PRESERVATION OF RUMINAL MICROORGANISMS FOR IN-VITRO DETERMINATION OF RUMINAL PROTEIN-DEGRADATION

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.