Effect of Liquorice extract on in vitro rumen fermentation parameters of diets containing different levels of concentrate

Plants produce an enormous array of phytochemicals arising from various biosynthetic pathways. More than 200,000 defined structures of phytochemicals have been recognized. The growing concerns over bacterial resistance to antibiotics and chemical residues in animal derived foods have led to use phytochemicals as alternatives to antibiotics, other chemotherapeutic agents, and chemical and growth promoting antibiotic feed additives. Phytochemicals exhibit antibacterial, antiviral and antifungal activities against a wide range of pathogenic and non-pathogenic microorganisms. The antimicrobial properties of phytochemicals are being explored to utilize as feed additives in livestock production system (Patra 2012). One of the plant secondary metabolites which researchers recently focus on beneficial effects is saponins. Saponin-containing plants and their extracts by suppressing the bacteriolytic activity of rumen ciliate protozoa could enhance total microbial protein flow from the rumen. Saponins also have selective antibacterial effects which may prove useful in, for example, controlling starch digestion (Wallace et al 2002). Extract of licorice root can be used to manipulate rumen fermentation due to its saponin content. To date, there are limited research on the effect of plant phytochemicals on ruminal microbial fermentation at low pH. Most research studied the effects of plant phytochemicals on high-forage diets at pH > 6.2. The effects of plant extracts on ruminal microbial fermentation are pH-dependent.Microbial populations and ruminal fermentation conditions in cattle fed high-concentrate diets may be very different from cattle fed high-forage diets, because of the type of substrate being fermented or the resulting pH. Therefore, the search for additives that will help to replace ionophores in high concentrate diets needs to be tested in a high-concentrate and low-pH environment. It was hypothesized that extract of licorice root could exert some antimicrobial activities in low pH. Hence, the objective of this experiment was to study the effect of Licorice extract on in vitro gas production parameters, pH, volatile fatty acids (VFA), N- ammonia concentration and protozoa populations in diets containing different levels of concentrate to forage ratio.

Dried licorice extract obtained from Zagros Company (Kermanshah). Two experimental diets with different forage to concentrate ratio (40 to 60% or 60 to 40 %) were formulated. Licorice extract was added to diets at three levels, 1 or 2 and or 3 milligram /liter of incubation media. The saponin content of Licorice extract was 134.75 mg/g of dired licorice extract. For in vitro gas production, rumen fluid was taken from two rumen fistulated Kordish rams. For measuring gas production, 200 mg of experimental diets with four levels of licorice extract (0, 1, 2 and 3 mg/L incubation medium) were incubated with 40 ml of buffered-rumen fluid for 120 hours. The cumulative produced gas was recorded at different times of incubation and gas production parameters were fitted with Blummel et al equation (2003). Organic matter digestibility (OMD) was estimated after 24 hours of incubation (Menke and Steingass 1988). N-ammonia concentration was measured based Broderickand Kang (1980). Rumen protozoa were identified according to the method of Dehority (2003). After 24 incubation, 5 ml of buffered rumen fluid was pipetted into a screw-capped test tube containing 5 ml of formalin. Thereafter, two drops of brilliant green dye (2 g brilliant green and 2 ml glacial acetic diluted to 100 ml with distilled water) were added to the test tube, mixed thoroughly and allowed to stand overnight at room temperature. Total and differential counts of protozoa were made with five replications. In vitro rumen concentration of volatile fatty acids (VFA) was measured by gas chromatography (Ottenstein and Bartley 1971). All in vitro gas production trials were carried out in three runs. Rumen fermentation parameters, protozoa population and organic matter digestibility date were analyzed in a factorial arrangement based on a completely randomized design and gas production data were analyzed in a factorial arrangement based on a complete randomized block design using Proc GLM of SAS software. The differences among treatments were evaluated using Tukey adjustment when the overall F-test was P ≤ 0.05. Trends were declared when 0.05 < P ≤ 0.10.

The results showed that the interaction of diets and licorice extract levels were not significant on in vitro gas production, rate of gas production, estimated organic matter digestibility N-ammonia concentration, pH, total protozoa population, Entodinium, Epidinium, Diplodinium, Eudiplodinium and Isotricha population and in vitro ruminal concentrations of total VFA, acetate, propionate, butyrate, isobutyrate, valerate and isovalerate. The effect of diets on gas production (P≤0.05) and estimated OMD (p < 0.01) was significant and high concentrate diets in compare to low concentrate diets had greater gas production and greater estimated OMD. Inconsistent to these results, adding saponin to diet containing low level of concentrate decreased gas production (Yogianto et al 2014) and adding saponin to diet containing high level of concentrate increased gas production (Aazami et al 2013). Furthermore, high concentrate diet in compare to low concentrate diet had lower organic matter diegestability (Yogianto et al 2014). Diets containing high concentrate had lower acetate and greater isovalerate concentrations (p < 0.01). Addition of licorice extract reduced estimated OMD and total protozoa population and Entodinium population (p < 0.01) and tended to increase total VFA concentration (P=0.09) and to decrease isovalrate concentration (P=0.06).

Based on this study, it is concluded that different levels of licorice extract had no effect on fermentation parameters of diets containing different concentrate: forage ratios. Further studies are necessary to determine the effectiveness of saponin-containing plants extracts on rumen microbial fermentation and digestion kinetics.