Browsing by Author "THUBE, HARSHALA A."
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ThesisItem Open Access EFFECT OF FIBROLYTIC ENZYMES SUPPLEMENTATION IN TOTAL MIXED RATION ON NUTRIENT UTILIZATION AND GROWTH PERFORMANCE IN KIDS(AAU, Anand, 2016) THUBE, HARSHALA A.; GUPTA, R. S.The present study was conducted in two phases to optimize the level of inclusion of fibrolytic enzymes in Surti kids. In Phase-I, in vitro studies were conducted to arrive at the optimum level of incorporation of fibrolytic enzymes in wheat straw based total mixed ration (TMR). On the basis of the results of vitro fermentation of TMR incorporated fibrolytic enzymes at different levels used as substrate, the optimum level of each was selected, for the in vivo study (Phase II). Eighteen Surti kids of similar body weight were randomly allotted in to two groups of nine each and were individually fed for 15 days under preliminary period and 120 days experimental period to meet their energy and protein requirements as per ICAR (1998) standards. The kids were fed TMR with no fibrolytic enzymes (T1); and TMR with 0.05% fibrolytic enzymes (T2) The average daily DM1 of kids in T1 and T2 groups during digestion trial was 659.33 ± 8.11 and 649.13 ±6.61 g and when expressed as kg/100 kg body weight was 4.44 ± 0.11 and 4.20 ± 0.0.05 and the same in terms of g/kg W0.75 was recorded as 87.11 ± 1.48 and 83.29 ± 0.70. The DM intake of experimental kids did not differ significantly (P>0.05) when expressed in terms of absolute intake or intake per 100 kg body weight. However, when expressed in terms of DMI/ kg metabolic body weight a significant difference was noted. The average digestibility coefficients (%) of nutrients in T1 and T2 groups were 60.83 ± 1.24 and 60.89 ± 0.99 for DM, 63.99±1.17 and 64.49±0.94 for OM, 64.17 ± 1.14 and 64.26 ± 0.88 for CP, 45.50 ± 2.08 and 49.90 ± 2.02 for CF, 71.77 ± 1.00 and 72.18 ± 0.66 for NFE, 54.77 ± 1.69 and 59.76±0.84 for NDF, 47.99 ±2.16 and 52.39 ± 2.03 for ADF and 69.45 ± 1.74 and 73.12 ± 1.26 for HC of experimental kids under T1 and T2 groups respectively. The cumulative DM I of experimental Surti kids was 77.40±1.07 and 77.10±0.68 kg/head for 120 days in T1 and T2 groups, respectively. The treatment groups did not differ from each other. The average conversion efficiency for DM (kg/kg gain) was 8.63 ± 0.22 and 7.73 ± 0.27, forCP (kg/kg gain) was 0.94 ±0.10 and 0.91 ± 0.09, for DCP 0.67 ± 0.03, and 0.62 ± 0.03 and for TDN (kg/kg gain) was 5.07 ± 0.20 and 4.59 ± 0.20 in T1 and T2 groups, respectively. The groups differed significantly (P<0.01) from each other. The average daily gain was 78.24 ± 2.25 and 86.36 ± 2.98 g in T1 and T2 groups, respectively. The treatment groups differed significantly (P<0.01) from each other. The increase in the body measurements viz. increase in body length and height at wither of Surti kids under T2 group was numerically higher in comparison to Surti kids under T1. However, the heart girth was significantly higher (p<0.05) in T2 group as compared to T1 (control). The respective values of T1 and T2 groups for rumen parameters: pH (6.34 and 6.20); total N (79.85 and 90.06 mg/dL); NH3-N (19.42 and 23.86 mg/dL), non protein nitrogen (12.72 and 15.42 mg/dL), TCA precipitable-N (38.18 and 44.01 mg/dL) and total volatile fatty acids (7.41 and 9.07 mM/dL), under the treatment groups differed significantly (P<0.05) from each other except for rumen pH. The blood samples collected during experimental feeding, the average total protein, average albumin, average globulin, glucose, cholesterol, triglycerides, BUN, urea and creafinine concentrations were 6.24 ± 0.33 and 6.02 ± 0.17 g/dL, 3.26 ± 0.08 and 3.29 ± 0.07 g/dL. 2.99 ± 0.28 and 2.73 ±0.16 g/dL. 50.65 ± 1.35 and 50.95± 1.09 mg/dL, 53.55 ± 2.81 and 53.60 ± 2.07, 37.95 ± 2.02 and 39.30 ±1.10 mg/dL 14.37 ±0.54 and 12.51 ± 0.39, 30.80 ± 1.16 and 26.80 ± 0.83 and 0.80 ± 0.02 and 0.74 ± 0.03 in T1 and T2 group, respectively, which did not differ from each other. The average values (U/L; mean ± SE) of alanine aminotransferase (ALT)/serum glutamic pyruvate transaminase (SGPT), aspartate aminotransferase (AST)/serum glutamate oxaloacetate transaminase (SCOT) and alkaline phosphatase (ALP) of experimental Surti kids in T1 and T2 groups were 31.25 ± 1.18 and 29.25 ± 0.57, 78.10 ± 1.98 and 80.00 ± 2.30, 69.78 ± 8.30 and 48.72 ± 3.25, respectively. The values were comparable between the two dietary treatment groups. These liver enzyme profiles are considered generally as indicators of alterations in body tissue and organs especially that of the pathological changes. The feed cost in terms of Rs/kg gain in T1 and T2 groups was Rs. 134.29 ± 3.33 and 127.25 ± 4.43, respectively. The feed cost per unit gain was statistically lower in group supplemented with fibrolytic enzymes than the control kids (P<0.05). The results indicated that inclusion of fibrolytic enzymes @ 0.05% in TMR (60:40 concentrate: roughage) for growing Surti kids resulted in 10.38% better growth, improved rumen fermentation, increased digestibility of CF and fibre fractions significantly and feed conversion efficiency with 5.53% reduction in feed cost per kg gain in weight as compared to control group.ThesisItem Open Access MOLECULAR CHARACTERIZATION AND DIVERSITY OF RUMEN METHANOGENS IN BUFFALO(AAU, Anand, 2008) THUBE, HARSHALA A.; PANDYA, P. R.Methane (CH4) eructated from ruminants represents a loss of 8 to 13% of the digestible energy ingested by the animal and contributes to global warming. Decreasing methane emissions from ruminant livestock is desirable in order to both, reduce greenhouse gases in the atmosphere and improve energy capture during digestion. Nutritionists have been trying for a long time to mitigate rumen methane emissions in order to enhance animal performance. The methods most commonly attempted involve elimination of ciliate protozoans which support methanogen populations, and addition of acetogenic bacteria to rumen fluid. The key steps for developing successful methane abatement strategies are likely to be steps which exploit natural processes in the rumen. Therefore, the specific role of an individual rumen methanogens is important as it has to compete with the other microbes of the eco-system for its survival. The enumeration of a specific species of methanogenic bacteria in the ecosystem (to quantify its role in rumen fermentation) is difficult with the conventional techniques due to large number of biochemical tests to be performed and imprecision of the technique even for the most predominant microbe present in the ecosystem. The application of molecular techniques has already revealed the enormous wealth of microbial diversity and putative novel species in the rumen. Present study aimed at exploring diversity of rumen methanogens in buffalo by molecular approach. Three adult buffalo were maintained on maintenance ration as per ICAR, 1998) feeding standards, continuously for three weeks. Samples of Rumen liquor (about 500 ml) were collected from three buffaloes at 0, 2 and 4 h after feeding by a suction pump using a flexible stomach tube. Samples were filtered through four layers of cheesecloth. The strained rumen liquor was used for the microbial study. The bacterial DNA was isolated following enzyme-chemical lysis method. The DNA stock samples were quantified using Nano-drop spectrophotometer at 260 and 280 nm using the convention that one absorbance imit at 260 nm wavelength equals 50 µg DNA per ml. The Ultra violet absorbance was checked at 260 and 280 nm for determination of DNA concentration and purity. Purity of DNA was judged on the basis of optical density ratio at 260:280 nm which was between 1.8 to 2.0 for all the samples indicating desirable purity. Concentration of DNA was estimated using the formula : Concentration of DNA (mg/ml) = OD 260 x 50 x Dilution factor. Quality and purity of DNA were also checked by agarose gel electrophoresis. Universal primer pair (Met 86F 5'ACA GGC CTA ACA CAT GCA AGT C-3'and Met 1340R 5'AGG GCG G (AT) GTG TAC AAG GC-3') for methanogens targeting 16S rRNA gene were used for amplification of DNA. The amplified product was visualized as a single compact band of expected size under UV light and revealed amplicons of 1200 bp size when documented by gel documentation system. The PCR products after purification were ligated in pDrive vector followed by transformation into competent cells (DH5-α strain) of E.coli. One hundred fifty white recombinant colonies were obtained out of which 133 were randomly selected, revived on another plates and screened for expected insert by colony PCR. Recombinant colonies were inoculated in Luria Broth for 16-18 hrs. Plasmid extraction from overnight culture was carried out by alkaline lysis method. The concentration of the plasmid was determined and was subjected to automated DNA sequencing on ABI PRISM® 310 Genetic Analyzer (Applied Biosystems, USA). Sequencing was carried out using BigDye Terminator v3.1 Cycle sequencing kit. Sequences obtained were in the range of 400 to 800 base pairs. Sequences after cleaning (removal of primer and vector sequence) were searched against BLASTn database to find similarity matches. Phylogenetic analysis was carried out by DNADIST with neighbor joining method and DNA Parsimony available at PHYLIP 3.5c package. The tree was mainly divided in to 4 major clusters viz. Methanomicrobia group, Thermoplasmata group, Methanobacteriales group, and Unidentified group. Out of 132 clones, 41.22% showed similarity with Methanomicrobia, with two sub groups Methanoculleus (51.85%) and Methanosarcina (48.15%) in DNA DIST. How ever, in DNA Parsimony analysis, over and above these two subgroups, a group of clones (38.89% clones) was observed which did not share high similarity with any known genus and grouped as unidentified Methanomicrobia. Eighteen (13.6%) clones grouped with Thermoplasmata group in both the trees. The smallest group was of Methanobacteriales with 12.12% and 3.8% clones in DNA DIST and DNA Parsominy analysis, respectively. Our library also uncovered a novel group of rumen archaeal sequences which includes 44 (33.33%) clones in DNA DIST and 57 (43.18%) clones in DNA Parsimony and did not form close cluster with any of cultured and uncultured sequences in both the trees and remained as unidentified methanogens which are supposed to be new. All sequences were submitted to Genbank and are available with the accession numbers EU794740-EU794854, EU814699-EU814714 in EMBL, GenBank and DDBJ Nucleotide Sequence Databases.