In vitro Fermentation Method as a Tool to Assess the Effects of Secondary Metabolites on Rumen Fermentation—A Brief Review

In vitro fermentation is a useful method to test the effects of secondary metabolites on the rumen fermentation. It can be a noninvasive method when the rumen fluid is collected from slaughterhouses from dead animals. The experimental design for this type of experiment is normally a factorial design where common factors tested are: incubation time, main energy source incubated, and additive concentrations (secondary metabolites). The data gathered from the incubations can be quite extensive and therefore the statistical power of the in vitro method can be very good. In this paper we will discuss the main characteristics (how the method works and which parameters are measured) of the in vitro fermentation method.


Introduction
Noninvasive methods to study novel feedstuff to be used in animal agriculture have currently been developed and improved. The in vitro fermentation system can be a noninvasive method which allows laboratory testing of novel feedstuff produced secondary metabolites in the rumen. It is derived from the methodology developed by Tilley and Terry (1963), with some adjustments. For a noninvasive approach rumen fluid is pooled from different donors and taken to the laboratory to start the incubation. Once completed, the incubation will yield vast datasets with information such as: pH, ammonia levels, methane concentration, volatile fatty acids, dry matter degradability, total gas production, profile of microbial population, amongst others (Calsamiglia et al., 2007;Makkar et al., 2006;Yang et al., 2010). The analysis of this information can reveal the effects of an individual chemical compound when incubated in the rumen.

Rumen Fermentation
The rumen, which is about one-seventh of the body mass of ruminants (McDougall, 1948), is maintained at relatively constant temperature (39 o C), buffered by salivary secretion, and is an ideal fermentation site for microbial ecosystems. During fermentation of feedstuffs by microorganisms; VFA, microbial cells, NH3, carbon dioxide (CO 2 ), CH 4 , adenosine triphosphate (ATP), and heat are formed. VFA and ATP are used as the available energy sources for the animal, while microbial cells are the significant source of quality protein entering the small intestine (Demeyer, 1981;Russel & Hespell, 1981). Non-utilized NH3, CH4, and heat production may represent the loss of energy and N for the ruminants (Demeyer, 1981). In order to obtain appropriate knowledge and strategies to manipulate rumen fermentation, it is important to understand the mechanisms of carbohydrate and protein metabolism, methanogenesis, and acetogenesis in the rumen.
Ruminant diets contain substantial amounts of carbohydrate polymers such as cellulose, hemicellulose, starch, pectin, xylan, and water-soluble carbohydrates mainly in the form of fructans (McDonald et al., 2011;Russel & Hespell, 1981). A description of the conversion of carbohydrate polymers to VFA in the rumen is provided: diets containing plant particles are attacked by microorganisms and carbohydrate polymers are then released from plant cell structural matrices. After this, the carbohydrate polymers are hydrolysed to simple sugars such as cellobiose, maltose, xylobiose, hexoses, and pentoses by extracellular microbial enzymes. Cellulose is catalyzed by β-1,4-glucosidases to cellobiose and further converted either to glucose or glucose-1-phosphate. Starch is  (Makkar & Vercoe, 2007). These groups of natural "growth promoters" constitute a good opportunity to help farmers to increase animal production. Also the consumer can benefit because of the reduction of the potential transmittance of resistance or harmful effects caused by traditional antibiotics, which are an alternative. Practical effects of supplementing these chemicals are described in the literature ranging from metabolic effects which optimize rumen fermentation through to changes in milk composition.  Parodi, 1999 Alexander et al. (2008) completed an in vitro study to identify plant extracts that modulate partitioning of degraded organic matter (OM) towards microbial protein synthesis, at the expense of gas production, and decrease protein degradation in the rumen. They found that that aqueous methanol extract of M. oleifera seed and aqueous extract of P. kurroa root may have potential as feed additives to increase the efficiency of utilization of energy and nitrogen in ruminant diets. In addition, a review of the potential of some chemicals to manipulate rumen fermentation, utilizing publications that used in vitro fermentation method, have indicated that garlic oil, cinnamaldehyde (the main active component of cinnamon oil), eugenol (the main active component of the clove bud), capsaicin (the active component of hot peppers), and anise oil, among others, may increase propionate production, reduce acetate or methane production, and modify proteolysis, peptidolysis, or deamination in the rumen (Calsamiglia et al., 2007).

The Utilization of the in vitro Fermentation Method and Some Literature Findings
Another work utilizing the in vitro fermentation method, found that a substantial amount (71-93%) of tannins soluble in aqueous acetone was released from leaves of some trees and shrubs on incubation in the in vitro medium for 48 h. It also concluded that he rumen liquor was not capable of degrading oligomeric condensed tannins (Makkar et al., 2006). Newbold et al. (1995) studied the importance of methanogenic bacteria associated with ciliate protozoa either by removing protozoa from whole rumen fluid (using defaunated rumen fluid to correct for the effects of centrifugation on bacteria) or by isolating the protozoa. Rumen fluid was withdrawn from sheep inoculated with either Polyplastron multivesiculatum, a co-culture of Isotricha prostoma plus Entodinium spp. or a mixed type B fauna of Entodinium, Eudiplodinium and Epidinium spp. They found that methanogenesis was highest in rumen fluid containing a mixed protozoal population of the following genera: Entodinium, Eudiplodinium and Epidinium, was lower in defaunated rumen fluid and lowest in rumen fluid containing either I. prostoma plus Entodinium or P. multivesiculatum. Also, methanogenic bacteria associated with rumen ciliates were apparently responsible for between 9 and 25% of methanogenesis in rumen fluid.
Lima Neto (2012) investigated the effects of plant fractions (Solanum lycocarpum St. Hil.) on the rumen fermentation utilizing the in vitro fermentation system. The leaf fraction of the shrub (10 g/Kg DM inclusion) had the strongest effect when added to the Lucerne and increased ruminal the Acetate: Propionate (P < 0.05). The author also found that when SL fractions were evaluated with incubated ryegrass hay, root and stem fractions decreased Ace: Prop (P < 0.05).