al., 2004), or the management of feeding (Chamberlain and Thomas, 1979; Verbiè et al., 1999). The amount of fermentable OM is a dominant factor influencing CP_M synthesis in the rumen because it depends on the availability of N and carbohydrates (NRC, 2001).

The association between high levels of DMI and high amounts of concentrates has raised interest in feeding TMR to dairy cows. Such rations offer the opportunity to stabilize highly efficient ruminal digestion (Coenen, 1996), but systematic studies on factors that determine the efficiency of CP_M synthesis with TMR feeding have not been reported.

The purpose of this study was to determine the extent of fermentation of different nutrients from TMR and to derive equations that allow for an identification of factors influencing efficiency of CP_M synthesis. When such prediction equations are to be calculated mathematically, many data are generally needed, as is also a wide range in nutrient content. Such a data set, including CP_M synthesis, is difficult to obtain with dairy cows because these studies depend on cannulated cows and are prohibitively costly. In vitro methods provide an easier, less costly way to describe fundamental processes of rumen physiology and to define precisely the conditions used in experiments (Czerkawski and Breckenridge, 1985).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Description and Preparation of Diets
Sixteen TMR were used (Table 1). They were composed to represent rations that are typical for the German feedstuff basis for dairy cows in different stages of lactation. Each TMR contained at least one silage. Corn silage, grass silage, or both were components in 15 of the 16 TMR. Silages from beet pulp, alfalfa, moist ensiled corn, and brewers’ grains also were included. In most diets, the concentrates were barley or solvent-extracted meals from soybean and rapeseed. Two TMR contained peas. Ingredients were individually weighed into plastic bags, mixed, and stored at –18°C until further handling. Before the beginning of the experiment, the TMR were thawed, dried at 65°C for 24 h, and ground through a sieve with 1-mm pore size in a hammer mill.

The apparatus used was a modified rumen simulation technique (Rusitec) described by Czerkawski and Breckenridge (1977) in which 6 reaction vessels had a capacity of 800 mL each. Three vessels were used per ration. Each feed container inside the vessel contained 2 nylon bags (~100 x 50 mm, pore size = 100 µm, Fa. Linker Industrie-Technik GmbH, Kassel, Germany) filled with 15.0 g of the TMR that had been dried for 24 h at 65°C. At 24-h intervals, one bag was replaced with a new one so that each bag was incubated for 48 h. At the start of the system for the first 24 h, one bag was filled with pooled rumen solids (~60 g) instead of TMR. We mixed 400 mL each of strained rumen fluid and artificial saliva (McDougall, 1948) and placed the mixture into each reaction vessel initially. Artificial saliva was continuously infused at a rate of approximately 600 mL/d using an 8-channel peristaltic pump (Ole Dich Instrumentmakers ApS, Denmark). Vertical movement of the feed containers was ensured by an electric motor with 10 to 12 strokes/min. The effluent was collected in 1-L glass bottles standing inside an ice-cold water bath.

For determination of CP_M synthesis, ¹⁵N was used as a tracer for labeling the microbial fraction. Artificial saliva contained 0.7 mmol of NH₄⁺/L from NH₄Cl with a declared ¹⁵N excess of 10 atom% (Chemotrade Chemiehandelsgesellschaft mbH Leipzig, Germany).

The removed bags containing the feed residue were washed with 2 x 40 mL of artificial saliva and squeezed moderately; the liquid was then returned to its respective vessel. The feed residues were dried at 65°C for 24 h.

Sampling, Preparation, and Analyses
An 8-d period of collecting samples of the feed residues, the liquid effluent, and the microbes followed a 7-d period of adaptation. As determined during the course of the study, this adaptation was necessary to achieve a plateau in ¹⁵N enrichment of microbes isolated from liquid effluent. To describe the dynamic of ¹⁵N enrichment, samples of microbes were isolated for 2 TMR (O and P; Table 1) on each day of incubation starting on d 1. Samples of the liquid effluent (320 mL) were taken daily, and the reference microbes were isolated by differential centrifugation according to Brandt and Rohr (1981; Suprafuge 22, Heraeus Instruments, Hanau, Germany). In brief, the suspension was centrifuged twice at 2,000 x g at 4°C for 5 min. Subsequently, the supernatant was centrifuged 3 times at 27,000 x g at 4°C for 15 min. In between, the microbes were resuspended with a saline solution (0.9% wt/vol). After the last centrifugation, the microbes were frozen at –18°C, freeze-dried, and crushed with pestle and mortar. For all other TMR, the microbes were isolated in the same way only in the period between d 7 to 15.

Microbes that were attached to the feed [solid-associated microbes (SAM)] were separated from the feed residues contained in the 2 nylon bags at the termination of the study, based on Minato and Suto (1978) and according to the method described by Carro and Miller (2002). The SAM were separated from the remaining fluid and further treated as described for the reference microbes.

The ¹⁵N enrichment of N in dry samples from NH₄Cl, feed residues, effluents, and isolated microbes was measured by emission spectrometry (NOI 7, Fischer Analysen Instrumente GmbH, Leipzig, Germany) after combustion in a coupled C/N analyzer (Vario EL, Elementar Analysensysteme GmbH, Hanau, Germany). To obtain a sufficiently high amount of N for the ¹⁵N analysis, ~1.0 mg of NH₄Cl, 15 to 30 mg of feed residues, and 2 to 5 mg of microbes were exactly weighed into tin capsules and made airtight prior to combustion. Thirty milliliters of effluent was taken, and ammonia N was transferred into a sulfuric acid solution via NaOH. This solution was then desiccated by lyophilization, and up to 50 mg of the dry residue was weighed into the tin capsules for combustion. The amount of effluent N obtained this way was not sufficient to reliably measure the ¹⁵N enrichment in this fraction for TMR A, B, and D.

The pooled samples of feed and feed residues were analyzed for DM, ash, CP, crude fiber, NDF, and ADF according to the official methods in Germany (Naumann and Bassler, 1976). The gross energy was measured by a bomb calorimeter (C7000, Janka & Kunkel IKA, Analysentechnik). Nitrogen content in the liquid effluent after centrifugation was determined by a Kjeltec auto 1030 analyzer from Tecator AB (Höganäs, Sweden) without a preceding hydrolysis.

Calculations
The extent of fermentation of crude nutrients, NDF, ADF, or gross energy (F_x; %) was determined as

[1]

where

input of x	=	input in the vessel with feed (g/d), and
output of x	=	output from the vessel with feed residue (g/d).

Fermentation of OM was also calculated after correction for OM originating from microbes attached to the feed residues. First, the amount of N originating from SAM [N_SAM (g/d)] was calculated as

[2]

where

15NFR	=	15N enrichment determined in feed residues (%),
NFR	=	amount of N in feed residues (g/d), and
15NSAM	=	15N enrichment in N of the SAM (%).

Second, the amount of OM originating from SAM in the feed residue (OM_SAM; g/d) was calculated as

[3]

where

NSAM	=	N originating from SAM (Equation 2; g/d),
N	=	analyzed N content of SAM (%) on an as-is basis,
12	=	analyzed concentration of ash in SAM (%), and
0.93	=	analyzed proportion of DM in the isolated SAM fraction.

Because of a small sample size, DM and ash content of SAM were analyzed in pooled samples of this fraction only.

The amount of microbial N (N_M in mg/d) leaving the vessel with effluent was calculated as

[4]

where

15Nin	=	15N amounts introduced into the vessel with buffer solution (calculated on the basis of daily buffer flow and analyzed 15N content in NH4Cl) and feed (assuming natural abundance = 0.3663% of total N; µg/d),
15Nout	=	15N amounts leaving the vessel with liquid effluent and feed residues (except 15N in reference microbes; µg/d), and
15NPlateau	=	15N in total N of the isolated reference microbes between d 7 and 15 (µg of 15N/mg of N).

The amount of CP_M (mg/d) was calculated as N_M x 6.25.

To describe the dynamics of ¹⁵N enrichment in the microbial N fraction in the 2 experiments with continuous sampling of microbes, the following equation (GraphPad Prism 4.02 for Windows) was used:

[5]

where

y	=	15N enrichment at given x (%),
x	=	time after onset of 15N administration via the artificial saliva (d),
a	=	minimum level of 15N enrichment (%),
b	=	plateau of 15N enrichment (%),
c	=	one-half of the plateau value of 15N enrichment (%), and
s	=	maximum slope.

In this equation, the expression log₅₀e^c describes the time needed to achieve one-half of the plateau response in ¹⁵N enrichment.

The software package SAS for Windows (version 8.2, 1999–2001, SAS Inst., Inc., Cary, NC) was used for the statistical analysis. Multiple linear regressions were calculated with Proc Reg to identify factors that might influence CP_M synthesis in the rumen. Dietary concentrations of OM, CP, crude fiber, and ether extract, as well as inclusion rates of both corn and grass silage, were considered as variables with the aim of minimizing the average coefficient of variation. As a measure of the contribution of each regressor, the P values of partial correlation (P_p) according the users’ guide of the SAS software were taken. The P_p described the influence of each factor on the target value. The global P value expressed the meaning of the sum of factors on the target value. Also, the values of r² and P will be given as measures for the goodness of fit of the complete equation.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
Dynamic of ¹⁵N Enrichment in Microbial N
With TMR O and P (Table 1), the course of ¹⁵N enrichment in microbial N was studied during the entire experimental period. As shown in Figure 1, a plateau in ¹⁵N enrichment of microbial N was achieved within 7 d after starting the ¹⁵N dosage with both TMR. The course was different, however, for the 2 rations. With ration P, which contained more ingredients and a higher energy value than ration O, the plateau was higher and was achieved earlier than with ration O. Based on these observations, we decided to use pooled samples of microbes from d 7 onward in all other experiments and to consider the ¹⁵N enrichment determined in these pooled samples as the respective plateau value.

View larger version (9K): [in this window] [in a new window]   Figure 1. 15N enrichment of the microbes isolated from the effluent depending on the duration of 15N application via the buffer solution. The equation is , where a = minimum of y, b = maximum of y, c = 1/2 maximum of y, and s = maximum slope.

View larger version (17K): [in this window] [in a new window]   Figure 2. Comparison of OM fermentation determined either uncorrected (light bars) or under consideration of microbes associated with the feed residues taken from the nylon bags (shaded bars; mean ± SD). Rations K, L, M, and N are not included because the 15N enrichment in residues from the nylon bags could not be determined.

View larger version (6K): [in this window] [in a new window]   Figure 3. Relationship between fermented OM (OMfer) and CP and NDF content of the 16 TMR.

[6]

where

OM_fer = fermented OM (%) and CP, NDF, and EE are measured in g/kg of DM of TMR.

The amount of CP_M synthesized ranged from 677 to 1778 g/d (Table 5). For TMR A, B, and D (Table 1), the CP_M synthesis could not be calculated because of a shortage in the amount of effluent N for isotope analysis. The correlation between fermented OM and the amount of CP_M synthesized/d was high (r = 0.75; Figure 4). However, the efficiency, expressed as grams of CP_M per kilogram of fermented OM or as grams of CP_M per megajoule of ME, ranged from 141 to 286 and 4.9 to 11.1, respectively (Table 5).

View larger version (8K): [in this window] [in a new window]   Figure 4. Relationship between fermented OM (OMfer) and the amount of microbial CP (CPM; n = 13 TMR).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

The 2 TMR used for this comparison were very different in nutrient and energy contents, but basically, the course of ¹⁵N enrichment over time was similar between them. The time needed to achieve one-half of the response in ¹⁵N enrichment was about one-half of 1 d shorter for the higher quality TMR P than for TMR O. Also, the plateau was slightly higher for TMR P than for TMR O; however, the comparison suggests that the choice of TMR does not greatly affect the response to ¹⁵N administration. We concluded that a plateau in microbial N would be achieved for all other TMR after 7 d of ¹⁵N infusion.

Microbial growth depends on the amount and availability of nitrogen and energy (Stern and Hoover, 1979; Hoover and Stokes, 1991); thus, CP_M synthesis was expressed in relation to OM fermented in the rumen. The correlation between fermented OM and the amount of CP_M synthesized per day was high (Figure 4). However, in comparison with other in vitro investigations (Abel et al., 1990; Mansfield et al., 1994; Carro et al., 1995; Yang et al., 2002), the extent of nutrient fermentation was low in the present study. Different factors may be responsible for such low fermentation. The fermentation of OM may be depressed when N is deficient for the growth of microbes (Hespell and Bryant, 1979). In the present investigation, we found a trend toward a higher fermentation of OM with increased CP supply to the vessel (Figure 3). Because most rumen bacteria prefer to use ammonia as the source of N for growth (Pilgrim et al., 1970; Salter et al., 1979), the degradation of feed protein may become an important factor influencing the N supply of microbes in this system. Ammonia concentration in the liquid effluent (Table 4), however, was not significantly correlated with CP content (r = 0.50) or OM fermentation (r = 0.55). It is unlikely that the amount of ammonia limited the microbial activity because a maximum microbial growth was reported for an ammonia concentration in rumen liquid between 20 and 100 mg/L (Okorie et al., 1977; Slyter et al., 1979; Pisulewski et al., 1981). For the in vitro system, Satter and Slyter (1974) estimated that an increase in ammonia concentration beyond 50 mg of NH₃N/L in liquid effluent had no effect on the CP_M synthesis. Their data suggest, however, that the limiting concentration is closer to 20 mg of NH₃N/L. In our study, in no case was the concentration <45 mg of NH₃N/L (Table 4).

Furthermore, the relatively high pH values (6.9 to 7.3) and the low flow rate of artificial saliva (565 mL/d; SD = 24) are not in line with the observed low level of nutrient fermentation. Some researchers have suggested that a high pH promotes the degradation of OM (Grant and Weidner, 1992; Calsamiglia et al., 2002; Yang et al., 2002) and that the degradation of DM was not affected within a wide range of dilution rate (Czerkawski and Breckenridge, 1977). Even the chosen pore size of 100 µm can be expected to have a positive effect on the degradation of DM and NDF when compared with 40 or 200 µm (Carro et al., 1995).

Perhaps the postincubation washing and squeezing procedure contributed to the differences in fermentation rate compared with other studies; these procedures may affect the loss of small feed particles from the bag. Also, microbes attached to feed particles could cause an underestimation of fermentation; however, the correction of OM fermentation for SAM could also not explain the low level of OM fermentation. Figure 2 shows that the extent of fermentation of OM was only between 1.2 and 6.0% higher after correction for SAM. The percentage of OM in feed residues originating from SAM amounted to at least 2%.

High variation in efficiency of the CP_M synthesis has often been described both in vivo (Armstrong, 1980) and in vitro (Abel et al., 1990; Calsamiglia et al., 1995; Bach et al., 1999; Carro and Miller, 1999; Colombatto et al., 2003). Factors that contribute to this variation are the feedstuffs used, their chemical composition, and the amount of feed, as well as the microbial population in the rumen (Armstrong, 1980). Many interrelations make it difficult to systematically investigate single factors with regard to their effects on microbial activity. In the present study, it appears that CP content of TMR was an important factor determining the amount of CP_M per unit of fermented OM (Table 6). Both the N content in the ration and the availability of N and energy are decisive for CP_M synthesis. The use of TMR enables an optimization of ruminal digestion because different sources of N and carbohydrates with different kinetics of degradation are offered at the same time. Moreover, the TMR used in this work contained between 12 and 21% CP in DM. According to Kluth et al. (2000), in this range of dietary CP content, no effect on the N balance in the rumen on efficiency of CP_M synthesis can be expected. Therefore, we conclude that the content and availability of N were not the limiting factors in the present study. This conclusion is supported by the lack of correlation between ammonia concentration in the system and OM fermentation as discussed previously. Demeyer and Fievez (2004) argued that the synthesis of rumen bacterial protein may be limited by amino acid supply in the rumen at high production levels and that the optimization of bacterial protein production may require knowledge of the nature of the limiting free amino acids. Under in vivo conditions, free amino acids are generally contained in rumen fluid only in low concentrations (Wallace, 1996). Nevertheless, it turned out in in vitro studies that some AA are essential for bacteria (Atasoglu et al., 2001, 2004) and that the growth of microbes can be promoted by supplementation of peptides or AA (Maeng et al., 1975; Argyle and Baldwin, 1989; Griswold et al., 1996; Kajikawa et al., 2002). Along this line, the CP effect on efficiency of CP_M synthesis may be caused by the supply of certain (limiting) amino acids by the TMR.

The supplementation of lipids caused a decrease in ruminal degradation (Palmquist and Jenkins, 1980; Jenkins and Fotouhi, 1990). Abel et al. (1990) and Doreau et al. (1991) reported only small effects of fat supplementation on the fermentation of nutrients; however, the equations determined in this study indicate that a higher content of EE increased the amount of OM fermented and also increased the efficiency of CP_M synthesis. This finding is in agreement with Jenkins and Fotouhi (1990), who reported that adding fat increased the efficiency of microbial yield.

Another factor that was identified as important for CP_M synthesis was the inclusion rate of corn silage in TMR (Table 6). In vivo studies have indicated that higher inclusion rates of corn silage in mixed rations increased the efficiency of CP_M synthesis, but the different inclusion rates were not taken into account (Givens and Rulquin, 2004). The efficiency in rations based on grass silage is reduced, probably as a consequence of rapid and asynchronous release of N relative to energy from carbohydrates (Siddons et al., 1985). In the present study, a significant monofactorial relationship between the efficiency of CP_M synthesis and the inclusion rate of corn silage was not observed (r = –0.26); however, the goodness of fit in multiple regression analysis could be improved by additional consideration of the maize silage inclusion rate in the regressions (Tables 6 and 7).

The efficiencies expressed in relation to ME as shown in Table 5 are on a lower level than data from studies with dairy cows (Gesellschaft für Ernährungsphysiologie, 2001), but they are difficult to interpret and to compare with in vivo data. First, CP_M was determined in vitro, and ME was calculated based on the digestibilities determined in standardized wether trials. Second, a comparison between the rumen and the Rusitec system must be made with care. The number of protozoa is extremely reduced in rumen simulation systems (Mansfield et al., 1995), and it is also possible that the bacterial population changes in comparison with the natural rumen (Slyter and Putnam, 1967). Feed is enclosed in nylon bags in the Rusitec, which may affect accessibility for microbes. Differences between fermentation in continuous culture and in vivo may also arise because of a lack of N recycling and absorption for products of metabolism, respectively (Mansfield et al., 1995); however, the degradation of OM, CP, and amino acids did not differ between dairy cows and a continuous rumen simulation system in the study of Hannah et al. (1986). Also, short-term studies with a modified, semicontinuous Rusitec in comparison with a natural rumen did not show significant differences in the fermentation, the concentration of short-chain fatty acids, and the number of microbes (Gizzi et al., 1998). Passage rates of fluid and feed, pH in the vessel, amount of feed per day, and the method of separation of microbes representing the total population are critical points in such comparisons and make it difficult to draw conclusions that apply to the feeding of cows.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
In vitro fermentation of OM from TMR depends on the contents of CP and NDF in the ration. The CP_M synthesis correlated with OM fermentation. Regression analysis did not identify dietary factors that can clearly explain the high variation in the efficiency of CP_M synthesis; however, among the factors studied, CP and ether extract content as well as the inclusion rate of corn silage in the ration helped to explain part of the variation. When ¹⁵N is used as a marker to study microbial growth in a Rusitec system fed with TMR, 7 d of continuous ¹⁵N infusion are needed to achieve the steady-state enrichment in microbial N. Sampling of microbes for ¹⁵N analysis should not start prior to this time.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 
This study was supported by the Studienstiftung des Deutschen Volkes with a doctoral scholarship for Jeannette Boguhn, which is gratefully acknowledged.

Received for publication April 15, 2005. Accepted for publication November 11, 2005.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION CONCLUSIONS ACKNOWLEDGEMENTS REFERENCES

 


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