Forages and pastures symposium: an update on in vitro and in situ experimental techniques for approximation of ruminal fiber degradation.

Static quantification measures of chemical components are commonly used to make certain assumptions about forage or feed nutritive value and quality. In order for modern nutrient requirement models to estimate intake and digestibility more accurately, kinetic measures of ruminal fiber degradation are necessary. Compared to in vivo experiments, in vitro (IV) and in situ (IS) experimental techniques are relatively simple and inexpensive methods to determine the extent and rate of ruminal fiber degradation. This paper summarizes limitations of these techniques and statistical analyses of the resulting data, highlights key updates to these techniques in the last 30 yr, and presents opportunities for further improvements to these techniques regarding ruminal fiber degradation. The principle biological component of these techniques, ruminal fluid, is still highly variable because it is influenced by ruminally fistulated animal diet type and timing of feeding, and in the case of the IV technique by collection and transport procedures. Commercialization has contributed to the standardization, mechanization, and automation of the IV true digestibility technique, for example, the well-known DaisyII Incubator. There has been limited commercialization of supplies for the IS technique and several review papers focused on standardization in the last 30 yr; however, the IS experimental technique is not standardized and there remains variation within and among laboratories. Regardless of improved precision resulting from enhancements of these techniques, the accuracy and precision of determining the indigestible fraction are fundamental to modeling digestion kinetics and the use of these estimates in more complex dynamic nutritional modeling. Opportunities for focused research and development are additional commercialization and standardization, methods to improve the precision and accuracy of indigestible fiber fraction, data science applications, and statistical analyses of results, especially for IS data. In situ data is typically fitted to one of a few first-order kinetic models, and parameters are estimated without determining if the selected model has the best fit. Animal experimentation will be fundamental to the future of ruminant nutrition and IV and IS techniques will remain vital to bring together nutritive value with forage quality. It is feasible and important to focus efforts on improving the precision and accuracy of IV and IS results.

In vitro and in situ techniques are important to studying ruminant nutrition because these procedures go beyond measures of components of a feedstuff in a laboratory by fermenting a sample in ruminal fluid. The in situ procedure was first described regarding ruminant nutrition in 1938 and in vitro in 1966 and both techniques have been refined over time to improve the reliability of results. This review focused on the state of knowledge 30 yr ago and significant discoveries that have impacted these techniques in the last 30 yr and shared a vision for future opportunities to refine these methods further. Commercialization of equipment and supplies has resulted in increased standardization of these methods; however, efforts should be made to continue to improve the standardization, and reliability of the results, of these procedures. Statistical analyses and data science applications are opportunities to expand these techniques to modern nutritional models and/or forecasting animal performance. The amount and kinetics of ruminal degradation estimate that in vitro and in situ techniques provide continue to be crucial to advance the efficiency and sustainability of ruminant animal production.

Effects of roughage type on particle separation, rumination, fiber mat characteristics, in situ degradation, and ruminal fermentation parameters in beef steers.

Six ruminally cannulated steers (average BW = 791 ± 71 kg) were used in a replicated 3 × 3 Latin square experiment to determine the effects of roughage type on rumination, fiber mat characteristics, and rumen fermentation variables. Three roughages were included at 7% (DM basis) in a steam flaked corn-based diet: cotton burrs (CB), wheat silage (WS), or corn stalks (CS). Steers were fitted with a sensory collar to record rumination behaviors in 2-h intervals at the beginning of the experiment. Each 30-d period consisted of 7 d of recovery, 14 d of diet adaptation, 7 d of rumination data collection (daily and bi-hourly average rumination), 1 d of rumen fluid collection, and 1 d of rumen evacuations. In situ degradation of individual roughages was determined for 4 d after period 3 evacuations. During rumen evacuations, ruminal contents were removed; the rumen fiber mat (RF) was separated from the liquid portion with a 2-mm sieve, weighed, and a subsample was dried. Data were analyzed using the MIXED procedure of SAS with steer as the experimental unit and roughage (CB, WS, and CS) as the main effect. Dry matter intake (DMI) was not different for CB and WS (P = 0.25) and greatest for steers consuming CS diet (P ≤ 0.01). Roughage type did not influence the weight of the RF dry matter (%; DM; P = 0.92), RF weight (P = 0.69), or RF:DMI ratio (P = 0.29). Daily rumination (min/d) did not differ among roughages (P = 0.40), but min of rumination/kg of DMI was greatest for CS (18.0 min), min/kg of NDF was greatest for WS (89.8 min; P = 0.02), and min/kg of peNDF was greatest for CS (132.4 min; P ≤ 0.01). Wheat silage had the greatest percentage of soluble and degradable DM. Rumen fiber mat did not differ for roughages, although rumination min/kg of DMI and peNDF was greatest for steers consuming CS and WS. In situ degradation determined that CB-R and CS-R had the greatest percentage of ruminal undegraded DM. Based on the objective of the experiment, roughage type did not influence daily rumination or fiber mat characteristics.

Inclusion of quebracho tannin extract in a high-roughage cattle diet alters digestibility, nitrogen balance, and energy partitioning.

Condensed tannins (CT) might improve animal and system-level efficiency due to enhanced protein efficiency and reduced CH4. This study evaluated the impact of quebracho tannin (QT) extract fed at 0%, 1.5%, 3%, and 4.5% of dry matter (DM), within a roughage-based diet on apparent digestibility of DM, organic matter (OM), fibrous fractions, and N retention and energy partitioning of growing steers (236 ± 16 kg BW). A Latin rectangle design with eight animals and four periods was used to determine the whole-animal exchange of CO2, O2, and CH4 as well as the collection of total feces and urine over a 48-h period, using two open-circuit, indirect calorimetry respiration chambers. Following the removal of steers from respiration chambers, rumen inoculum was collected to determine ruminal parameter, including volatile fatty acids (VFA) and ammonia. Animals were fed a 56.5% roughage diet at 1.7% BW (dry matter basis). Dry matter and gross energy intakes were influenced by the level of QT inclusion (P ≤ 0.036). Digestibility of DM, OM, and N was reduced with QT inclusion (P < 0.001), and fiber digestibility was slightly impacted (P > 0.123). QTs altered the N excretion route, average fecal N-to-total N ratio excreted increased 14%, and fecal N-to-urinary N ratio increased 38% (P < 0.001) without altering the retained N. Increased fecal energy with QT provision resulted in reduced dietary digestible energy (DE) concentration (Mcal/kg DM; P = 0.024). There were no differences in urinary energy (P = 0.491), but CH4 energy decreased drastically (P = 0.007) as QT inclusion increased. Total ruminal VFA concentration did not differ across treatments, but VFA concentration increased linearly with QT inclusion (P = 0.049). Metabolizable energy (ME) was not affected by the QT rate, and the conversion efficiency of DE-to-ME did not differ. Heat energy decreased (P = 0.013) with increased QT provision likely due to changes in the DE intake, but there was no difference in retained energy. There were no differences for retained energy or N per CO2 equivalent emission produced (P = 0.774 and 0.962, respectively), but improved efficiency for energy retention occurred for 3% QT. We concluded that QT provided up to 4.5% of dry matter intake (about 3.51% of CT, dry matter basis) does not affect N and energy retention within the current setting. Feeding QT reduced energy losses in the form of CH4 and heat, but the route of energy loss appears to be influenced by the rate of QT inclusion.

Perennial peanut (Arachis glabrata Benth.) contains polyphenol oxidase (PPO) and PPO substrates that can reduce post-harvest proteolysis.

BACKGROUND: Studies of perennial peanut (Arachis glabrata Benth.) suggest its hay and haylage have greater levels of rumen undegraded protein (RUP) than other legume forages such as alfalfa (Medicago sativa L.). Greater RUP can result in more efficient nitrogen utilization by ruminant animals with positive economic and environmental effects. We sought to determine whether, like red clover (Trifolium pretense L.), perennial peanut contains polyphenol oxidase (PPO) and PPO substrates that might be responsible for increased RUP. RESULTS: Perennial peanut extracts contain immunologically detectible PPO protein and high levels of PPO activity (>100 nkatal mg(-1) protein). Addition of caffeic acid (PPO substrate) to perennial peanut extracts depleted of endogenous substrates reduced proteolysis by 90%. Addition of phenolics prepared from perennial peanut leaves to extracts of either transgenic PPO-expressing or control (non-expressing) alfalfa showed peanut phenolics could reduce proteolysis >70% in a PPO-dependent manner. Two abundant likely PPO substrates are present in perennial peanut leaves including caftaric acid. CONCLUSIONS: Perennial peanut contains PPO and PPO substrates that together are capable of inhibiting post-harvest proteolysis, suggesting a possible mechanism for increased RUP in this forage. Research related to optimizing the PPO system in other forage crops will likely be applicable to perennial peanut.