Comparison of two in vitro fermentation gas production methods using both rumen fluid and faecal inoculum from sheep

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1 Animal Feed Science and Technology (2005) Comparison of two in vitro fermentation gas production methods using both rumen fluid and faecal inoculum from sheep Zora Váradyová, Miroslav Baran, Imrich Zeleňák Institute of Animal Physiology, Slovak Academy of Sciences, Šoltésovej 4-6, Košice, Slovak Republic Abstract Two in vitro gas production apparatuses, glass syringes (GS) and pressure transducers (PT), used for measurement of gas production (GP), were compared using rumen fluid (RF) and fresh faeces (FF) as sources of inocula in order to study fermentation patterns of feeds. The RF and FF from ruminally fistulated Merino sheep were mixed with McDougall s buffer and added to fermentation vessels (either fermentation bottles or syringes) containing substrate, being meadow hay (MH), wheat straw (WS), amorphous cellulose (AC) and barley grain (BG), which were evaluated in separate incubations at 39 ± 0.5 C for 96 h. Total GP was recorded after 6, 12, 24, 36, 48, 60, 72, 84 and 96 h of incubation. Volatile fatty acids (VFA) and in vitro dry matter degradability (IVDMD) were determined at the end of the incubation. Initial gas production was higher with PT versus GS, but rates and extent of gas production in the two apparatuses were well correlated (r = for total gas production and r = 0.765, 0.919, for rates of gas production at 3, 18 and 30 h, respectively). The IVDMD and total VFA production did not differ between PT and GS. Total gas production by PT and GS were correlated to both IVDMD (R 2 = 0.685, for PT and GS, respectively) and stoichiometrically predicted gas volumes (R 2 = 0.938, for PT and GS, respectively). However, results obtained by RF and FF inocula differed, with rate and extent of gas production being much lower with FF versus RF, and data from the two inocula were generally poorly related (r = 0.581, 0.809, 0.355, 0.271, 0.109, 0.558, 0.887, 0.909, 0.210) for total gas production, rates of gas production at 3, 18 and 30 h, IVDMD, total VFA produced and molar proportions of acetate, propionate and n-butyrate, respectively). Both PT and GS are suitable to estimate gas production and results from one apparatus Abbreviations: GS, glass syringes; PT, pressure transducer; RF, rumen fluid; FF, fresh faeces; GP, gas production Corresponding author. Tel.: ; fax: address: varadyz@saske.sk (Z. Váradyová) /$ see front matter 2005 Elsevier B.V. All rights reserved. doi: /j.anifeedsci

2 82 Z. Váradyová et al. / Animal Feed Science and Technology (2005) may be extrapolated to those from the other. However, this is not possible for data obtained using faecal inocula versus rumen fluid Elsevier B.V. All rights reserved. Keywords: In vitro gas production technique; Rumen fluid; Faeces; Fermentation; Sheep 1. Introduction The gas production technique is a useful method to estimate rate and extent of feed degradation (Blümmel and Ørskov, 1993; Beuvink et al., 1992; Groot et al., 1996; Cone et al., 1997). Gas production (GP) provides more detailed information on fermentation kinetics of ruminant feeds than can be achieved by estimating disappearance of feed from fermentation media (France et al., 1993) and a number of different in vitro GP techniques have been developed. These techniques have been described in reviews by Stern et al. (1997) and Getachew et al. (1998). Use of glass syringes as vessels for incubating feeds with rumen fluid and monitoring accumulated gas (Menke et al., 1979; Menke and Steingass, 1988) is a well-known in vitro GP method. Alternatively, transducers can be used to monitor the pressure of accumulating gases during fermentation in batch culture and a number of different transducer approaches have been developed (Pell and Schofield, 1993; Theodorou et al., 1994; Cone et al., 1996). Use of fresh faeces from cows and sheep, rather than rumen fluid, has been investigated as an alternative inoculum (Aiple et al., 1992; Nsahlai and Umunna, 1996; El-Meadaway et al., 1998; Omed et al., 2000) and its potential in the in vitro GP technique has been reported by Harris et al. (1995), Mauricio et al. (2001) and Cone et al. (2002). The aim of this study was to compare glass syringes with pressure transducers for measurement of GP using either rumen fluid or fresh faeces as inoculum. 2. Materials and methods 2.1. Rumen fluid (RF) and fresh faeces inocula (FF) Rumen fluid was collected from three rumen-fistulated Merino sheep (6 years of age; 50 ± 2.0 kg) about three hours after the morning feeding, transferred to the laboratory in a water bath preheated to 39 ± 0.5 C, squeezed through four layers of gauze, combined among sheep and purged with CO 2. Sheep were housed separately in pens and fed a diet consisting of 700 g/kg meadow hay and 300 g/kg barley grain with free access to water. Fresh faeces was collected directly from the rectum of the sheep immediately after sampling of rumen fluid, and placed in a pre-warmed thermos flask filled with CO 2. The FF, 20 g, was diluted in 140 ml of McDougall s (1948) buffer and stirred with a glass rod. The resulting suspension was strained through four layers of gauze, and remaining solids were re-suspended in 140 ml of McDougall s buffer and homogenised. The homogenate

3 Z. Váradyová et al. / Animal Feed Science and Technology (2005) was strained through two layers of gauze, mixed with the first strained solution and kept at 39 C under CO 2. All manipulations with each bottle took only a few minutes Pressure transducer method (PT) The 120 ml serum bottles used as fermentation vessels for the pressure transducer method (PT) of the in vitro gas production technique (IVGPT) had 25 ml of McDougall s (1948) buffer added to each fermentation bottle, containing 0.25 g of substrate, 12 h before inoculation. The fermentation bottles were filled up with CO 2, closed with a butyl rubber stopper and aluminium-sealed and stored at 4 C. Before inoculation, fermentation bottles were warmed to 39 C in an incubator. A syringe fitted with an 18 gauge needle was used as a punch to a rubber stopper on the fermentation bottle and 10 ml of RF or FF inoculum was injected into each bottle, which were placed in the incubator. The metering system of PT consisted of a three-way valve, a mechanical manometer fitted to a transducer (Premagas, Slovakia), a gas-tight-syringe and needle. The three-way valve was connected to the transducer to measure pressure in the serum bottles and a gas-tight syringe measured the volume of gas produced. The third port was connected with a needle, by a hose, which was used to punch the rubber stopper on the serum bottle. The volume of accumulated gas was measured at 6, 12, 24, 36, 48, 60, 72, 84 and 96 h of incubation, and the accumulated gas volume was released after each reading Glass syringe method (GS) The 50-ml glass gas-tight syringes (Sigma, St. Louis, MO, USA) used as fermentation vessels for the glass syringe method of the IVGPT had 25 ml of McDougall s (1948) buffer added to each syringe, containing 0.25 g of substrate, 12 h before inoculation. Pistons were lubricated with vaseline, air bubbles in syringes removed, the plastic clip closed and syringes placed on a stand in a refrigerator at 4 C. Before inoculation, syringes were warmed to 39 C in the incubator. A 10 ml syringe was used to inoculate RF or FF (10 ml) into each fermentation glass syringe through the plastic clip. The glass syringes were then placed on a stand in the incubator for the 96-h incubation. Throughout the incubation, the temperature in the incubator was maintained at 39 ± 0.5 C. According to the change in the syringe piston position, gas production was measured at 6, 12, 24, 36, 48, 60, 72, 84 and 96 h. After each recording, the clip was opened and the piston moved to the 35 ml position Substrates The substrates used were meadow hay (MH), wheat straw (WS), amorphous cellulose (AC) and barley grain (BG). The MH, WS and BG were ground through a mm screen, bulked, and stored in sealed plastic containers until required. Five replicate fermentation bottles or glass syringes of each substrate were used for experimental groups with RF (inoculum + buffer + substrate) and five were also used for the controls with RF (inoculum + buffer, no substrate). Five replicate bottles or glass syringes of each substrate were also used for experimental groups with FF (inoculum + buffer + substrate) and five were also used for the controls with FF (inoculum + buffer, no substrate).

4 84 Z. Váradyová et al. / Animal Feed Science and Technology (2005) VFA analysis and determination of IVDMD The volatile fatty acids (VFA) in the medium at the end of the incubation period were determined by gas chromatography (Cottyn and Boucque, 1968) using crotonic acid as the internal standard with Perkin-Elmer 8500 gas chromatograph. In vitro dry matter degradability (IVDMD) was determined from the difference of substrate weight before and after incubation. Contents of the fermentation bottles or syringes were transferred into a centrifuge tube and centrifuged at 3500 g for 10 min, and residues were washed twice with distilled water, re-centrifuged and dried to 105 C constant weight (Mellenberger et al., 1970) Calculations and statistical analysis Gas accumulation volumes were determined from the recorded pressure or volumes of gas production. Cumulative gas production profiles were constructed by summing the regression-corrected gas volume for each measurement time (France et al., 1993). Rates of gas production (ml gas/g DM incubated per hour) at 3, 18, and 30 h were calculated from the determined gas volumes at 6, 12, 24, and 36 h from the equations: Rate 3h=gasvolume at 6 h/6 Rate 18h=(gasvolume at 24 h gas volume at 12 h)/12 Rate 30h=(gasvolume at 36 h gas volume at 24 h)/12 Gas production, VFA and IVDMD data were analysed by analysis of variance. Effects included in the model were inocula method substrate and the interactions between parameters (Graphpad InStat, GraphPad Software Inc., San Diego, CA, USA). The VFA data were also used to stoichiometrically calculate gas volumes (Blümmel et al., 1999). Regression analysis was used to determine the relationship between predicted and observed total gas volumes. Estimates of total gas produced, rates of gas production at 3, 6, 18 and 30 h, IVDMD, total VFA produced and molar proportions of acetate, propionate and n-butyrate by apparatus and the inoculum were compared by determining Pearson correlation coefficients between estimates from the apparatus, and estimates from inocula. 3. Results 3.1. Rates and extent of gas production The effect of apparatus (GS versus PT), inoculum and substrate on gas production profiles is shown in Fig. 1a d. With the exception of meadow hay, the initial volume of gas produced was higher with PT versus GS. Similar profiles were usual between the apparatuses, but the lower initial gas volume from GS shifted this profile relative to PT. Rate and extent of gas production was much lower with FF versus RF. Rate of gas production from RF was higher than with FF (Fig. 2a d), and the biggest differences occurred at the first calculated time point (i.e., 3 h). In addition, rate of gas production from RF, and from FF with PT, was higher as compared with GS inocula. In

5 Z. Váradyová et al. / Animal Feed Science and Technology (2005) Fig. 1. Cumulative gas production measured by the mechanical manometer (PT) and glass syringes (GS) methods for meadow hay (a), wheat straw (b), amorphous cellulose (c) and barley grain (d) incubated with rumen fluid (RF) and fresh faeces (FF) inocula. the case of MH (Fig. 2a) differences between PT and GS methods were lower versus other substrates (Table 1) IVDMD and VFA production There was no effect of apparatus on IVDMD (Table 2). GS produced slightly more VFA with MH versus PT, and there was a small increase in the molar proportion of acetate with MH and WS, and in the molar proportion of propionate with WS, in GS (Table 2). IVDMD and total VFA production were lower with FF versus RF (Table 2), and there were differences (but not consistent between substrates) in molar proportions of acetate and propionate Relationship between predicted and observed gas volumes The VFA and gas production data were used to determine relationships between stiochiometrically calculated (Blümmel et al., 1999) and measured gas volumes of the substrates (Fig. 3). Good relationships occurred with both apparatuses (r 2 = 0.938, Sy.x = 4.86 for PT and r 2 = 0.934, Sy.x = 4.99 for GS).

6 86 Z. Váradyová et al. / Animal Feed Science and Technology (2005) Relationship between estimates produced by different apparatuses and inocula Correlation coefficients between total gas production apparatus, and IVDMD and stoichiometrically predicted gas volumes are in Table 3. Total gas production by both PT Fig. 2. Calculated rates of gas production (ml/g DM) at 3, 18, and 30 h from determined gas volumes at 6, 12, 24 and 36 h by mechanical manometer (PT) and glass syringes (GS) methods for meadow hay (a), wheat straw (b), amorphous cellulose (c) and barley grain (d) incubated with rumen fluid (RF) and fresh faeces (FF) inocula.

7 Z. Váradyová et al. / Animal Feed Science and Technology (2005) Fig. 2. (Continued ). and GS methods were related to both IVDMD (R 2 = for PT and R 2 = for GS) and stoichiometrically predicted gas volume (R 2 = for PT and R 2 = for GS). Correlations between in vitro fermentation data occurred when PT was compared to GS (Table 4).

8 88 Z. Váradyová et al. / Animal Feed Science and Technology (2005) Table 1 Rate and extent of gas production when substrates were incubated with either rumen fluid (RF) or fresh faeces (FF) in glass syringes (GS) or bottles with a pressure transducer (PT) Substrate Apparatus Inoculum Gas volume Rate of gas production (ml/g DM h) (ml/g DM) 3h 18h 30h Meadow hay Wheat straw Amorphous cellulose Barley grain PT RF GS RF PT FF GS FF PT RF GS RF PT FF GS FF PT RF GS RF PT FF GS FF PT RF GS RF PT FF GS FF PT RF GS RF Control PT FF GS FF S.E.M Significance ns, not significant. * P<0.05. ** P<0.01. *** P< Apparatus (A) ** ns ns Inoculum (I) *** *** *** *** Substrate (S) *** *** ** ns A I *** ns ns ns A S *** *** ns ns I S *** *** ** *** A I S *** ** ns 4. Discussion 4.1. Comparison of two in vitro fermentation gas production methods (PT and GS) The initial volume of gas produced was higher with the pressure transducer versus syringe apparatus, but GP curves were generally similar. When using PT, more gas was added during bottle preparation (i.e., flushing with CO 2, sealing, warming and inoculation), which would result in a pressure increase, possibly explaining profile differences between apparatuses.

9 Z. Váradyová et al. / Animal Feed Science and Technology (2005) Table 2 Fermentation and degradation of substrates incubated with either rumen fluid (RF) or fresh faeces (FF) in either glass syringes (GS) or bottles with a pressure transducer (PT) Substrate Apparatus Inoculum IVDMD Total VFA Molar proportion of VFA (mm) Acetate Propionate n-butyrate Meadow hay Wheat straw Amorphous cellulose Barley grain PT RF GS RF PT FF GS FF PT RF GS RF PT FF GS FF PT RF GS RF PT FF GS FF PT RF GS RF PT FF GS FF PT RF GS RF Control PT FF GS FF S.E.M Significance ns: not significant. * P<0.05. ** P<0.01. *** P< Apparatus (A) ns ns ns ns ns Inoculum (I) *** *** *** *** *** Substrate (S) *** *** *** *** *** A I *** *** ns ns A S ns *** *** I S *** ** *** *** *** A I S ** ** Table 3 Correlation coefficients between total gas production measured with different apparatus and the estimated IVDMD and stoichiometrically predicted gas volume Apparatus Pressure transducer Glass syringe IVDMD Stoichiometrically predicted gas volume

10 90 Z. Váradyová et al. / Animal Feed Science and Technology (2005) Fig. 3. Relationship between stiochiometrically calculated and measured gas volumes by mechanical manometer (PT) and glass syringes (GS) of four substrates incubated with rumen fluid and fresh faeces. Although the fully automated system described by Cone et al. (1996) is very sensitive, relatively good results can be obtained by simpler techniques (Beuvink et al., 1992; Prasad et al., 1994; Váradyová et al., 1998). The accuracy of both methods used here depends on the accuracy of reading the gas volume in the syringes or reading the manometers. It is clear that GS requires expensive gas-tight syringes with capacity limitations and a laborious requirement for time-consuming measurements, which can be a disadvantage. Also, the syringes are narrow and it is difficult to place more than 500 mg of sample into Table 4 Correlation coefficients and significance of correlation of in vitro fermentation data obtained with either a pressure transducer or glass syringes, or using either fresh faeces or rumen fluid as inoculum Parameter Comparison Pressure transducer with glass syringes Rumen fluid with fresh faeces Total gas volume *** Rate of gas production (3 h) ** ** Rate of gas production (18 h) *** Rate of gas production (30 h) *** IVDMD *** Total VFA *** Molar proportion acetate *** *** Molar proportion propionate *** *** Molar proportion n-butyrate *** ** P<0.01. *** P<0.001.

11 Z. Váradyová et al. / Animal Feed Science and Technology (2005) them. When larger amounts of samples are incubated, or substrates have a high potential GP, more gas is produced which requires frequent push-back of the piston, which can also be a source of error. However with PT, if gas is not recorded and released frequently enough, the accumulated pressure may negatively affect microbial fermentation and cause errors in gas measurement. The accuracy of the PT method is indicated by the good relationship between gas pressures and gas volumes that are linearly related over a wide range of pressure readings (i.e., R 2 > 0.97; Theodorou et al., 1994). For use in gas production studies, the pressure transducer has a range of kpa, although more scattering was observed with data points at the higher range of pressure readings (over 100 kpa) than at the lower range (up to 40 kpa). Comparison of PT and GS suggested advantages and disadvantages of each. While they both gave similar gas production profiles, there was a higher initial gas volume with PT, but rates of gas production and total gas produced, as well as IVDMD and VFA production were well correlated. Both predicted gas volumes similar to those calculated stoichiometrically, and the data produced from one apparatus can be extrapolated, with caution, to the other Comparison of the two inoculum sources (RF and FF) When using in vitro techniques, variables, such as diet of the donor animal, adaptation of the microbial population, time of collection of ruminal fluid after feeding and degree of anaerobiosis attained in incubation may all affect the gas production profile (Grant and Mertens, 1992; Cone et al., 1996). Comparison of gas production with RF and FF confirmed that FF produced less gas than RF (Goncalves and Borba, 1996; Cone et al., 2002). According to Mauricio et al. (2001) lower fermentation (i.e., less gas production) with FF is a result of factors, such as provision of substrates to the rectum with lower nutritive value, shorter retention times, lower populations of bacteria or absence of protozoa, compared to the rumen. Results from this experiment show that gas production with FF was higher for MH and BG versus WS and AC. These differences in total gas production among substrates indicate that FF may not have the potential to be used for poor quality substrates, such as WS. El-Meadaway et al. (1998) also used faeces as a source of microbial inoculum in gas production studies using the method of Menke et al. (1979), concluding that cattle faeces had the potential to be used instead of rumen fluid, but not for poor quality forages, such as barley straw. Amorphous cellulose also seems to be an unsuitable substrate for determining IVDMD with FF, and the lower gas production of AC with FF demonstrates the lower cellulolytic activity of FF versus RF. The lower volumes of gas with FF is probably due to differences in microbial activity, or diversity of types and number of microorganisms between inocula. Total VFA production after 96 h of incubation for all substrates was higher with RF versus FF. The molar proportion of propionic acid varied among substrates, although the acetic:propionic ratio was similar for RF and FF, except for BG. These differences in VFA, due to different sizes and activities of microbial populations in the inocula, were similar to findings of El-Meadaway et al. (1998). Fresh faeces from sheep and cattle have successfully replaced rumen inocula for in vitro digestibility in some studies (Harris et al., 1995; Akhter et al., 1999). However, our results

12 92 Z. Váradyová et al. / Animal Feed Science and Technology (2005) show that IVDMD of substrates differed with RF and FF, and that results were poorly correlated. Use of FF decreased IVDMD versus RF, especially for WS and AC. Reduction of IVDMD for straws with faecal inoculum was also observed by Omed et al. (1989), athough El-Shaer et al. (1987) and Nsahlai and Umunna (1996) reported that IVDMD determination with 3% fresh faeces was similar to that obtained using RF. It is possible that an increased concentration of FF might result in increased estimates of IVDMD and gas production, such that results obtained by RF and FF could be more similar. However, the FF used here had no relationship to the degradative and fermentative capacity of RF and, on the basis of this evidence, FF cannot be used to replace RF. However, predictive relationships between RF and FF have been observed, and a means of mapping the gas production profile produced by RF using data obtained with FF has been proposed (Dhanoa et al. (2004). 5. Conclusions Although computerized systems for measuring gas production kinetics are very sensitive and precise, the non-automated methods are less expensive, relatively simple to handle and do not require sophisticated equipment. Despite some limitations to use of simple methods, both the pressure transducer and gas syringe methods used here to measure IVGPT were suitable to determine fermentation kinetics of feeds, and results obtained from both apparatuses were well correlated. Both apparatuses also produced volumes of gas that were well related to stoichiometrically predicted gas volumes. There were large differences in in vitro fermentation with fresh faeces versus rumen fluid from sheep, and no relationship between the data occurred. If fresh faeces is to be used as an alternative inoculum for in vitro gas production procedures, further work is required to determine preparation methods that enable its results to be related to data obtained from rumen fluid. Acknowledgements The study was supported by funds from Grant Agency for Science of the Slovak Academy of Sciences (2/3058/23) and ŠPVT (2003 SP 51/ / ). References Akhter, S., Owen, E., Theodorou, M.K., Butler, E.A., Minson, D.J., Bovine faeces as a source of microorganisms for the in vitro digestibility of forages. Grass Forage Sci. 54, Aiple, K.P., Steingass, H., Menke, K.H., Suitability of a buffered faecal suspension as the inoculum in the Hohenheim gas test. J. Anim. Physiol. Nutr. 67, Beuvink, J.M.W., Spoelstra, S.F., Hogendorp, R.J., An automated method for measuring time-course of gas production of feedstuffs incubated with buffered rumen fluid. Neth. J. Agric. Sci. 40, Blümmel, M., Ørskov, E.R., Comparison on in vitro gas production and nylon bag degradability of roughage in prediction feed intake in cattle. Anim. Feed Sci. Technol. 40,

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