The effect of adding different levels of Ziziphora clinopodioides to the diet on the in vitro digestion and fermentation activity of anaerobic bacteria and fungi in the rumen of fattening Arabi lambs

The ruminants have a stomach with four separate compartments, in the two parts of which, rumen and reticulum, because of the action of their bacteria, protozoa, and other populations such as anaerobic fungi, the energy of plant cell wall are available to the host. The main products of fermentation include volatile fatty acids (VFA) and microbial crude protein, which provide about 80 percent of the energy and 60 to 85 percent of the animal protein requirements, respectively. Therefore, achieving the best efficiency of digestion and microbial fermentation in rumen is very important. On the other hand, the production of methane, carbon dioxide, and ammonia in the rumen causes a loss of energy and protein, an increase in greenhouse gases, and environmental pollution. Therefore, nutritionists, by manipulating ruminal fermentation processes, are looking for ways to reduce energy and protein loss in the form of methane and ammonia and to increase the efficiency of fermentation and synthesis of microbial protein in rumen and increase fiber digestion. For this purpose, several additives such as organic acids, yeasts, enzymes, buffers, and antibiotics have been used. In recent decades, medicinal plants have attracted a lot of attention due to their ability as an alternative to growth stimulants and antibiotics in animal and poultry nutrition. In this regard, the plants of the Lamiaceae (mints) family, which are considered as a medicinal plant with high antimicrobial properties, have been considered. The perennial species of Ziziphora clinopodioides belongs to this family, Lamiaceae. The Ziziphora clinopodioides is widespread in the north, center, northwest, south, and northeast of Iran. Some of the most important active ingredients in this plant are Pulegone, Piperitenone, Yomogi alcohol), DL-Menthol, Carvacrol, Piperitenone, γ-Terpinene, and Carnoon. Since the activity of rumen anaerobic fungi and bacteria is important in the digestion of feed and considering that there is no information about the effect of adding this plant to the diet on the activity of bacteria and fungi isolated from the rumen, the latency is very limited. Therefore, the aim of this study was to investigate the effect of using Ziziphora clinopodioides in the diet of fattening lambs as a supplement, on digestion and fermentation activity of their rumen bacteria and fungi.

 Initially, the best amount of Ziziphora clinopodioides supplement in the diet was determined by the two-steps digestion method. Therefore, six treatments containing zero (control), 0.2%, 0.4%, 0.6%, 0.8%, and 1% powdered Ziziphora clinopodioides plants were added to a standard diet of fattening lambs. Then, based on the results, three treatments containing zero (control), 0.2%, and 0.4% were selected among them to determine the effect of Ziziphora clinopodioides supplement on the activity of ruminal anaerobic fungi and bacteria. The rumen fluid required for the two-steps digestion experiment and the specific culture medium of bacteria and fungi were prepared from four male lambs fed a fattening lamb diet with similar ingredients and compositions to the diet of in vitro section, through a stomach tube. Then, anaerobic bacteria and fungi were isolated and purified from their ruminal fluid. 

 Concentration of ammonia nitrogen and pH in the specific culture medium of rumen fungi at the first, third, and sixth days of incubation and the average concentration of the whole period, and also at 24, 48 and 72 hours after initiation of incubation of experimental diets with bacteria, were not affected by experimental treatments. The in vitro digestibility of dry matter, neutral detergent fiber (NDF), and acid detergent fiber (ADF) increased significantly (P<0.05), as an influence from the addition of Ziziphora clinopodioides supplements to the diet of fattening lambs. The highest and lowest dry matter, NDF, and ADF digestibility was observed in the control diet and diet containing 0.4% Ziziphora clinopodioides , respectively; and there was a numerical and non-significant difference between diets containing Ziziphora clinopodioides . The digestibility of dry matter and NDF of experimental diets by ruminal anaerobic fungi on the first, third, and sixth days of incubation, and ruminal bacteria at 24, 48 and 72 hours after initiation of incubation, and the average of the whole experimental period increased significantly under the influence of Ziziphora clinopodioides supplementation in the diet of fattening lambs. There was no difference between diets containing Ziziphora clinopodioides in the activity of anaerobic rumen fungi and bacteria in the digestion of diets. Increasing and improving the digestibility of diet nutrients may be due to changes in the microbial population and improved digestion and fermentation activity of them as a result of secondary compounds of Ziziphora clinopodioides. It has been suggested that some secondary metabolites, such as phenolic compounds (also found in Ziziphora clinopodioides ), selectively inhibit the activity of amylolytic and proteolytic bacteria, without affecting other groups such as cellulolytic bacteria. Therefore, this selective effect leads to the competitive elimination of some species in favor of other species, therefore, the raw materials of the environment are provided for the activity of other species, perhaps this mechanism is a factor in improving digestibility in diets containing Ziziphora clinopodioides . consistent with  the results of the present experiment, increasing the digestibility and fermentation of sugarcane top or diets have been reported by adding Ziziphora clinopodioides to the diet of lambs or thistle (such as Ziziphora clinopodioides containing tannins) in the diet of calves. The supplementation of the Trachyspermum plant (has thymol and, γ-Terpinene similar to Ziziphora clinopodioides ) to diet also was increased the digestion and fermentation, which is consistent with the present experiment.

 Overall, the use of Ziziphora clinopodioides supplement in the diet of fattening lambs improved the digestive activity of anaerobic fungi and bacteria isolated from their rumen.