In situ and in vitro techniques for studying rumen fermentations: methodology and applications

Abstract The general aim of this thesis was to study rumen fermentation processes using in situ and in vitro digestibility and in vitro gas production techniques. The thesis includes the results of five experiments. The first experiment investigated the validity to reduce the amount of feed sample incubated in filter bags with DaisyII (from 0.50 to 0.25 g feed/bag). This hypothesis was verified by considering the results of two separate trials (conducted on 4 and 7 feeds, respectively) in which the digestibility values provided by DaisyII were compared with those achieved using a conventional batch culture. Results evidenced that, by using the lower sample size (0.25 g feed/bag), digestibility values provided by DaisyII resulted more similar, less variable and better correlated with those achieved with the batch culture. The second experiment was conducted on 11 feeds and compared the digestibility values achieved: i) in situ and in vitro using two different kinds of bag (nylon and filter) with a different pore size (40 and 25 µm); ii) in situ, placing the bags in the rumen environment, and in vitro using rumen fluid collected from intact cows by a suction technique. Results of the experiment showed that the use of filter bags induced a systematic, but predictable, underestimation of digestibility values compared to the nylon bags. Rumen fluid collected from intact cows provided digestibility values directly proportional to those obtained in situ. This latter result is relevant, as the validity of using rumen fluid collected from intact cows can be of interest for many research centers, in order to overcome ethical and public concerns related to the use of surgically treated animals. The third experiment was conducted on the same feeds analysed in the second experiment and it was aimed to compare the energy value of feeds estimated using three different equations: i) one equation based on feed chemical composition and in situ digestible NDF at 48 h of incubation (MENRC); ii and iii) two equations based on feed chemical composition and in vitro gas production at 24 h of incubation (MEMenke and MEUCD). A further aim was to evaluate the validity of reducing the in situ incubation time from 48 to 24 h. Results of the trial evidenced that, when MENRC estimates were taken as reference, MEUCD equation provided feed energy estimates more accurate and precise compared to MEMenke. However, the accuracy and precision of MEMenke estimates increased when four feeds rich in protein (CP > 16% DM) were excluded from the analysis. As the in situ digestibility values achieved at 24 and 48 h were highly correlated and showed a comparable reproducibility, the reduction of in situ incubation time from 48 to 24 h seems can be proposed for reducing the cost of the tests. The fourth experiment was aimed to compare the effects of two different systems of gas release (venting at fixed times or at fixed pressure) on gas production (GP) kinetics provided by two feeds with a different degradability (meadow hay and corn meal), using an innovative GP system. Results of the experiment showed that the venting system exerted significant effects on GP kinetics. The effects were particularly accentuated for corn meal and for venting at fixed times. Therefore, when venting at fixed times is applied, the headspace volume of GP system, the venting frequency and the amount of fermentable matter incubated must be carefully balanced to avoid the generation of high pressures, which could alter GP kinetics. However, venting at fixed pressure is preferable to that at fixed times, as it can strongly reduce these shortcomings. The fifth experiment aimed to evaluate the effects of increasing dosages of two antioxidants (BHT and a red chicory extract) on some in vitro rumen parameters, when they are incubated with meadow hay and corn meal. Results of the experiment showed that the two antioxidants exerted only small effects on rumen degradability, GP kinetics and volatile fatty acids profile. At the highest dosage, the red chicory extract showed to improve the efficiency of nitrogen utilization in the rumen, by increasing the amount of nitrogen captured by rumen microbes and utilized for microbial protein synthesis