In vitro evaluation of Salinomycin addition in wheat straw based total mixed diets on rumen fermentation, methanogenesis and dry matter degradability in buffalo.

Aim: The aim of the current study was to evaluate the effect of salinomycin in vitro on methanogenesis and rumen fermentation. Materials and Methods: Different levels of (0,10, 15 and 20 ppm) salinomycin were checked for their effect on in vitro methanogenesis and rumen fermentation on three wheat straw based diets i.e. low fiber diet (LFD, 40R:60C), medium fiber diet (MFD, 50R:50C) and high fiber diet (HFD, 60R:40C). Evaluation of salinomycin was carried out using in vitro gas production technique. Methane production and individual fatty acids were estimated by Gas Chromatography. Results: Results of different levels of salinomycin on in vitro methanogenesis indicated that the maximum methane reduction (38.14% in term of mM/gDM) was noticed in HFD at 20 ppm level. IVDMD showing increasing trend with an increasing concentration of salinomycin with HFD and LFD, while shown decreasing trend with MFD respectively. Protozoal population significantly decreased by addition of salinomycin in all diets. Conclusion: The results of salinomycin evaluation in the current study can be implicated to mitigate the methane production, thus saving the feed energy loss and the accumulation of green house gases in environment.


Introduction
to be effective in enhancing both rate of gain and feed efficiency in feedlot cattle [3,4,5].It is also effective in Ionophores are the treatment of coccidial infections of poultry and commonly used in the diets of monogastric and cattle [6].poultry for increasing the feed conversion efficiency Ionophores inhibit gram positive bacteria, which and prevention of some diseases.Commonly used subsequently alters ruminal fermentation.When the gram negative bacteria predominate in the rumen, less Salinomycin is a polyether methane is produced and more propionate is produced antibiotic and produced by a strain of streptomyces as reported by several workers [7,8].Methane is one albus (ATCC 21838).The main physiochemical of the most potent GHG which is produced from characteristics of salinomycin are, molecular weight, enteric fermentation in ruminants.Therefore, present 750; molecular formula, C H O ; melting poin, 42 70 11 study was planned to investigate the effect of 0 112.5 to 113.5 C; pKa, 6.40; cation affinity, salinomycin supplementation in different diets on K>Na>Ca,Mg; cation transport activity, Na>K> rumen fermentation and methanogenesis in vitro.Ca,Mg [1,2].Salinomycin has been shown the activity

Materials and methods
against gram-positive bacteria, including mycobacteria and some filamentous fungi.However, no Preparation of Diets: To study the effect of activity has been found against gram-negative bacteria salinomycin on rumen fermentation and methane or yeasts [1].
production three diets were prepared with different A number of study have found that salinomycin roughage concentrate ratio i.e. high fiber diet (HFD, antimicrobial compounds that are ionophores are monensin, lasalocid, laidlomycin, salinomycin and narasin.60R:40C), medium fiber diet (MFD, 50R:50C) and syringe (containing no substrate, but only the low fiber diet (LFD, 40R:60C) and milled to pass inoculum and buffer) from total gas produced in the through 1 mm sieve and used as substrate.Source of syringe containing substrate and inoculum.roughage and concentrate were wheat straw and For methane estimation, representative of gas normal farm concentrate having about 20 % crude was sampled from the headspace of syringe in an protein and 70% total digestible nutrients (TDN).The airtight syringe and injected into Nucon-5765 gas chromatograph equipped with flame ionization concentrate part comprised of maize (33%), GNC detector (FID) and stainless steel column packed with (21%), mustard cake (12%), wheat bran (20%), Porapak-Q (length 6';o.d.1/8" i.d. 2 mm; mesh range deoiled rice bran (11%), mineral mixture (2%) and salt 80-100).The gas flow rates for carrier gas (nitrogen), (1%).
hydrogen and air were 30, 30 and 300 ml/min, Treatments and experimental design: Four levels respectively.Temperature of injector oven, column i.e. 0ppm, 10ppm, 15ppm and 20ppm of salinomycin 0 oven and detector were 40, 50 and 50 C, respectively.were added to the different wheat straw based HFD, For methane estimation, each gas sample (250µl) was MFD and LFD diets.All the treatment combinations manually injected using Hamilton airtight syringe.were arranged in 4 x 3 factorial arrangements in Methane content in sample was calculated by randomized block designs with three replicates.Set external calibration, using a certified gas standard was also incubated devoid of substrate with and mixture of 50% CH and 50% CO (Spantech without additive, which served as blanks for particular calibration gas, Surrey, England).treatment and values were corrected for different Methane production (ml) = Total gas produced parameters with these blanks.
(ml) x % methane in the sample

Preparation of Inoculums and In Vitro Gas
In vitro true Dry matter degradability: To estimate Production: Rumen liquor (RL) was obtained from true DM degradability of feed sample of each syringe fistulated adult male buffaloe (Bubalus bubalis) kept containing residues after incubation was estimated as on standard diet (60 parts roughage: 40 parts per method [10].concentrate) before one hour of morning feeding into a pre-warmed insulated flask and brought into the Proximate analyses and cell wall constituents: laboratory.Permission for taking rumen liquor from The proximate analysis of substrate was carried out as male fistulated buffaloes already taken from Animal per the methods of [11].The cell wall constituents of ethics committee of institute.Then rumen liquor substrates were determined according to suggested filtered through four layers of muslin cloth and the method [10].required amount of strained rumen liquor used as a Estimation of ammonia nitrogen and individual source of inoculum.
volatile fatty acid (IVFA): After the end of The incubation medium was prepared as per incubation, fermented material was transferred into previously described method [9].In vitro studies were centrifuge tubes and centrifuge at 3000 rpm for ten carried out in 100ml calibrated glass syringes minutes, than supernatant of each syringe including containing 200±10 mg of milled (1mm) three type that of blank was collected in vials used for ammonia wheat straw based diet [9].nitrogen (NH -N) and individual volatile fatty acid 3 Plungers of syringes applied with petroleum (IVFA).jelly for smooth movement and stop any leakage.The For NH -N estimation, 5 ml of supernatant was 3 30 ml incubation medium was dispensed taken in tube mixed with 12 ml 1 N NaOH and steam anaerobically in each syringe and before incubation, passed using KEL PLUS-N analyzer (Pelican, India) different levels of salinomycin was added by small and the NH evolved was collected in conical flask with 25% meta-phosphoric (4 ml) prepared in 1N After 24 h incubation, gas production was recorded sulphuric acid and kept for 3-4 h at ambient with the displacement of piston during incubation.The temperature [12].Thereafter, it was centrifuged (3,000 gas produced due to fermentation of substrate was rpm for 10 min) and clear supernatant was collected 0 calculated by subtracting gas produced in blank and stored at -20 C until analyzed.IVFAs were estimated using gas chromatograph (Nucon 5700, LFD at 10ppm level, while decreased slightly with India) equipped with flame ionization detector (FID) increasing concentration of salinomycin in case of and stainless steel column (length 4'; o.d 1/8''; i.d MFD.A reduction in methane production (ml/gDM, 2mm) packed with Chromosorb-101.mM/gDM, mM/gDDM) was seen in all the diets with Temperature of injection port, column and addition of salinomycin.Methane production percentage 0 decreased with increasing concentration of salinomycin detector was set at 200, 180 and 210 C, respectively.in all diets.Results showed the highest methane The flow rate of carrier gas (nitrogen) through the reduction (38.14, 30.14 and 27.68%) at 20 ppm level column was 40 ml/ min and the flow rates of hydrogen in HFD, MFD and LFD, when expressed in mM/gDM gas and air through FID were 30 and 300 ml/ min.respectively.Similar effect of salinomycin on methane respectively.
production was also found by other workers [15,16].

Sample (2µ) was injected through the injection
The propionic acid concentration (mM/l) increased port using Hamilton syringe (10µ).Individual VFA's with increasing dosage in all the three diet.The of the samples were identified on the basis of their increase in propionic acid concentration was 48.43% retention time and their concentration (mM) at 15 ppm level in MFD, 19.95% at 20 ppm level in calculated by comparing the retention time as well as HFD and slightly changes in LFD.A non significant the peak area of standards after deducting the (P<0.05)change in acetate concentration was observed corresponding blank values.in all diets.Acetate concentration increased in almost Protozoa counting: The stained protozoa was all cases except HFD, although the increase was non diluted (if needed) and counted by Haemocytometer significant.A significant effect of salinomycin on as per prescribed method [13].
butyrate concentration was seen in present study.Slight change in A/P ratio was observed in all cases Statistical analysis: Experimental data of different except 20 ppm level in HFD, where the maximum parameters were analyzed in complete randomized reduction (47.20%) was found.The similar results block design for analysis of variance and standard were observed during salinomycin supplementation error of means [14].
were consistent with the earlier findings of numerous

Results and Discussion
researchers [17][18][19].A non significant (P<0.05)reduction in NH -N was observed due to salinomycin The physical and chemical composition of all the 3 three diets was given in table 1. Results of different supplementation.The concentration of ammonia levels of salinomycin on in vitro rumen fermentation nitrogen increased with increasing concentrations of and methanogenesis were shown in Table 2.
salinomycin in HFD and LFD while, decreased in Effect of salinomycin on IVDMD was significant MFD.The maximum reduction was (20%) found in for all three diets.In HFD, digestibility was maximum MFD at 15 ppm level.increased (10.18%) at 20 ppm level than 7.80% in In present experiment, a significant (P<0.05)

Ingredient of concentrate
In vitro evaluation of Salinomycin addition in wheat straw based total mixed diets on rumen fermentation

3 syringe
into 100ml syringes individually and mixed containing boric acid solution having mixed indicator uniformly.Syringes were closed using clamps and and titrated against N/100 H SO .39±0.5 C for 24h in temperature Individual volatile fatty acids (IVFAs) were controlled incubator cum shaker.estimated by taking 1 ml of the supernatant treated Total gas production and methane estimation: