Different levels of clove and savory essences on fermentative parameters in gas production technique and their antimicrobial effects on Peptostreptococcusanaerobius bacteria isolated from rumen

 Since the legislation of European Union has prohibited the use of growth-promoting antibiotics such as monensin, scientists have been interested in alternatives to manipulate rumen fermentation. The use of growth-promoting antibiotics in animal feeds is banned in Europe due to potential risks such as the spread of antibiotic resistance genes or the contamination of milk or meat with antibiotic residues. Recently, essential oils have been increasingly evaluated to replace or facilitate reductions in the use of antibiotics. The most effects of plant essential oils, especially cloves and savory oils, are their antioxidant effects and their effects on the metabolism of ruminal microbes. The antiprotozoal effects of clove extract have been proven in the studies in vitro by gas production technique. Few studies have been done on the effects of clove oil, especially the savory oil, on the digestive properties in the country. Also, no study was found on the interactions of these essential oils in the experiments in vitro. Thus, the aim of this study was investigated to evaluate the effects of clove and savory oils on gas production and in vitro fermentation process and estimation of gas production parameters of feedstuffs (alfalfa hay and barley grain).

Experimental treatments were included control (basal feeds without additive), basal feeds supplemented with three levels of clove oil (0, 250, 500 mg) and three levels of savory oil (0, 250, 500 mg) per kg of DM in a rumen culture. Ruminal fluid was collected from two fistulated sheep (49.5±2.5 kg). All samples isolated from the rumen were withdrawn 2 h after the morning ration had been consumed. Collected rumen contents were strained through four layers of cheesecloth and brought immediately to the laboratory. Gas production technique was used to detect the fermentation parameters of the treatments. About 200 mg of basal diet (alfalfa hay and barley grain, 1:1, with clove and savory oils) were incubated in 100ml glass syringes and 30ml of incubation liquid were added and were incubated in 39 C° water bath. The gas production was measured in 2, 4, 6, 8, 16, 24, 48, 72, 96, 120h. Three parallel syringes of each treatment were prepared in this experiment in a completely randomized design in a factorial arrangement. They were used to measure the gas production parameters (fermentable fraction (b) and rate (c) of gas production) cumulative gas production, organic digestibility and metabolizable energy of treatments until 120 h. In the present study, digestible organic matter, metabolizable energy, and production of volatile fatty acids were estimated based on the presented equations.In the second experiment, Peptostreptococcus anaerobic was isolated from the ruminal fluid, cultured in the medium of BAAA) Bile EsculinAzideAgar(, and evaluated by different levels of cloves and fennel essences in a randomized complete design with sixteen treatments and three replicates using Duncan test at level 0.05. Experimental levels in this experiment including: 20, 35 and 40 µl of cloves essence, 20, 35 and 40 µl of fennel essence, 20 µl of fennel essence + 20 µl of cloves essence, 20 µl of fennel essence + 35 µl of cloves essence, 20 µl of fennel essence + 40 µl of cloves essence, 35 µl of fennel essence + 20 µl of cloves essence, 35 µl of fennel essence + 350 µl of cloves essence, 35 µl of fennel essence + 40 µl of cloves essence, 40 µl of fennel essence + 20 µl of cloves essence, 40 µl of fennel essence + 35 µl of cloves essence, 40 µl of fennel essence + 40 µl of cloves essence, control.Statistical analysis of data was performed by SAS statistical software (9.1 version). Duncan's multiple test range was conducted in level 5%.

High levels of savory oil were increased gas production in the first hours (p<0.05), but clove oil was reduced gas production in 8-24 hours (p<0.05). Although, few data have been published on the interaction effects of the use of the essential oils, as well as on the effects of savory oil on fermentation parameters. The amount of gas production (b) tend to be decreased (P=0.06) due to the simultaneous use of savory and clove oils. Different levels of savory oil had no effect on estimated organic matter digestibility, metabolizable energy, and volatile fatty acids. But, with increasing the levels of clove oil, the amount of estimated organic matter digestibility, metabolizable energy tend to be decreased (P=0.09). In the microbial experiment, after incubation of peptostreptococcus anaerobic in medium of Bile AesculinAzideAgar and adding different levels of cloves and fennel essences was observed that all levels of treatments were significant (P<0.05); the highest growth of bacteria was related to treatment 11 (35 µl of fennel essence + 350 µl of cloves essence). This result showed ammonia-producing bacteria known peptostreptococcus anaerobic is inhibiting by different levels of clove and savory essences and finally resulting in the decrease of rumen fluid Ammonia nitrogen.

 According to our results we can conclude thatusing savory oil improves ruminal fermentation in vitro and with increasing the levels of savory oil, the amount of gas production, the organic matter digestibility, the metabolizable energy and volatile fatty acidsconcentration were decreased. Also in this study, were shown the co-effects of simultaneous use of savory and clove oils on the reduction of gas production. Therefore the effects of essential oils should be considered. For further studies, it is suggested that the effect of essential oils along with various sources of energy, such as pectin, starch, and various protein sources, should be studied in vitro and in vivo. Also, consideration of other changes in fermentation products such as ammonia nitrogen, methane and the composition of the microbial population in these experiments can also be of particular importance.