Effect of individual protozoa , Isotricha intestinalis and Metadinium medium , on ruminal fermentation and methane production in vitro

The effect of rumen protozoa I. intestinalis and M. medium on the fermentation of lucerne hay and maize grain substrates was evaluated in vitro using rumen fl uid from monofaunated wethers. I. intestinalis had a positive effect on the extent of feed degradation, especially in maize-containing substrates, but this effect was offset by an increase in methane production as compared to fauna-free or mixed-fauna fermentations. M. medium had no major infl uence on substrate degradation or production of fermentation products. The fermentation ability of both species, however, was differently infl uenced by the presence of fauna-free rumen fl uid originated from defaunated or faunated animals.


INTRODUCTION
Mixed rumen protozoa are positively associated with increases in feed degradation and methane production.However, the contribution of individual protozoal species is less known.The fermentation characteristics and methane production of two metabolically different protozoa, I. intestinalis and M. medium, were evaluated in vitro.

MATERIAL AND METHODS
Six wethers fi tted with rumen cannulae were used as donors of rumen fl uid.Two animals had a regular mixed protozoal population.The other four animals had previously been defaunated following the method of Jouany and Senaud (1979); two of them were kept free of fauna and the remaining two animals were each inoculated with I. intestinalis or M. medium.These animals were kept defaunated or monofaunated for more than one year.All animals were fed a maintenance diet twice daily.

Experiment 1
In a fi rst experiment, rumen fl uid from defaunated, monofaunated, and faunated animals was used to ferment lucerne hay or maize grain.Whole rumen contents were collected before the morning feeding and strained through a polyester monofi lament fabric (250 µm mesh aperture) under a stream of CO 2 to remove solids.Rumen fl uids were mixed in a 1:3 ratio with an anaerobic buffer solution (Goering and Van Soest, 1970) kept at 39°C under O 2 -free CO 2 gas.This rumen fl uid-buffer mixture was utilized immediately to inoculate fermentation vials containing lucerne hay or maize (300 mg) as substrates and incubated anaerobically at 39°C for up to 24 h.Vials without substrate were used as controls.At the end of the incubation period gas production was measured with the aid of a pressure transducer and a sample collected for analysis of constituents by gas chromatography.Vial contents were centrifuged; supernatants were processed for analysis of soluble fermentation products and pellets used for estimation of dry matter degradation (DMD).The experiment was repeated in time.

Experiment 2
Experiment 2 was designed to minimize the confounding effect of donor animal on the infl uence of fermentation activities by protozoa.Rumen fl uid from defaunated, monofaunated, and faunated animals was obtained as above for Experiment 1. I. intestinalis and M. medium cells were collected by a low-speed centrifugation, and then resuspended in rumen fl uid supernatant from defaunated or faunated animals and used to ferment an lucerne:maize (70:30) mixed feed.Rumen fl uid from defaunated and faunated animals was also centrifuged and the supernatant and pellet obtained were mixed.Vials without substrate were used as controls.Measurements were as in Experiment 1.
Data was statistically analysed by one-way analysis of variance using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC).

RUMEN PROTOZOA FERMENTATION CHARACTERISTICS RESULTS AND DISCUSSION
The fermentation characteristics of the protozoal inocula differed depending on the substrate (Table 1).For lucerne hay there was no difference in DMD among inocula.However, in monofaunated inocula, gas production decreased while total VFA production increased as compared to fauna-free and mixed fauna treatments.This favourable shift in the partition of fermented organic matter between VFA and gas in monofaunated inocula could be benefi cial to the host in terms of available energy.Unexpectedly, methane production was lower in the mixed fauna inoculum.For the high starch, maize grain substrate, rumen fl uid containing I. intestinalis was the most effi cient inoculum.Increases in DMD (P<0.05) were correlated to higher production of gas, methane and VFA (Table 1).However, the amount of VFA and methane produced by unit of substrate disappearance was not different among inocula (P>0.05).The addition of individual protozoal species to fauna-free rumen fl uid from defaunated animals increased the production of methane for I. intestinalis, and induced a numerical increase in the amount of DMD for both species as compared to defaunated rumen fl uid controls (Table 2).In contrast, total gas and VFA production remained unchanged.When protozoa were combined with fauna-free rumen fl uid from conventional animals, the presence of I. intestinalis improved substrate degradation markedly without a concomitant increase in methane production.In contrast, M. medium presence did not affect DMD but decreased (P<0.1)methane emissions.RANILLA M.J. ET AL.

CONCLUSIONS
The decrease in methane emissions that follows the elimination of protozoa from the rumen was not observed in vitro using rumen inocula from animals with a stable microbial population.I. intestinalis had a positive effect on the extent of degradation, especially in maize-containing substrates, but this effect was counterbalanced by an increase in methane production.In contrast, M. medium did not stimulate methane production.Monofaunation is not an intermediate state between defaunation and faunation; the fermentation profi les of monofaunated rumen fl uids were characteristic for each species.The presence of distinct protozoal populations certainly induce changes in the ruminal microbial ecosystem that help to explain the differences observed in this work among inocula, differences that cannot be attributed to protozoa alone.

Table 1 .
Fermentation characteristics of rumen fl uid from animals harboring single, mixed, and no

Table 2 .
Fermentation characteristics of I. intestinalis and M. medium suspended in fauna-free rumen fl uid from defaunated or faunated animals (Experiment 2)