Screening of plants from diversified natural grasslands for their potential to combine high digestibility, and low methane and ammonia production.

A total of 156 plant species from 35 botanical families collected from diversified grasslands in the French Massif Central were screened in vitro for their potential to combine high nutritive value for ruminants and a reduced impact on the environment. The vegetative part of plants were analyzed for their chemical composition and incubated in a batch system containing buffered rumen fluid at 39°C for 24 h. The gas production and composition were recorded, and the fermentation end-product concentrations in the incubation medium and the in vitro true organic matter digestibility (IVTOMD) were determined. The results were expressed relative to perennial ryegrass (PRG) values used as a reference. We observed that no relationship between methane (CH4) and volatile fatty acids (VFA) was evidenced for 12 plants, the fermentation of these plants producing significantly less CH4 for a similar level of VFA production. In all, 13 plants showed 50% less CH4 production per unit of organic matter truly digested (OMTD) than PRG. Among these plants, two reduced CH4 by more than 80% and four species had an IVTOMD higher than 80%. The underlying modes of action seem to be different among plants: some result in an accumulation of H2 in the fermentation gas, but others do not. In terms of nitrogen (N) use efficiency, the fermentation of 37 plants halved the ratio between ammonia (N-NH3) and plant N content compared with PRG, of which six showed a complete absence of N-NH3 in the medium. Among these plants, four maintained the IVTDMO at values not significantly different from PRG (P>0.05). Considering the multi-criteria selection, 16 plants showed simultaneously a reduction of more than 80% in N-NH3 production and 30% in CH4 emission per unit of OMTD compared with PRG, including three with an IVTOMD higher than 80%. Overall, the botanical families that reduced simultaneously CH4 and N-NH3 most efficiently were the Rosaceae, Onagraceae, Polygonaceae and Dipsacaceae. The Onagraceae also gave high values for IVTOMD.

Occurrence of associative effects between grasses and legumes in binary mixtures on in vitro rumen fermentation characteristics.

When animals are fed a grass and legume mixture, digestive interactions can occur in the rumen between the substrates contained in the different plants, and the response of the animal to the combination of forages can differ from the balanced median values of their components considered individually. Our objective was to assess the associative effects between temperate forages in 8 grass-legume binary combinations on in vitro rumen fermentation characteristics to highlight synergies or antagonisms in terms of nutritional and environmental impacts. Two grasses (cocksfoot, CF; ryegrass, RG) and 4 legume species (alfalfa, A; white clover, WC; red clover, RC; sainfoin, SAN) were incubated alone and in grass-legume mixture (1:1, wt/wt) in batches containing buffered rumen fluid during 3.5 and 24 h. For each substrate and each incubation time, parameters describing the degradation of the energetic and nitrogenous compounds and their partition into fermentation end products were measured. Data were subjected to ANOVA using a mixed procedure to test quadratic contrasts. At 3.5 h of incubation, many quadratic effects were observed. The presence of A, WC, or RC in mixtures quadratically increased the NH(3)-N production (up to +28% when compared with the calculated value, P < 0.001), whereas the presence of condensed tannin (CT) activity in SAN considerably decreased it (up to -67%, quadratic effect, P < 0.001) and the N disappearance. To a lesser extent, the presence of SAN reduced the proportion of methane in the gas produced (up to 7%, quadratic effect, P = 0.018). Generally, the degradation of OM and NDF was not stimulated by mixing grass and legume, except for SAN. However, the presence of SAN severely impaired the NDF digestion at the early phase of fermentation. At 24 h of incubation, few associative effects were observed in comparison with those observed at 3.5 h of incubation, but the effect of CT of SAN on N metabolism was still clearly present. This study shows that, among the grass-legume mixtures tested in vitro, only the presence of SAN can interact with the grasses to reduce the degradation of proteins and the production of methane but with a transitory negative effect on fiber digestion.

Two-step freezing procedure for cryopreservation of rumen ciliates, an effective tool for creation of a frozen rumen protozoa bank.

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25 degrees C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kisidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of -30 degrees C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2 degrees C/min and 2.5 degrees C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.