The in vitro gas Production and Ruminal Fermentation of Various Feeds using Rumen Liquor from Swamp Buffalo and Cattle

The objective of this study was to evaluate the effects of various feeds using rumen fermentation in inoculum of buffalo and cattle by using the in vitro gas technique. Incubations were carried out using rumen fluid obtained from rumen-fistulated swamp buffalo and cattle during which rice straw was fed on ad libitum as a main feed with minimal amount of concentrate (concentrate mixture: 12% CP and 76% TDN) feeding. The fermentation kinetics from twelve feeds commonly used in ruminant was studied using in vitro gas production technique. Rumen fermentation parameters substrate disappearance and Volatile Fatty Acids (VFA) production were determined after 96 h of incubation. The results revealed that high potentials for gas production were obtained in concentrate, upper-leaf cassava hay, cassava hay, Trichanthera gigantea, lower-leaf cassava hay, Plia fran leaf (Pluchea indica) and were highly degradable in the rumen of both species, especially significantly higher in swamp buffalo than in cattle fluid. However, rumen NH₃, VFAs, acetate and acetate to propionate ratio in cattle were higher than in buffalo. However, the propionate production resulted higher in buffalo (p<0.001) than in cattle. Therefore, the results indicate that twelve feeds in total gas production and C₃ in buffalo were higher than in cattle liquor accept for rumen NH₃-N, total VFAs, C₂, C₄ and C₂: C₃ concentrations.

INTRODUCTION

The comparison of rumen metabolism of buffalo and cattle are quite challenging and interesting in order to understand the rumen microbial activities of these ruminant species under the same feeding or under different conditions. Earlier, Wanapat (1989) found that buffaloes could utilize feed more efficiently, particularly with low quality roughages with the digestibility of feed 3-5% higher than in cattle. Furthermore, Wanapat et al. (2000) suggested that buffaloes had higher population of cellulolytic bacteria, fungal zoospores, lower protozoal population and a greater recycle nitrogen to the rumen than in cattle. Nevertheless, Franzolin et al. (2010) observed that no affect of energy or nitrogen sources on rumen protozoa counting in buffalo and cattle. While Misra et al. (2002) also observed higher dry matter intake in buffaloes than in cattle fed on sorghum stover and supplemented with urea. On the other hand, Pradhan et al. (1997) observed that dry matter intake per unit metabolic body size was lower in buffalo than in cattle.

Calabro et al. (2008) have carried out in vitro studies with rumen fluid incubated with common feedstuffs for ruminants; it was found that gas production was lower for inoculum derived from buffalo than for samples from the rumen of cattle. However, the amount of dry matter intake was different between the buffaloes and cattle according to feeding system (Franzolin et al., 2010). Currently, some feed additives that are capable of influencing fiber fermentation and digestion in ruminants were developed (Lee and Ha, 2003; Patra, 2011). Their by-products could be economically used as potential fibrous, energy and protein sources in ruminant nutrition (Aghajanzadeh-Golshani et al., 2010), while legumes are important in order to design feeding strategies for ruminant animals on low quality roughages (Kiraz, 2011; Chanthakhoun et al., 2011). Moreover, Sunvold et al. (1995) reported that the in vitro gas production technique were useful for study the differences in fermentation patterns, micro-organisms between animal species inoculum.

However, the understanding and research data of the rumen fermentation used end-products of these ruminant species with different feed sources has been limited. Therefore, the aim of this study was to evaluate the effects of various feeds using rumen fermentation in inoculum of buffalo and cattle by using the in vitro gas technique.

MATERIALS AND METHODS

Crop residues and selected roughages as substrates: Twelve feeds were used for in vitro assay, namely: (Tua-mun, Phaseolus calcaratus), cassava chip, rice straw, sorghum, urea treat rice straw, corn cob, concentrate, upper-leaf cassava hay, cassava hay, Trichanthera gigantea, plia fran leaf (Pluchea indica), lower-leaf cassava hay. These feeds were collected from the farm at Khon Kaen university, North-East of Thailand. Duplicate fresh samples (0.5 kg replicate) were dried in a hot, dry air force oven at 65°C for 72 h and weighed. The samples were then ground to pass through a 1 mm screen for in vitro incubation and chemical analysis. The samples were analyzed for DM, CP and ash content by procedures of AOAC (1990). NDF and ADF were assayed using the method of Van Soest et al. (1991). Chemical composition is presented in Table 1.

In vitro gas production and fermentation technique: Rumen fluid was collected before the morning feeding from two ruminally fistulated swamp buffalo (Bubalus bubalis) and cattle (NativexBrahman) with live weight 400±5 and 320±5 kg, respectively.

Table 1:	Chemical composition of local feed resources used in in vitro gas production incubated in rumen liquor of buffalo and cattle
DM: Dry matter, OM: Organic matter, CP: Crude protein, NDF: Neutral-detergent fiber, ADF: Acid-detergent fiber, CT: Condensed tannins

The animals were offered rice straw on ad libitum and fed at 0.5% body weight of concentrate (12% CP and 76% TDN). The animals were fed twice daily, water and mineral licks were available ad libitum for 14 days. Rumen fluid was taken from the middle part of the rumen. The 1000 mL rumen liquor was obtained from each of the buffalo and cattle before the morning feeding. The rumen fluid was filtered through four layers of cheesecloth into pre-warmed thermos flasks. Preparation of artificial saliva was done according to Menke and Steingass (1988). Artificial saliva was prepared and rumen fluid was mixed in a 2:1 ratio to prepare fermentation solution. The serum bottles with the mixture of substrate treatments were pre-warmed in a water bath at 39°C for 1 h before filling with 30 mL of rumen inoculum's mixture. During the incubation, the gas production was recorded at 0, 2, 4, 6, 8, 10, 12, 18, 24, 36, 48, 60, 72 and 96 h. Cumulative gas production data were fitted to the model of Orskov and McDonald (1979) as follows:

y = a+b(1-e^(¯ct))

where, a is the gas production from the immediately soluble fraction, b is the gas production from the insoluble fraction, c is the gas production rate constant for the insoluble fraction (b), t is incubation time (a+b) is the potential extent of gas production and y is gas produced at time “t”.

Determination of fermentation parameters: The sample inoculum was collected at 0, 4, 6, 8 and 12 h post feeding and were divided into 2 portions; the first portion was centrifuged at 16,000x g for 15 min and the supernatant was stored at -20°C for VFA analysis using HPLC (Samuel et al., 1997) and the second portion was used for NH3-N analysis using the micro-Kjeldahl methods (AOAC, 1990).

Statistical analysis: For curve fitting, the 1 mL of gas produced per g in time profiles were fitted to the model y = a+b(1-e^(¯ct)) (Groot et al., 1996).

The fermentation characteristics and the fitted parameters were subjected to analysis of variance to detect the inoculum of buffalo and Cattle rumen fluid and various treatment effects; in the model the treatmentxinoculum interaction by SAS (1996).

RESULTS AND DISCUSSION

Chemical composition of crop residues and roughages: Chemical compositions of crop residues and roughages are presented in Table 1. The crude protein content of the crop residues and roughage ranged from 2.7 to 24.3%. Rice straw had the lowest crude protein content while the upper-leaf cassava hay had the highest crude protein content. Similar crude protein content was observed in Trichanthera gigantea (21.1%) and cassava hay (21.6%). However, crude protein of their substrate in this area was similar to those of Wanapat (2009).

Gas production, kinetic analysis of gas production: Cumulative gas production for each of the substrate treatments are presented as gas production and values for the kinetics of gas production models for substrates studied are given in Table 2. There were not significantly difference (p>0.05) in the intercept (a), the fermentation of the soluble fraction of buffalo and cattle. However, there were not significantly different among treatments and species (p>0.05).

Table 2:	In vitro fermentation characteristics of the substrates with buffalo and cattle rumen fluid
T1: Tua-mun (Phaseolus calcaratus), T2: Cassava chip, T3: Rice straw, T4: Sorghum, T5: Urea treat rice straw, T6: Corn cob, T7: Concentrate, T8: Upper-leaf cassava hay, T9: Cassava hay, T10: Trichanthera gigantea, T11: Lower-leaf cassava hay, T12: Plia fran leaf (Pluchea indica); MSE: Mean square error; NS: Non significant; *: p<0.05; ***: p<0.001

These results were similar to other values as reported by Blummel and Orskov (1993) while Khazaal et al. (1993) reported negative values for various substrates when using mathematical models to fit gas production kinetics. This was probably due to either a deviation from the exponential cause of fermentation or delays in the onset of fermentation due to microbial colonization. It is well known that the value for absolute a (a), described ideally, reflects the fermentation of the soluble fraction.

Whereas, gas production from the insoluble fraction (b) of buffalo and cattle ranged from 62.8 to 178.0 and 56.3 to 138.9, respectively and were significantly different among treatments and species (p<0.05). The potential extent of gas production (a+b) of buffalo and cattle ranged from 63.5 to 168.6 and 53.6 to 132.0, respectively and were significantly different among treatments and species (p<0.05). Gas production rate constants for the insoluble fraction (c) of buffalo and cattle were not significantly different among treatments (p<0.05). Cumulative gas production at 96 h of buffalo and cattle were significantly different (p<0.01) among treatments and were higher in buffalo than in cattle. However, these were no significant interaction between treatments and species (p = 0.05).

Table 3:	Ruminal fermentation end-products parameters measured
T1: Tua-mun (Phaseolus calcaratus), T2: Cassava chip, T3: Rice straw, T4: Sorghum, T5: Urea treat rice straw, T6: Corn cob, T7: Concentrate, T8: Upper-leaf cassava hay, T9: Cassava hay, T10: Trichanthera gigantean, T11: Lower-leaf cassava hay, T12: Plia fran leaf (Pluchea indica); MSE: Mean square error; NS: Non significant; *: p<0.05; ***: p<0.001

On the other hand, Chumpawadee et al. (2006) reported that the kraphanghom, rice straw, corn stover, chinese spinach and cavalcade hay were highly digestible in the rumen while, Khazaal et al. (1995) reported that protein fermentation did not lead to much gas production. In addition, fibrous constituents also negatively influenced in vitro gas production (Melaku et al., 2003). In the present study, it was found that high potentials for gas production were observed in concentrate, upper-leaf cassava hay, cassava hay, Trichanthera gigantean, lower-leaf cassava hay, plia fran leaf (Pluchea indica) and were highly digestible in buffalo than in cattle fluid.

In vitro fermentation products: The effect of twelve substrates on in vitro fermentation NH₃ and VFAs were given in Table 3. There were significantly difference (p<0.01) in the NH₃, total volatile fatty acid and butyrate, while the proportions of NH₃, VFAs, acetate, butyrate and acetate to propionate ratio, were significant different (p<0.01) found in cattle than in buffalo. Therefore, this may be due to cattle were able to utilize concentrate better than buffalo. However, Beever and Mould (2000) who reported that high forage diets were higher C₂ and C₄ whike high starch diets were higher C₃ although C₂ was still the predominant VFA. Thus, under earlier experiment, cattle and swamp buffaloes also showed differences in rumen bacterial, protozoal population and fungal zoospore counts (Wanapat et al., 2000). Under similar experimental condition, Malakar and Walli (1995) found that the average counts of anaerobic bacteria were significantly higher for buffalo than for cow.

CONCLUSIONS

Based on this experiment, it could be summarized that there were differences in gas production, fermentation end-products using rumen liquor from swamp buffalo and cattle. These feeds were highly degradable in the rumen of both species. The differences found could contribute to the capability of feed utilization in the two species. However, more in vivo digestion and feeding trials should be investigated further.