Design and application of a stratified continuous rumen fermentation system

In the first half of the twentieth century several ruminal in vitro systems were developed. These systems can be classified as \u201cbatch culture\u201d or \u201ccontinuous culture\u201d. The continuous culture systems (CC) had a quite complex evolution process, and those nowadays used are classified as RUSITEC, Dual Flow Continuous Culture (DFCC) or Single Flow Continuous Culture (SFCC). The diverse CC are characterized by having different design and different working settings, such as: volume and shape of the fermentation vessel, stirring device, artificial saliva, solid and liquid flow and dilution rate, volume and origin of the rumen inoculum, feeding practice and device. On the bases of the characteristics of the already existing ruminal continuous cultures, a stratified continuous culture has been developed at the Laboratory of Nutrition of the Department of Agricultural and Environmental Sciences of University of Udine. This system has the peculiarity of a simple design and of being built by using cheap and readily available materials. The CC consists of 8 x 2 L glass bottles, warmed at 39\ub0C and placed on a water proof magnetic stirrer. The bottles are closed with a rubber stopper, provided with an insert for the inflow of artificial saliva, and have the outflow at the base. A first trial has been made to test the stratified continuous culture. In particular, the capability of maintaining protozoa populations and reduce the accumulation of undigested material was investigated in two separated experiments. In Experiment 1 was evaluated the effect of two stirring frequencies (continuous vs intermittent) and two dilution rates (D, 1.29 vs 1.04) on the pH values and on the counts of the protozoa in the fermentation fluid. Experiment 2 aimed to test the effects of varying the daily diet amount (15, 20, 25, 30 g DM/d of a diet with 50:50 forage: concentrate ratio) on the organic matter digestibility and on the fermentative patterns. In 2 fermentation runs of 8 d each, bottles were inoculated with 450 ml of rumen fluid and 1050 ml of artificial saliva, which was then pumped at a rate of 78 ml/h. The tested CC reached stable conditions of fermentation after some days of adaptation and maintained an acceptable concentrations of the protozoa population till the end of the trial. The results of the second experiment suggest that the amount of feed administered to the fermenters has to be properly chosen to limit the accumulation of undigested materials in the glass bottles and maintain the fermentation patterns within acceptable ranges. A second trial was organized with the aim to understand the effect of urea administration on the fermentation parameters; the trial consisted in two experiments. In the first experiment, a specified dose of urea was used to simulate the regular release of nitrogen in the rumen in a continuous fermenter environment. The aim was to study the rumen metabolism at controlled defined conditions of release of nitrogen, to identify the conditions of the optimal matching between nitrogen release and the rumen bacterial degradation of the carbohydrate fraction. The experiment consisted of 2 runs of 7 days each (4 adaptation days and 3 measuring days). The fermenters were supplied with 12 g DM/d of a diet containing corn silage (30% DM), hay (10% DM), dry corn (50% DM) and a mineral vitamin supplement (10% DM), divided in two equal portions at 09:00 and 15:00. The urea supplement was administered in 1, 2, 3 or 4 doses at 1 hour intervals. The second experiment, performed at the Ruminant Nutrition Laboratory of Utah State University, investigated the effects of supplementing slow-release urea (SRU; Optigen, Alltech, Nicholasville, KY) in 3 lactation dairy diets with different forage-to-concentrate ratios (F:C) on in vitro ruminal fermentation characteristics. The experiment was performed in 4 independent runs of continuous cultures. Each fermenter was offered a diet of 20 g DM/d in 2 equal portions at 08:00 and 20:00 h. In both experiments variations of pH values were detected with the administration of urea at doses usually utilized in ruminant diets and in the range of rumen acidity. In experiment 1, high concentrations of rumen ammonia increased propionate with a tendency of reducing the acetate:propionate ratio. Moreover, in experiment 2, the substitution of fiber from forages with that of byproducts in diets determined a reduction of the ammonia accumulation with a reduction in buffering the rumen; a reduction of the acetate concentrations and an increase in propionate concentrations and a reduction of the methane yield. The third trial aimed to test the effects of increasing the dose of an active dry Saccharomyces cerevisiae on rumen metabolism in a rumen continuous culture system. The fermenters were supplied with 12 g DM/d of a diet containing corn silage (20% DM), hay (10% DM), alfalfa hay pellet (20% DM), and a compound feed (50% DM), divided in 2 equal portions at 9:00 and 16:00. At each feeding time, all the fermenters received a supplement (0.025 g per feeding) consisting of a commercial yeast extract and/or soybean meal in differing ratios Y:S (0:500, Y0; 5:495, Y5; 50:450, Y50; 500:0, Y500). In the present trial, for several measurements, such as pH, ammonia, and some protozoa numbers, a significant difference between control and yeast inclusion treatments were detected, but there was no dose-treatment effect, even though the doses of yeast followed an exponential rate of increase. A possible explanation of the lack of differences between yeast doses could be because of the favorable in vitro conditions for their survival and multiplication of the yeast (e.g. high pH values, reducing conditions, etc.) which could have favored the colonization of the fermentation bottles to a similar level, independently of the initial dose of yeast. In general, the stratified continuous fermenter was able to reproduce the ruminal environment in a way to detect even small differences in the parameters measured as a consequence of the diets tested. In the experiments were measured mainly the basic fermentative parameters (pH, VFA, NH3, digestibility), with the future aim to integrate these analysis with technics of molecular biology, to better study the ruminal microbial population