Loading...
Thesis
Browse
91 results
Search Results
ThesisItem Open Access COMPARISON OF DIFFERENT MILK REPLACERS ON GROWTH PERFORMANCE OF MALABARI KIDS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2018-12-30) N. SASIKALA; Sabin GeorgeAn experiment was conducted to study the effect of feeding different milk replacers in Malabari kids on their growth performance for the period of two months. Twenty four healthy Malabari kids of four days of age were selected and divided into four groups of six each, as uniformly as possible with regard to age and body weight and were allotted randomly to different experimental rations. Experimental rations were T1 (control, dam’s milk), T2 (Milk replacer based on whey powder), T3 (Milk replacer based on groundnut oilcake) and T4 (Milk replacer based on coconut oilcake). Dam’s milk and milk replacers were fed to kids at the rate of 1/6th and 1/8th of the body weight during first month and second month respectively. Kid starter and fresh green grass were offered to the kids from 15th day onwards to all the experimental kids throughout the experimental period. Data on body weight gain, body measurements, daily dry matter intake, feed conversion efficiency, haematological and serum biochemical parameters such as haemoglobin, plasma total protein, serum glucose, urea nitrogen, serum calcium and phosphorus, faecal consistency score, incidence of diseases and techno economics of production were employed for evaluation of different treatments. Average daily gain and total weight gain were 63 g and 3.98 kg, 62 g and 3.91 kg, 49 g and 3.07 kg and 47 g and 2.96 kg, respectively for kids fed T1, T2, T3 and T4. Average daily gain and total weight gain were significantly higher in T1 and T2 compared to the kid fed T3 and T4. Over all mean of body length at two months ranged from 37.75 to 39.92 cm. Mean heart girth ranged from 35.50 to 36.33 cm. Mean height at withers ranged from 36.83 to 39.92 cm. Body measurements showed no significant difference between the treatments. Total dry matter intake of kids was 6.93, 5.23, 4.80 and 4.88 kg, respectively for T1, T2, T3 and T4. Total dry matter intake was significantly higher in T1 than T2, T3 and T4. Feed conversion efficiency for kids fed T1, T2, T3 and T4 was 1.73, 1.34, 1.56 and 1.65, respectively. The haematological and serum biochemical parameters such as haemoglobin, plasma total protein, serum calcium, phosphorus, glucose, blood urea nitrogen did not show any significant difference between the treatments and the values were within the normal range reported for the species. Faecal consistency score was comparatively higher in the first fortnight than the following fortnights and the values were similar between the treatments. Out of the total cases of diarrhea, incidence was more in kids fed T4 (30 per cent). Respiratory illness was noticed higher in kids fed T3 and T4 than T1 and T2. Majority of the cases of bloat was noticed in T4 group. The feed cost per kg body weight gain for the kids fed T1, T2, T3 and T4 were Rs. 580.02, 139.60, 176.94, 179.78, respectively. Feed cost / kg gain was significantly lower in kids fed with T2 followed by T3, T4 and T1 groups. From the results obtained on the present study it could be inferred that kids in T2 had similar growth performance as that of control (T1) group. Feed cost per kg gain in T2, T3 and T4 was better than T1. Therefore it could be concluded that milk replacer based on whey powder can be given to the kids as an alternative to milk without compromising the growth performance.ThesisItem Open Access HEPATOPROTECTIVE EFFECT OF ECLIPTA PROSTRATA (L.) L. LEAVES ON EXPERIMENTALLY INDUCED AFLATOXICOSIS IN BROILER CHICKEN(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, 2018-11-30) PRIYA. K; Preethy JohnThe study was aimed to investigate the hepatoprotective effect of E. prostrata (Kayyonni) leaf powder on experimentally induced aflatoxicosis in broiler chicken. The leaf powder was subjected to preliminary phytochemical screening to find out the active principles present in it. Aflatoxin was produced in maize using the culture Aspergillus flavus NRRL 6513. The maize culture powder yielded 143.48 ppm of aflatoxin. This mouldy maize was incorporated in experimental feed to arrive 500 ppb of aflatoxin. Sixty Cobb400 day old broiler chicks weighing 50 ± 5 g were randomly divided into six groups comprising 10 birds in each group. The birds were maintained under deep litter system and provided with ad libitum water and feed throughout the experimental period. All the birds were vaccinated as per the standard schedule. Aflatoxicosis was experimentally induced in all groups except T1 and T3 by giving 500 ppb of aflatoxin B1 (AFB1) from eighth day of age onwards. The group T1 was kept as normal control and T2 as toxic control. T3 was fed with E. prostata leaf powder at 0.2 per cent level. The leaf powder of E. prostrata was given to T4, T5 and T6 at dose rates of 0.05, 0.1 and 0.2 per cent respectivelyBody weight was recorded at weekly intervals and the blood was collected from the wing vein on days 7, 21 and 42. Serum was separated and used for the estimation of biochemical parameters such as aspartate transaminase (AST), creatine kinase (CK), cholesterol and total proteins. On the day 42, all the birds were sacrificed; detailed post- mortem examination was conducted. Liver samples were taken to estimate antioxidant parameters such as lipid peroxidation (LPO) and reduced glutathione (GSH). Representative liver samples were also taken and preserved with 10 per cent neutral buffered formalin for histopathological examinationThe preliminary phytochemical screening of E. prostrata leaf powder revealed the presence of steroid, tannins, flavonoids, diterpenes, tripterpenes and saponin.Treatment with E. prostrata leaves powder revealed hepatoprotection in dose dependent manner which is indicated by significant (P<0.05) reduction in the level of serum AST and increase in the level of cholesterol and total protein. The oxidative stress induced by aflatoxin in liver was reduced to a great extend as indicated by the increased level of reduced glutathione and decrease in the lipid peroxidation. Histopathological examination of liver showed regenerative changes in a dose dependent manner when compared with that of normal control group. Thus, it could be concluded that E .prostrata leaf powder had marked antioxidant and hepatoprotective effect on experimentally induced aflatoxicosis in broiler chickenThesisItem Open Access Development of protocol for management of hoof problems in crossbred cattle(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2018-09-03) POOJA U; Anil K.SThe present study was conducted to identify predisposing factors of hoof problems in crossbred dairy cattle and to develop a protocol for the management of hoof problems in crossbred cattle of Kerala. A total of 45 private farms in Thrissur and Palakkad districts and 2 farms under KVASU were taken for the study. The study covered small scale farms(1-2 animal), medium scale farms(3-10 animals) and large scale farms(>10animals) of both districts and two organized farms viz, University livestock farm, Mannuthy, and Cattle breeding farm, Thumburmuzhy. Hoof problems were identified using In stall lameness indicators and severity was assessed using locomotion score and body condition score of the animal. The large scale farms had maximum incidence (41.2 per cent) followed by medium (26.7 per cent) and small scale farms (20.0 per cent). The incidence in organized farms was thirteen per cent. The incidence of hoof problem was more in large-medium land holdings (56.3 per cent) farms where as in marginal-small farms incidence was less (10.3 per cent). The incidence of hoof problem in crossbred HF and crossbred Jersey were 31.7 per cent and 16.7 per cent respectively. The effect of age and body weight of the animal was found to be significant (p0.05). The animal reared on concrete floor had more incidence of hoof problems (46.4 per cent) compared to stone pavement which was (20 per cent). In slippery/pitted floor hoof disorders for cattle was 36.8 per (p<0.05). The incidence of hoof problem was more with asbestos as roof material (59.1 per cen). As frequency of animal bathing increases the incidence of hoof problem was found more (46.7 per cent). The average lactation yield per day was less than five litres in all animals with hoof problem and also in early stage of lactation hoof problem was found more. The animal with parity two (61.5%) is found to have more incidence hoof disorders compared to one and three. The 75 percent of animal having hoof problems was also suffering from anestrum (p<0.05). The incidence of hoof problem was only 13.6 per cent in farms with provision of foot bath whereas it was 44 per cent in farms without footbath although. The occurrence of hoof problem was more in rainy season (70.2 per cent) than in summer and post monsoon. In 2.3 per cent of farms culling was done due to hoof problems. The incidence of hoof problem was 20.7 per cent in farms where hoof trimming was carried out and 44.4 per cent incidence in farms without hoof trimmi. The average locomotion score in private farms was 4.31±0.96 whereas in organized farms it was 3.97±0.133. The average body condition score was 1.82±0.099 for private farms and for organized farms was 2.38±0.140 (p<0.05) . From this study it can be concluded that hoof disorders are a serious problem in crossbred cattle under field conditions and all efforts are needed to control by providing adequate inputs.ThesisItem Open Access A COMPREHENSIVE APPROACH FOR DIAGNOSIS OF LEPTOSPIROSIS IN DOMESTIC PIGS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2018-09-30) P. S. RESHMA; M. MiniLeptospirosis is a worldwide zoonotic disease that in pigs primarily causes reproductive disturbances. Samples collected from Centre for Pig Production and Research (CPPR), Mannuthy and selected private farms in Thrissur were used for the present study. Serum samples (n=103) were tested using microscopic agglutination test (MAT) and an overall seropositivity of 35.92 per cent could be detected. The serogroup Pomona (45.95 per cent) was the most prevalent among the 37 positive samples followed by serogroups Grippotyphosa (24.32 per cent), Canicola (13.51 per cent), Icterohaemorrhagiae (10.81 per cent) and Tarassovi (5.41 per cent). Samples of serum (n=56), whole blood (n=52) and aborted foetus (n=7) when tested using PCR for the presence of lipl32 gene of Leptospira, DNA of three whole blood samples and one aborted foetus amplified the gene producing an expected 767 bp amplicon. Latex agglutination test (LAT) and indirect fluorescent antibody test (FAT) was standardised using seven-day-old reference cultures of Leptospira interrogans serovar Pomona as positive controls at 10-fold dilutions. However, none of the abortion samples tested using LAT and FAT were positive. Attempts for isolation from positive samples were also made, but Leptospira could not be isolated from any of the foetal membrane, liver, kidney samples from the PCR positive animals. Hence in the present study, a seroprevalence of 35.92 per cent for leptospirosis among pigs in Thrissur district was detected using MAT, and PCR was found to be the most sensitive method for directly detecting the presence of the organism in clinical samples, compared to LAT, FAT and culture.ThesisItem Open Access EVALUATION OF EFFLUENT TREATMENT METHODS IN DAIRY FARM(Kerala Veterinary and Animal Sciences Mannuthy, Thrissur, 2018-09-30) JONATHAN JAMES MOONNUMACKAL; A. KannanA study was conducted to evaluate the efficacy of different effluent treatment methods in dairy farm and to analyze cost effectiveness of effluent treatments. An initial survey was conducted among hundred farms of Thrissur district using a well structured questionnaire and data was collected regarding the farming system and their effluent management. The farms were categorised into small, medium and large. Six farms from each category were selected randomlyfor effluent collection in two different season namely summer and monsoon. Effluent was collected in the morning at 8am and evening at 5pm and pooled together. Representative sample (15 litres) of pooled effluent was taken from each farm and was subjected for different treatment protocols. After the end of each treatment water quality parameters were analysed. From the data of survey, it was found that there was a positive correlation between number of animals (96 per cent) and total water used (98 per cent) with effluent output. Effluent subjected with different treatment protocols (C- control, T1- Screening, T2- sedimentation, T3- Filtration and T4- chlorination). After the end of each treatment, the water were subjected to evaluation of quality parameters like colour, Total Dissolved Solids (TDS), pH, turbidity, Biological Oxygen Demand (BOD), Chemical Oxygen Demand (COD), Total Coliform count (TCC) and E. coli count. Data were compared with Indian Standards for reuse in dairy farm.The colour (Hazen) of water indicated that control (958.50 ± 4.18) was significantly higher than the rest of treatments. There was no significant difference between colour value of water from T3 (266.80 ± 5.8) and T4 (249.13 ± 8.66). T3 (filtered water) and T4 (chlorinated water) were lesser due to the respective treatments. The TDS (mg/L)of water from treatments T3 (1404.47 ± 31.68), T2 (2358.63 ± 34.52), T4 (2408.05 ±48.23) and T1 (3354.55 ± 29.65) with increasing trend were significantly lower than that of control (5161.22 ± 28.94). T2 (2358.63 ± 34.52) and T4 (2408.05 ± 48.23) were not significantly different from each other and T4 was significantly higher than that of T3. The pH of water from control (8.20 ± 0.07)and T1 (8.19 ± 0.05), T3 (7.40 ± 0.05)and T4 (7.26 ± 0.06)were significantly not different from each other where T2 (6.98 ± 0.10)was significantly different from all other treatments. The turbidity (NTU) of water from control (718.80 ± 6.86)and T1 (669.36 ± 10.16), T2 (76.38 ± 2.02)and T3 (42.26 ± 1.53) were significantly different from each other where T3 (42.26 ± 1.53) and T4 (38.00 ± 1.74)were not significantly different from each other. The BOD 5 (mg/L) of water from control (154.30 ± 1.31), T1 (131.75 ± 1.31), T2 (77.16 ± 1.31), T3 (52.41 ± 1.31) and T4 (41.44 ± 1.31) were significantly different from each other. The COD (mg/L) of water from control (1447.83 ± 15.04), T1 (1222.08 ± 19.40), T2 (672.00 ± 6.62), T3 (426.80 ± 10.37) and T4 (316.50 ± 8.51) were significantly different from each other. The TCC (MPN/100 ml) of water for control, T1, T2, T3 and T4 were 22.25 ± 0.27, 21.44 ± 0.27, 12.93 ± 0.27, 11.12 ± 0.27 and < 1.80 ± 0.27respectively.Control and T1 were having highest and T4 was having least TCC. The E. coli count (MPN/100 ml) of water for control, T1, T2, T3 and T4 were 22.22 ± 0.27, 21.13 ± 0.27, 12.93 ± 0.27, 11.12 ± 0.27and <1.80 ± 0.27respectively.Control and T1 were having highest and T4 was having least E. coli count.Effect of effluent treatments between the farm size revealed that parameters like pH, turbidity, COD and BOD 5 showed significant difference between treatment and farms and parameters like colour, TDS, TCC and E. coli had no significant difference between treatment and farms. The pH value of water from small farm (8.40 ± 0.12) was not significantly different from medium farm (8.32 ± 0.12)but significantly different from large farm (7.94 ± 0.12) in the control group. The turbidity (NTU) of water was significant different between treatments and farms. There was no significant difference between T3 and T4 in small, medium and large farms. The COD (mg/L) of water in small, medium and large farms, control (small- 1401.08 ± 26.05, medium- 1378.05 ± 26.05 and large- 1564.16 ± 26.05)were significantly higher (p<0.05) than that of T4, T3, T2 and T1. The BOD 5 (mg/L) was significant difference between treatments and farms. In small, medium and large farms, control was significantly higher (p<0.05) than that of T4, T3, T2 and T1.`Effect of effluent treatment between the seasons revealed that for all the water quality parameters like colour, TDS, pH, turbidity, COD, BOD5, TCC and E. coli count had no significant difference between treatment and seasons. The water quality parameters colour, TDS, pH, turbidity, Total Coliform Count and E. coli count were within the limits of Indian standards IS 10500. COD and BOD 5 were slightly higher than the limits of the Indian standards (IS 10500), so this water must be treated for reuse. The overall results in the study indicated that T4 having better efficacy compared to all other treatments. The results of the study indicated that the wastewater generated in the dairy farm can be effectively treated using the four stage treatment (C- control, T1- Screening, T2- sedimentation, T3- Filtration and T4- chlorination) in this study and the reclaimed water can be reused in the dairy farms for cleaning the shed, toconserve water and to reduce the water body contamination. The cost of the fourth treatment is comparatively higher however effectiveness of effluent treatment indicated that T4 is better in comparison with other treatments.ThesisItem Open Access POLYMORPHISM OF CANDIDATE GENES CONTROLLING TEMPERAMENT TRAITS AND THEIR ASSOCIATION ON REPRODUCTION TRAITS IN VECHUR CATTLE(Kerala Veterinary and Animal Sciences Mannuthy, Thrissur, 2018-09-30) ANANDAKUMAR PONNALA; Manoj M.Temperament in cattle plays an important role in animal safety, production and reproduction performance. Bos indicus species are known for their aggressive behaviour than Bos taurus. The present study was conducted to identify the polymorphism of candidate genes controlling temperament traits and their effects on reproductive performance of Vechur cattle. The study comprised of 68 Vechur cattle that were divided into four temperament groups based on their subjective temperament score, social separation test, docile test and handler’s/milker’s score test. Available reproductive performance data of animals were collected from Vechur cattle conservation project to study the following traits - age at first service, age at first calving, service period, conception rate and number of services per conception. Six candidate genes - Tyrosinase (TYR), Dopamine receptor D4 (DRD4), Monoamine Oxidase A (MAO A), Cannabinoid receptor 1 (CNR1), Regulator of G-protein signaling 2 (RGS2) and Plexin A2 (PLXA2) - for temperament were selected. Three selected SNPs from each gene were screened using PCR-RFLP/ PCR-SSCP and further confirmed by sequencing. Out of these 18 SNPs, 9 were present in the Vechur herd. The genes MAOA, TYR, CNR1 and PLXA2 contain two, two, three and two SNPs, respectively. Except TYR 6416322 SNP remaining eight SNPs, coat colour (p=0.003) and sex (p=0.001) were significantly associated with handler’s/milker’s temperament score of handler three (H3). MAOA 105380513 SNP C to T transition was fixed in Vechur population. The average service period of four temperament classes (docile, restless, nervous and aggressive) were 251.02 ± 24.04, 168.57 ± 19.28, 230.59 ± 21.75 and 243.05 ± 32.26 days, respectively. Second restless group had significantly (p=0.039) lower average service period in comparison to all other groups. However, age at first service, age at first calving, conception rate and number of services per conception were not significantly associated with temperament score in the present study. There were significant correlations among different reproductive traits. Results of this study revealed that the selected genes were having significant effect on temperament and reproduction traits, which may be confirmed by further research using larger population and more accurate temperament score. This would enable us to incorporate temperament as an important trait in the selection programmes for genetic improvement of Vechur cattle.ThesisItem Open Access MANAGEMENT OF OXIDATIVE STRESS IN CHRONIC VALVULAR HEART DISEASE IN DOGS(Kerala Veterinary and Animal Sciences Mannuthy, Thrissur, 2018-10-30) REVATHI K; Madhavan Unny. N.Oxidative stress is considered to be one of the major contributing factors in the development of cardiac diseases. Sixty nine dogs presented with symptoms of cardiac and respiratory ailments were screened for cardiac diseases. Based on clinical, electro cardio graphic, radiographic and echo cardio graphic examination, cardiac diseases were confirmed in 34 dogs. Types of cardiac disorders diagnosed in the study were mitral valve insufficiency (76.47%), dilated cardiomyopathy (17.65%) and tricuspid valvular insufficiency (5.88%). Twenty dogs that belonged to stage C as per American College of Veterinary Internal Medicine consensus statement on guidelines for the diagnosis and treatment of canine chronic valvular heart disease were selected and randomly divided into two groups of ten animals each. The selected animals were subjected to detailed clinical examination, blood pressure measurement, electro cardio graphic, radiographic and echo cardio graphic examination and assessment of oxidative stress parameters on the day of presentation. Animals that belonged to group I and II were treated with furo semide, enalapril and pimobendan for two months. In addition, animals of group II were supplemented with coenzyme Q10. Oxidative stress parameters were rechecked on 30 th and 60 th day of treatment. All animals werereassessed by echo cardiographic and blood pressure measurement on 60 th day.Higher occurrence of valvular heart diseases was recorded in small breeds. Maledogs were more affected compared to females. The average age was 9.73 years. Inappetance, dyspnoea, murmurs on auscultation, lethargy, exercise intolerance,dull heart sounds and syncope were the major clinical signs and findings.Electro cardio graphic changes recorded were atrial fibrillation, ST coving, first degree AV block, second degree AV block, ‘P’ mitrale, sinus tachycardia and ventricular premature complexes. Left atrial enlargement and cardiomegaly were the predominant radiographic changes noticed. Clinical response was more evident in group II animals compared to group I animals. At the end of the study period, a significant fall in total antioxidant status and a slight decrease in mean values of lipid peroxides was recorded in group II. No statistical significance was recorded in the ejection fraction, fractional shortening and E point to septal separation values within the treatment groups.ThesisItem Open Access EFFECT OF SODIUM ALGINATE EDIBLE COATING WITH CLOVE (SYZIGIUM AROMATICUM) BUD OLEORESIN ON QUALITY OF BROILER CHICKEN BREAST FILLETS(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2018-11-23) JAMES, LIJIN; Nayar, RenukaThe present study was carried out in the Departmentof Livestock Products Technology, College of Veterinary and Animal Sciences, Pookode to evaluate the effect of edible coating of broiler chicken breast using sodium alginate incorporated with varying concentrations of clove (Syzigiumaromaticum) bud oleoresin and to assess the shelf life of coated as well as uncoated breast fillets under aerobic packaging in chiller storage (4±1°C). Coated breast fillets were made by coating with solution containing sodium alginate (two per cent) and glycerine (one per cent) incorporated with different concentrations of clove, T 1- sodium alginate coating alone, T2- three per cent clove incorporated, T3- four per cent clove incorporated and T4- five per cent clove incorporated and compared with control (C). Physico-chemical properties such as drip loss and cooking loss showed significantly (p<0.01) lower values for control when compared to treatments on all days of storage. There was a significant (p<0.01) increase in drip loss in all the samples throughout the storage period. pH values of both control and treatments increased significantly at one per cent level.Water holding capacity did not differ significantly in control and treatment groups on all days of storage except on day 0. TBARS number was significantly (p<0.01) lower for C than treatments on day 0. From day 3 onwards T 1 showed significantly (p<0.01) higher values than C and other treatments till day 12.Tyrosine values for treatment fillets significantly (p<0.01) increased across storage except in control where there was no significant difference. Except on day 3, C had significantly (p<0.01) lower ‘L’ values when compared to treatments. C and T 1 showed a significantly (p<0.01) higher ‘a’ values when compared to other samples on all days of storage. On storage, redness values significantly (p<0.01) reduced for C. There was significant (p<0.01) difference in ‘b’ values between samples on days 0, 9 and 12, with C showing the lowest value and T 4, the highest. On storage, there was no significant difference between the values in any of the sample. On all other days, except on day 0, shear force values were the highest for control and significantly (p<0.01) lower for treatments.There was a significant (p<0.01) decrease in hardness for all the samples across storage. Phenolic values of 3, 4, and 5 percent solutions were assessed as 0.552± 0.13, 0.879 ± 0.19 and 1.38 ± 0.21 µg TAE /ml of coating solutions, respectively. DPPH (2, 2’- diphenyl-1-picryl hydrazyl) assay revealed radical scavenging activity of 3, 4, and 5 per cent clove oleoresin containing coating solution (1 in 100 dilution) to be 21.82± 0.18 per cent, 27.27± 0.32per cent and 35.45 ± 0.21 per cent, respectively. Radical scavenging activity of sodium alginate coating solution (1 in 100 dilution) containing 0.5µg of phenolics as tannic acid equivalents was found to be 4.55 ± 0.15 per cent. The same for clove oleoresin (1 in 200 111 dilution) containing 25µg of phenolics as tannic acid equivalents was found to be 56.19 ± 0.23 per cent. Aerobic plate counts of all treatments from day 6 onwards, were significantly (p<0.01) lower than that of control, with the lowest counts for T 4. Psychrotrophic count was significantly (p<0.05) lower for T 2, T 3 and T 4 when compared to C and T 1. Proximate analysis revealed no significant difference between the samples with respect to fat, protein, ash, carbohydrate and energy levels. Sensory attributes for raw fillets like appearance and texture values showed no significant difference between control and treatments and also across storage, whereas odour scores for control and T1 reduced on day 9 and 12 where T2, T3, and T4 showed higher values. Appearance, texture and juiciness scores for cooked fillets did not vary significantly between samples. Flavour, after-taste and overall acceptability scores for clove incorporated fillets was higher when compared to control and T 1. Shelf life of control (C) and sodium alginate alone treated (T1) samples was found to be 15 days. Clove incorporated sodium alginate coated samples, T 2, T 3 and T 4 had a better shelf life and lasted till 18th day of chiller storage. Clove oleoresin at 3-5 per cent can be used for extending the shelf life of alginate coated breast fillets with good sensory attributes and can be considered as a preservation method with natural antimicrobial or antioxidant agents.ThesisItem Open Access IN VITRO HEPATIC METABOLISM OF THE PHYTOBIOTIC 1,8- CINEOLE IN DOMESTIC FOWL(COLLEGE OF VETERINARY AND ANIMAL SCIENCES, POOKODE WAYANAD, 2018-08-13) D. M., MAHESH; Juliet, SanisChickens reared under intensive systems are likely to be exposed to feed additives and various xenobiotics. With the ban of antibiotics as in-feed growth promoters (AGPs), there is requirement of alternative method to improve the bird’s performance capacity and also cope well with harsh conditions during rearing period. Phytobiotics, especially essential oils (EOs), are a new class of feed additives that have attracted attention due to their attributed antimicrobial and growth promoter properties. 1,8-cineole is a naturally occurring monocyclic monoterpene ether with an aromatic and camphor like odour and is one of the essential oil (EO) components used in poultry feed as phytobiotic. Even though the metabolism of 1,8-cineole is well studied in rats, rabbits, koala and brush tail possum and humans, its fate in poultry is not yet reported. Cytochrome P450 enzymes (CYP) are a group of monooxygenases playing a significant role in the biotransformation of several kinds of xenobiotics. CYP3A enzymes have been reported as responsible for metabolism of 1,8-cineole in rat and human liver microsomes. Avian and other mammalian species have distinctly different potential capacities of metabolizing drugs. The difference in the metabolic pathway and metabolic enzymes involved in biotransformation of drugs is one of the factors contributing for species variability in pharmacological responses. Although mammalian and avian CYPs are not strictly orthologs, some substrates were found to work for both groups. Hence, the present study was conducted in vitro to identify the cytochrome P450 enzyme orthologs involved in the biotransformation of 1,8-cineole in chicken hepatic S9 and microsomal fractions. The approaches used included the use of specific cytochrome P450 (CYP3A4) inhibitors and the correlation of prototype substrate activities with the formation of the hydroxylated metabolite of 1,8-cineole. Nifedipine and phenacetin were used as specific substrates for the CYP3A and CYP1A isoforms and ketoconazole was used as an inhibitor. Twelve day old Gramasree layer chicks were purchased from hatchery unit, Instructional Livestock Farm Complex, College of Veterinary and Animal Sciences, Pookode and were reared under standard management practices for 12 weeks. The chicks were fed with standard layer feed diet for starter (0-8 weeks) and grower (8-12 weeks) as per Bureau of Indian Standards, 2007 and had free access to water ad libitum until the experiment. A high performance liquid chromatography method was validated and applied for the determination of nifedipine, phenacetin and their formed metabolites nifedipine oxide and acetaminophen in hepatic microsomes and S9 fractions. Determination of nifedipine and nifedipine oxide were carried out at a flow rate of 1 mL/min using the mobile phase consisting of methanol and water in the ratio of 65:35. Phenacetin and its metabolite acetaminophen were determined at a flow rate of 1 mL/min by using the mobile phases consisting of methanol: water (70:30) and water: acetonitrile (85:15) respectively. Analysis of 1,8-cineole and its metabolite was done by gas chromatography mass spectrophotometry(GC-MS). The extraction efficiency was also determined for nifedipine, nifedipine oxide, phenacetin, acetaminophen and 1,8- cineole by comparing the peak areas from drug free samples spiked with known quantities of drug in the range of concentration of calibration curves and standard solutions with suitable solvent in which it is soluble, injected directly into analytical column. Chickens of ninety days age were euthanized by complete cranial decapitation followed by exsanguination. The liver was immediately collected and washed with icecold 1.15 per cent of potassium chloride solution. Both the body and liver weight were recorded. A portion of the collected livers (major lobe) was processed at 4 ºC for the generation of S9 and microsomal fractions. In vitro incubation studies were carried out in S9 and for with and without NADPH generating system with nifedipine, phenacetin and 1,8-cineole in presence and absence of the inhibitor ketoconazole at predetermined time intervals. The enzyme activities for the CYP isoforms were correlated with the formation of the metabolites. Analysis of various drug metabolisms were done using Graphpad prism 5 software and nonlinear regression analysis of drug disappearance versus time was done with mycurvefit. No mortality of the birds was recorded when reared strictly under intensive system. The birds at the end of 12th week attained an average body weight of. 771.33 ± 64.96 g. The nifedipine and its metabolite were best separated with retention times of 8.02 and 6.01 min respectively. The drug phenacetin and its metabolite had a retention time of 4.2 and 5.5 min respectively. Ketoconazole was detected at 220 nm with a retention time of 1.9 minute using the mobile phase mixture of methanol: 0.1% formic acid (90:10, v/v). The retention time of 1,8-cineole and its metabolite were 7.9 and 13.1 min respectively. An average obtained liver weight of 19.79 ± 2.88 g was obtained which was approximately 2.56 per cent of the average body weight for Gramasree birds. The microsomal protein in the present study was 28.42± 0.780 per gram of fresh weight tissue. Incubation of 1,8-cineole in the hepatic S9 fraction with a protein concentration of 7 mg/ml up to 180 min did not exhibit any significant decrease in the concentration of the parent compound compared to samples at ‘zero’ min. On the contrary, 1,8 cineole was significantly metabolized by the microsomal fraction with a linear decrease in the concentration of the drug. This could be due to the difference in the metabolic enzymes present in the cytosolic and microsomal fractions. No significant difference in metabolic activity was noted between the male and female birds. The formation of nifedipine oxide at different incubation times indicated the existence of chicken CYP ortholog of the human enzyme studied. Phenacetin, also metabolized by CYP1A2 isoenzyme was used as a specific substrate probe for determining its activity. Phenacetin was not metabolized by the hepatic cytosolic enzymes till 180 minutes. However, both phenacetin and 1,8-cineole was metabolised in the S9 fraction 6 hours post incubation indicating low CYP activity in the hepatic S9 fraction. . The study indicated that CYP3A isoform catalysed the hydroxylation of 1,8- cineole in chicken liver microsomes with the formation of 2α-hydroxy-1,8-cineole. The human isoenzyme CYP3A specific inhibitor ketoconazole significantly inhibited the metabolisms of nifedipine and 1,8-cineole in chicken liver microsomes. Since ketoconazole is also reported as a nonspecific inhibitor of CYP 1A in chickens, the role of CYP 1A2 in the metabolism of 1,8-cineole in chicken in the present study cannot be ruled out. Further, the rate of 1,8- cineole biotransformation is low in chickens when compared to biotransformation in humans and rats in vitro.
