Nd. Luchini et al., PRESERVATION OF RUMINAL MICROORGANISMS FOR IN-VITRO DETERMINATION OF RUMINAL PROTEIN-DEGRADATION, Journal of animal science, 74(5), 1996, pp. 1134-1143
Ruminal microorganisms, preserved either lyophilized or frozen, were c
ompared with freshly strained ruminal fluid for proteolytic activity a
nd as inoculum source for determination of ruminal protein degradation
rates by the inhibitor in vitro method. Dialysis and glycerol additio
n had no effect on the proteolytic activity of preserved microorganism
s. Net release of NH3 and total amino acids from protein using the flu
id plus particle-associated microorganisms was higher than that found
using the fluid-associated microorganisms alone. Method of inoculum pr
eservation altered total proteolytic activity, but harvesting bacteria
using centrifugal force greater than 5,000 x g did not increase prote
olytic activity of the pellet. The proposed method for harvesting and
preserving microorganisms consisted of centrifuging strained ruminal f
luid at 5,000 x g (30 min at 4 degrees C), stirring the pellet in a 50
:50 (vol/vol) solution of glycerol-McDougall's buffer for 15 min, and
then storing at -20 degrees C. Protein degradation rates in incubation
s with preserved microorganisms were four to eight times slower than w
hen using fresh ruminal fluid; however, feed proteins were ranked simi
larly for degradation rate. Preincubating the preserved microorganisms
reduced blank concentrations of NH3 and total amino acid and increase
d protein degradative activity of the preserved inoculum. Degradation
rates with preincubated, preserved inocula were similar to those obtai
ned using fresh ruminal fluid. These results indicated that mixed rumi
nal microorganisms can be preserved by freezing and, after a preincuba
tion period of 6 h, used as the inoculum source for in vitro estimatio
n of ruminal protein degradation.