All protocols and procedures followed in this study were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) of Ministry of Environment, Forests and Climate Change, Government of India, New Delhi.
2.1. Preparation of substrates with Red Sea weeds
Mixed substrate comprising of concentrate to roughage ratio of 50:50 on dry matter (DM) basis was used as control (CON). The anti-methanogenic potentials of Kappaphycus alvarezii and Gracilaria salicornia (GS) were evaluated using in vitro gas production system. Mixed substrates were supplemented at 0% (control), 1% (KA1), 2 (KA2), 4 (KA4), 6 (KA6), and 8 (KA8) of Kappaphycus alvarezii; and, 1 (GS1), 2 (GS2), 4 (GS4), 6 (GS6), and 8 % (GS8) of Gracilaria salicornia, respectively. Each treatment was comprising of 6 replicates.
Rumen liquor sampling
Four rumen fistulated animals kept on standard diets was used as donor animals. Rumen liquor was collected before feeding in the morning. After collection rumen liquor samples were screened with 4 layers of muslin cloth in a preheated thermos at 390 C and were transported to laboratory for inoculation of substrates. In vitro gas production was studied as per the procedure of Menke and Steingass (1988).Accurately weighed 200 mg of each substrates in triplicate was put into the graduated 100 ml calibrated glass syringe (Haberle Labortechnik, Lonsee-Ettenchie, Germany). The medium consisting of buffer and rumen liquor in 2:1 ratio (Menke and Steingass, 1988) was dispensed (30 ml) into the syringes by an automatic dispenser (OPTIFIX, Walter Graf & Co., Wertheim, Germany). Fermentation syringes were then kept in an incubator at 39°C with periodical shaking. Thus, total numbers of samples incubated were 2 (seaweeds)×6 (treatments) 7 (incubations)×3 (triplicate)=252. Besides, four blank syringes containing no substrate were also run during each incubation. The fermentation was terminated after 96 h of incubation.
Proximate and nutrient composition
Samples of wheat straw, fodder maize, Kappaphycus alvarezii and substrates were analyzed for proximate principles, fibre constituents and minerals. Proximate analysis (dry matter (DM), organic matter (OM), crude protein (CP), crude fiber (CF), and nitrogen free extract (NFE)) of the samples were estimated as per AOAC (2005) and cell wall constituents (neutral detergent fiber (NDF), acid detergent fiber (ADF), hemicellulose and cellulose) were estimated as per Van Soest et al. (1991). For determination of the concentration of minerals (calcium, magnesium, iron, copper, zinc, manganese and Iodine), samples of feedstuffs and seaweeds were subjected to wet digestion (HNO3: HClO4: H2SO4- 3:2:1) using Kelplus- KES 12L R system till it became clear and the digested samples were diluted with double distilled water (DDW) and passed through Whatman filter paper No. 42 and final volume was made to 25 ml. The contents of different minerals in the feed sample were analyzed using atomic absorption spectrophotometer (Hitachi- 5000 series). The calibration curve for determination of different minerals was prepared using a blank and working standard solution of different minerals. The calibration was periodically verified by analyzing a standard at the frequency of 20 readings. If the recovery was outside the limits, the analysis was stopped and the system was recalibrated. Content of P in feedstuff and seaweed was determined by using a colorimetric method (AOAC, 2005)
Measurement of total gas and methane production
For each substrate, cumulative gas production (CGP) was calculated as the amount of gas production (ml) from the substrate minus gas production from blank divided by the weight of substrate. Gas production (ml/g DM) after 6, 12, 24, 48, 72 and 96 h of incubation was recorded by piston displacement. For methane estimation, 100 μl of gas sampled from headspace (after 24h of incubation) of the syringe was injected into a Nucon-5765 gas chromatograph equipped with Porapak Q column and flame ionization detector (Agarwal et al., 2008). A mixture of 50% carbon dioxide and 50% methane (Spancan; Spantech Products Ltd, Godstone, UK) was used as standard.
2.3. Estimation of volatile fatty acids (VFA)
Estimation of volatile fatty acids was done by using Nucon-5765 gas chromatograph (AIMIL, New Delhi, India) equipped with a double flame ionization detector and the glass column (4 ft length and 1/8 inch diameter) packed with chromosorb 101 as per method described by Cottyn and Boucque (1968) and later modified by Agarwal et al. (2008). The gas flows for nitrogen, hydrogen and air were 30, 30 and 320 ml/min, respectively. Temperature of injector oven, column oven and detector was 2700C, 1720C and 2700C, respectively. Standard VFA mixture was prepared by mixing stock solutions (each of 25 mg/ml concentration) of standard VFAs and water (acetic acid, 1.68 ml; propionic acid, 0.48 ml; butyric acid, 0.24 ml; distilled water, 7.24 ml) to obtain final concentration of acetic acid, 7.0; propionic acid, 1.62; butyric acid, 0.68 mM/100 ml. Fermentation liquor samples were prepared by adding 0.2 ml of 25 % metaphosphoric acid per ml of liquor, allowing it to stand for 2 h followed by centrifugation at 7000 ×g for 10 min. Supernatant was used for estimation of VFA.
2.4. Ammonia nitrogen estimation
Ammonia nitrogen was estimated by the method of Weatherburn (1967). Briefly, to the sample (suitable quantity), 5.0 ml of solution A (1 g phenol and 5 mg sodium nitroprusside) was added to which 5.0 ml of solution B (0.5 g sodium hydroxide 0.84 ml sodium hypochlorite) was added and mixed thoroughly. The tubes were incubated at 39oC for 15 min for colour development. Samples were then read spectrophotometrically at 625 nm against a reagent blank. In a similar way standard tubes (ammonia nitrogen concentration ranging from 1.0 to 10 μg/ml) were processed and a calibration curve was plotted. Concentration of the unknown sample was calculated by the standard curve.
2.5. In vitro true digestibility (IVTD) of substrates
After 24 h of incubation, the content of the syringes was transferred to spout-less beaker by repeated washings with 100 ml of neutral detergent solution. The flask content was refluxed for 1 h and filtered through pre-weighed Gooch crucibles (Grade 1). The dry matter content of the residue was weighed and in vitro true digestibility of feed was calculated as follows:
(Initial DM of feed taken for incubation - DM residue)
True digestibility (TD %) =––––––––––––––––––––––––––––––––––––––––––––––X 100
(Initial DM of feed taken for incubation)
2.6. Statistical analysis
Cumulative gas production recorded at different hours of incubation were fitted to the following model to determine gas production kinetics: Y = b × (1-e –c * t); where ‘y’ is the cumulative volume (ml) of gas produced at time ‘t’ (h), ‘b’ the asymptotic gas volume (ml) and ‘c’ the rate constant of gas production (% h-1). The constants b and c were determined using the nonlinear procedure of SAS (2001). Substrate specific times were defined by the half time (t-1/2) of asymptotic gas production. Halftime (h) of gas production (t-1/2) [i.e., the time (h) when half of the asymptotic gas volume (b; ml) was produced] was calculated as: t1/2 = ln 2/c.
Data of the study were subjected to analysis of variance using the General Linear Model (GLM) procedure of the Statistical Software Package (SPSS for windows, V21.0; Inc., Chicago, IL, USA). The effect of green fodder replacement with corn silage on blood biomarker was tested using the following model:
Where; Yijk is the dependent variable, μ is the overall mean of the population, Si is the mean effect of the source of seaweed, Dj is the mean effect of dose of seaweed, (S×D)ij is the effect of the interaction between source and dose, and eijk is the unexplained residual element assumed to be independent and normally distributed. Individual animals were used as the experimental unit for all data. The pair-wise comparison of means was carried out using “Tukey’s honest significant difference (HSD) test”. Significance was determined at P<0.05 and the values are presented in the tables. Treatment means were separated using Duncan’s Multiple range Test and were considered significant at P<0.05.