The in¯uence of diet of the donor animal on the
initial bacterial concentration of ruminal ¯uid and
in vitro gas production degradability parameters
S. Nagadi, M. Herrero, N.S. Jessop
*Institute of Ecology and Resource Management, The University of Edinburgh, West Mains Road, Edinburgh EH9 3JG, UK
Received 3 December 1999; received in revised form 10 July 2000; accepted 24 July 2000
Abstract
Six sheep were fed twice a day on a different ratio of sheep pellets and hay (20:80 (diet 1), 40:60 (diet 2) and 80:20 (diet 3)) in a replicated Latin square design to study the effect of the host diet on the bacterial concentration of ruminal liquor and in vitro gas production degradability parameters of cellulose, glucose and hay. Bacterial DM, bacterial absorbance and the volume of gas produced in the absence of substrate increased as the ratio of sheep pellet to hay increased. The gas production degradability parameters obtained from ®tting data to the model GasB 1ÿexpÿC tÿLag) were also affected by changing the ratio of sheep pellets to hay in the diet of donor animals. For each substrate, incubation with ruminal ¯uid taken from sheep fed on diet 2 or 3 gave higher (P<0:05) asymptotic values `B' (except for hay), rates `C' of gas production and lower Lag times (cellulose and hay only) than when incubated in the ruminal ¯uid taken from sheep fed on diet 1. The digestibility of NDF from cellulose and hay was not affected by diet. Bacterial DM was strongly related to the absorbance of ruminal ¯uid and the volume of gas produced in the absence of substrate (R20:99,P<0:001). Results suggest that changing the ratio of concentrate to hay reduces the initial bacterial concentration and affects the gas production degradability parameters but the estimation of bacterial DM either from bacterial absorbance or volume of gas produced without substrate was not affected by changing the diet of donor animal.#2000 Elsevier Science B.V. All rights reserved.
Keywords:Gas production; Degradability; Forage quality; Rumen microbes; Techniques 87 (2000) 231±239
*Corresponding author. Tel.:44-131-535-4141; fax:44-131-667-2601. E-mail address: neil.jessop@ed.ac.uk (N.S. Jessop).
1. Introduction
The in vitro gas production technique has the ability to characterise feeds not only by the quantity of digestible carbohydrate they provide but also by the rate at which these nutrients are released. Such characteristics are of central importance to understanding the dynamics of rumen fermentation. For this system to be useful for routine evaluation of forages, it must produce results with high precision and repeatability. One factor which can in¯uence the in vitro gas production pro®les, is the microbial activity of the inoculum (Jessop and Herrero, 1998). This could be affected by the frequency of sampling of ruminal liquor (Nagadi et al., 1999), the time of collection (Menke and Steingass, 1988; Pell and Scho®eld, 1993; Cone et al., 1996) and the extent of dilution with buffer (Pell and Scho®eld, 1993; Rymer et al., 1999).
The diet of the host animal in¯uences the chemical environment within the rumen and subsequently the microbial population of ruminal ¯uid (Weiss, 1994). Several studies have indicated that the diet of donor animal in¯uences both the total gas production (Trei et al., 1970; Menke and Steingass, 1988) and the gas production pro®les (Bonsi et al., 1995; Cone et al., 1996; Huntington et al., 1998; Das and Singh, 1998).
The diet of donor animal differs between research groups that use the in vitro gas production technique. Pell and Scho®eld (1993); Theodorou et al., 1994 and BluÈmmel and Becker (1997) use ruminal ¯uid taken from donor animals fed on hay only, others use ruminal ¯uid taken from host animals fed on a particular ratio of hay to concentrate (Menke and Steingass, 1988; Beuvink et al., 1992; Cone et al., 1996) although the ratio varies between groups and the composition of the concentrate is rarely speci®ed. These differences in the diet of donor animals might be one of the factors that affects microbial activity or concentration and subsequently the gas production pro®les and causes differences in gas production data between laboratories (Moss et al., 1998). Preliminary results have shown that the relationship between bacterial DM and bacterial absorbance can be used to predict the microbial activity of the inoculant (Nagadi et al., 1999). However, it is not known if this relationship is affected by the diet of the host animal.
The aims of this study were to examine the effect of varying the ratio of hay to concentrate in the diet of the host animal on bacterial concentration and gas production degradability parameters, and, whether the relationship between bacterial absorbance at 600 nm and bacterial DM varies across diets.
2. Materials and methods
2.1. Animals and diet
The experiment was conducted as two (replicated) 33 Latin squares using six ruminally ®stulated Suffolk sheep in three periods. In each period sheep were fed twice a day (at 8.00 and 17.00 h) on one of each of diet 1 (200 g sheep pellets and 800 g hay, diet 2 (400 g sheep pellets and 600 g hay) or diet 3 (800 g sheep pellets and 200 g hay). The hay contained 631 g NDF and 77 g CP (N6:25) per kg DM. The sheep nuts contained 251 g NDF, 54 g sugar, 235 g starch and 170 g CP per kg DM.
The sheep were given three weeks to adapt to each diet before and between successive periods. Water was available to the sheep ad libitum.
2.2. Inoculum preparation
A sample of rumen contents containing both solid and liquid material was taken from each sheep before morning feeding and collected in pre-warmed vacuum ¯asks. In the laboratory ruminal ¯uid was ®ltered through two layers of muslin cloth and the solid material was squeezed lightly. The strained ruminal ¯uid was mixed (1:2 v/v) with anaerobic medium as described by Menke and Steingass (1988). Once prepared, the suspension of micro-organisms was kept at 398C with CO2bubbling through for approx.
20 min before addition to syringes.
2.3. Measurement of bacterial DM
Bacterial DM was measured directly by centrifuging 30 ml of the buffered rumen ¯uid at 1000g(to remove protozoa and fungi and particulate matter) for 5 min at 308C. The pellet was discarded and the supernatant was re-centrifuged at 26000g for 15 min at 48C. The supernatant was discarded whilst the bacterial pellet was resuspended in NaCl solution (9 g/l) and centrifuged again. The supernatant was discarded and the bacterial pellet was dried at 808C to constant weight for determination of bacterial DM (Henning et al., 1991). Bacterial DM was also measured indirectly by reading the absorbance at 600 nm (Wells and Russell, 1996) of ruminal ¯uid that had been diluted ®fty fold with buffer.
2.4. Gas production measurements and analytical procedures
2.5. Statistical analysis
Analysis of variance (Genstat, 1997) was used to compare bacterial DM, bacterial absorbance, BGV and estimated gas production degradability parameters between different diets. Least signi®cant differences were calculated from the standard error of the differences between means. Regression was used to study the relationship between bacterial DM, absorbance and BGV.
3. Results
The effect of sheep diet on the bacterial DM content of ruminal ¯uid, bacterial absorbance and BGV are summarised in Table 1. Ruminal ¯uid from sheep fed on diet 3 had the highest (P<0:05) mean values of bacterial DM, bacterial absorbance and BGV followed by ruminal ¯uid obtained from sheep fed on diet 2 and 1, respectively.
The effect of sheep diet on gas production degradability parameters, NDF digestibility and ®nal pH of glucose, cellulose and hay are presented in Table 2. It can be seen that ruminal ¯uid taken from sheep fed on diet 2 or 3 resulted in an increased (P<0:05) asymptotic gas production for cellulose and glucose (B), total gas production from the fer-mentation of hay (AB) and fractional rate of gas production (C) from the fermentation of glucose, cellulose and hay when compared with the ruminal ¯uid taken from sheep fed on diet 1. The lag phase before the fermentation of carbohydrate begins (Lag) for cellulose and hay was signi®cantly shorter when incubated in ruminal ¯uid taken from sheep fed on diet 2 and 3 than that incubated in diet 1. The gas produced from the fermentation of soluble material (A) for hay was signi®cantly higher when incubated in ruminal ¯uid obtained from sheep fed on diet 2 and 3 than that incubated in diet 1 whereas the asymptotic gas production from the fermentation of NDF (B) was not effected by the diet of donor animal. The NDF digestibility (NDFD) of cellulose and hay was not signi®cantly in¯uenced by source of ruminal ¯uid whether taken from sheep fed on diet 1, 2 or 3. Cellulose, glucose and hay incubated in ruminal ¯uid taken from sheep fed on diet 3 had the highest (P<0:05) ®nal culture pH followed by those incubated in diet 2 and 1, respectively. The relationship between bacterial DM and bacterial absorbance is shown in Fig. 1. Bacterial DM was linearly related (R20:99,P<0:001) to bacterial absorbance such that
Bacterial DM mg=10 ml of strained rumen fluid 863 SE 4:5 Bacterial Absorbance
Table 1
The effect of donor diet on bacterial DM, bacterial absorbance and the blanks gas volumea
Donor diet Bacterial DM (mg/10 ml) Bacterial absorbance (A) Blanks gas volume (ml)
1 73.9a 0.085a 11.5a
2 119.0b 0.137b 18.7b
3 155.0c 0.179c 24.5c
SED 5.35 0.007 0.98
Table 2
The effect of donor diet on gas production parameters, NDF digestibility and ®nal pHa Substrate Gas production
parametersb
Donor dietc
1 2 3 SED
Glucose B(ml) 14.6a 17.4b 17.7b 0.51
C(/h) 0.143a 0.204b 0.206b 0.0038
Lag (h) 0 0 0 ±
Final pH 6.56a 6.59b 6.61c 0.002
Cellulose B(ml) 84.7a 90.7b 91.1b 0.49
C(/h) 0.073a 0.101b 0.102b 0.0029
Lag (h) 5.2a 4.5b 4.4b 0.10
NDFD (g/kg DM) 999 1000 998 1.1
Final pH 6.53a 6.57b 6.59c 0.003
Hay A(ml) 4.7a 7.1b 7.0b 0.20
B(ml) 30.0 31.5 31.9 1.7
C(/h) 0.028a 0.037b 0.040b 0.0017
Lag (h) 5.9a 5.3b 5.2b 0.08
AB(ml) 34.7a 38.6b 39.0b 0.89
NDFD (g/kg DM) 531 538 541 6.9
Final pH 6.61a 6.64b 6.70c 0.006
aThe letters a, b, c in the same row denote a signi®cant difference (P<0:05) between means.
bAis the gas produced at 4 h andB,Cand Lag are parameters obtained by ®tting the gas production data, corrected forA, to the equation:
gasB 1ÿeÿC tÿLag
whereBis the asymptotic gas production from fermentation of NDF,Cis the fractional rate of gas production and Lag is the time taken for gas production fromBto begin.
c1 Ð sheep pellets:hay20:80; 2 Ð sheep pellets:hay40:60; 3 Ð sheep pellets:hay80:20.
Fig. 2 shows the relationship between bacterial DM and BGV. There was a linear relationship (R20:99,P<0:001) between bacterial DM and BGV such that
Bacterial DM mg=10 ml of strained rumen fluid 6:34 SE 0:085 BGV ml
4. Discussion
The primary objective of this work was to study the in¯uence of donor diet on initial microbial concentration and gas production degradability. Results clearly showed that bacterial DM, bacterial absorbance and BGV increased as the ratio of concentrate (sheep pellet) to hay increased in the donor animals' diet and that gas production degradability parameters of cellulose, glucose and hay were affected when incubated in ruminal ¯uid taken from sheep fed on diet 1. These differences might be attributed to the high ratio of low quality hay to low ratio of concentrate in diet 1 of the donor animal in¯uencing the chemical environment within the rumen resulting in lower microbial concentrations (Weiss, 1994). The correspondingly low initial microbial concentration in strained ruminal liquor (Table 1) was below that recommended by Jessop and Herrero (1998) and Nagadi et al. (1999). These results are in agreement with Menke and Steingass (1988) who recommended the diet of the host animal to be 50±60% hay and 40±50% concentrates since they observed a 25% reduction in the total gas production when rumen ¯uid was taken from sheep fed on only straw and no concentrate. They referred to the low level of micro-bial activity in the ruminal ¯uid of the donor animals fed on straw since, in their study, BGV (regarded as a measure of microbial activity) was around 3 ml compared to 12±16 ml from ruminal ¯uid taken from animals receiving a diet of hay and concentrates.
The results of this study also indicate that host diet affects the ®nal pH although differences were small. Dietary effects on gas production degradability (B,Cand Lag) and ®nal pH observed in this study are in agreement with some earlier works. Trei et al. (1970) studied the in¯uence of inoculum source on in vitro gas production and found that
Fig. 2. The relationship between bacterial DM (mg/10 ml of strained rumen ¯uid) and the volume of gas produced in the absence of substrate (n18).
total gas production was greater when the inoculum was obtained from grain fed steers as compared to that from hay fed steers. Bonsi et al. (1995) studied the effect of rumen ecology on in vitro gas production of four sun-dried fodder trees incubated in ruminal ¯uid taken from sheep fed on teff straw alone or supplemented with either Sesbania sesban or Leucaena leucocephala. They reported that the total and the rate of gas production from fodder trees were lower when incubated in ruminal ¯uid taken from sheep fed on teff straw as compared with that taken from sheep fed on eitherS. sesbanor
L. leucocephala. Cone et al. (1996) tested the in¯uence of source of ruminal ¯uid on the rate of gas production and found that the rate of gas produced from the incubation of maize cob mix in ruminal ¯uid taken from sheep fed on 800 g hay and 200 g concentrate was higher than that incubated from sheep fed on hay only.
Huntington et al. (1998) studied the effect of host diet on the gas production pro®les of grass hay and high temperature dried grass. They found that grass hay and high temperature dried grass incubated in ruminal ¯uid taken from a silage-based diet increased the asymptotic value, time dependent rate and ®nal pH but that Lag time, total VFA and OMD were not affected by the diet of donor animal. Recently, Das and Singh (1998) studied the effect of varying the level of berseem supplementation in the diet of donor animals on in vitro gas production of wheat straw. They noted that both the total and the rate of gas production were increased by increasing the level of berseem in donor diet up to 30%.
Jessop and Herrero (1998) proposed that if microbial activity was low, this would become a limiting factor and a signi®cant proportion of degraded carbohydrate would be incorporated into new microbial matter rather than being fermented to products that gave rise to gas production. The reduced asymptotic gas production (B) but similar NDFD observed in this study when the concentration of bacterial DM and BGV of ruminal ¯uid taken from sheep fed on diet 1 were below the minimum values of microbial activity recommended in earlier work (90 mg/ml and 15 ml, respectively, Nagadi et al., 1999; Jessop and Herrero, 1998) would support this assumption.
The coef®cients relating bacterial DM to both bacterial absorbance (863 s.e. 4.5 mg bacterial DM in 10 ml strained ruminal ¯uid per unit of absorbance) and BGV (6.34 s.e. 0.085 mg bacterial DM in 10 ml strained ruminal ¯uid per ml BGV) obtained in this study were very close to those (bacterial absorbance (868 s.e. 39 mg bacterial DM in 10 ml strained ruminal ¯uid per unit of absorbance) and BGV (589 s.e. 0.043 mg bacterial DM in 10 ml strained ruminal ¯uid per millilitre BGV) observed in earlier work (Nagadi et al., 1999). This indicates that the estimation of bacterial DM by reading the absorbance at 600 nm of ruminal ¯uid diluted 50-fold or from BGV is not affected by the diet of donor animal. Moreover, the above relationship between bacterial DM and bacterial absorbance could be used for monitoring the effect of donor diet on microbial concentration of ruminal liquor and whether or not such liquor provides suf®cient microbial matter for the in vitro gas production technique.
5. Conclusions
should be taken to ensure that the inoculum used contains suf®cient microbial activity. As a guide, a ratio of 60% hay and 40% concentrates in the donor animal's diet is recommended although the quality of the hay and composition of the concentrate will in¯uence this. The relationship between bacterial DM and bacterial absorbance can be used to determine whether the diet of the host animal allows the attainment of a suf®cient microbial concentration in ruminal ¯uid or not since results suggest that the diet of donor animal does not affect the relationships between bacterial DM, bacterial absorbance, bacterial DM and BGV.
Acknowledgements
We are grateful for the skilled technical assistance of G.F. Allan and the statistical advice of Mr. E.A. Hunter, Biomathematics and Statistics, Scotland.
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