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ThesisItem Open Access PHYTOCHEMICAL ANALYSIS OF MEDICINAL PLANTS AND THEIR EFFECT ON OXIDATIVE STATUS, RUMEN ENZYMES AND BIOCHEMICAL PROFILE OF GOAT(Assam Agricultural University, Khanapara, Guwahati, 2017-07) THAKURIA, PURABI; Nath, RitaIn this study, three medicinal plants viz. Artocarpus heterophyllus (Jackfruit), Carica papaya (Papaya) and Terminalia bellerica (Bhumura) were investigated for the presence of phytochemicals and their effect on the rumen enzymes, oxidative status and blood biochemical profile was evaluated. The preliminary phytochemical screening of Artocarpus heterophyllus, Carica papaya and Terminalia bellerica revealed the presence of phytochemicals such as tannin, saponin, flavonoid, terpenoid, alkaloid, glycosides and protein in methanolic extract. Quantitative analysis revealed that the highest concentration of tannin (%) was found in C. papaya (0.638 ± 0.05) followed by T. bellerica (0.562 ± 0.09) and A. heterophyllus (0.253 ± 0.06). However, the flavonoid concentration (mg QE/g) was highest in T. bellerica (0.825 ± 0.001). But there was no significant (P<0.01) difference in saponin concentration among all the three plant leaves and the highest concentration of saponin (mg/100g) was found in T. bellerica (2.67 ± 0.07). The antioxidant activity in terms of DPPH (2, 2- diphenyl-2- picryl hydrazyl hydrates) radical scavenging activity was highest in A. heterophyllus (70.23 ± 0.0.50 %) followed by C. papaya (65.57 ± 0.27 %), T. bellerica (62.70 ± 0.06 %) and Vitamin C (27.04 ± 0.01 %) respectively at concentration of 110 µg/ml. Similarly, in superoxide radical scavenging activity A. heterophyllus showed highest radical scavenging activity (83.35 ± 0.09 %) followed by C. papaya (81.08 ± 0.04 %), T. bellerica (75.36 ± 0.14 %) and vitamin C (14.50 ± 0.05 %) at concentration of 110 µg/ ml. Reductive ability was highest in A. heterophyllus (0.137 ± 0.001) compared to standard Vitamin C (0.134 ± 0.001) respectively at concentration of 110 µg/ml. On the otherhand, the highest nitric oxide free radical scavenging activity (%) was recorded in vitamin C (56.16 ± 0.03) followed by T. bellerica (40.43 ± 0.12), A. heterophyllus (36.05 ± 0.57) and C. papaya (26.01 ± 0.23). The highest concentration of hydroxyl radical scavenging activity (%) was found in C. papaya (76.64 ± 1.11) followed by T. bellerica (83 ± 0.08) and A. heterophyllus (70.36 ± 0.17) when compared with vitamin C (13.12 ± 0.01) at 110 µg/ml concentration. The vitamin C content was highest in C. papaya (39.71 ± 0.24 mg/100g). In the present study, eighteen (18) Assam Hill (local) goats of 3-4 months of age were procured and reared in the Experimental Animal Shed, Department of Veterinary Physiology, C.V.Sc., AAU, Khanapara, Guwahati-22. The animals were divided into three groups. One as control, receiving substrate ration, T1 with plant leaves @ 5% level and T2 @ 10% level of total mixed ration (TMR). The duration of the experiment was 90 days. Rumen liquor analysis revealed that out of the three rumen fibrolytic enzymes, Carboxymethylcellulase showed better enzyme activity in T2 group. The blood glucose level (mg/dl) was within the normal physiological range but apparently decreased in T1 and T2 groups indicating hypoglycemic effect of the plant leaves. The serum total protein level (g/dl) was also within the normal range and the level was significantly (P<0.01) higher in T2 group. The level of blood creatinine (mg/dl) increased significantly (p<0.01) in 0 to 90 days from 1.02 ± 0.08 to 1.80 ± 0.08 in all the experimental groups of animals but the observed values were within the normal range. The values recorded for blood urea nitrogen concentration (mg/dl) in all the groups of animal during different days of experiment were found to be apparently higher but analysis of variance revealed no significant differences during the respective days of experiment. The ALT and AST values (U/L) were found to be apparently higher but the values differed non-significantly amongst all groups during the respective days of experiment and the values were within the normal range indicating no harmful effect of the plant leaves on liver. The values recorded for GGT (U/L) in the present experiment were apparently decreased from 37.10 ± 1.35 to 35.91 ± 1.07 in 0 to 90 days but analysis of variance revealed no significant differences during the respective days of experiment. The overall mean value of GGT in T2 group was insignificantly lower than the values recorded in T1 group which indicated normal hepatic condition of the animal. The serum calcium concentration (mg/dl) recorded during 0 to 90 days of experiment in all the groups of goat increased significantly (p<0.01) from 9.17 ± 0.16 to 11.73 ± 0.15. An increase level of phosphorus (mg/dl) was also recorded in T2 group. The serum sodium concentration (mEq/L) was also within the normal physiological range and no significant differences were observed between the respective days of experiment in both T1 and T2 group. The observed value of serum potassium (mEq/L) increased significantly (p<0.01) from 0 to 90 days of experiment in control, T1 and T2 groups. The iron value ((μg/dl)) recorded in the present experiment increased apparently from 0 to 90 days and insignificantly higher values were observed in T2 group (173.69 ± 1.95 μg/dl) as compared to T1 and control groups. The average body weight (kg per animal) recorded in the present experiment showed significant trend (P<0.01) from 0 to 90 days of experiment. The blood malondialdehyde values (μmol/L) in control group increased significantly (P<0.01) from 0.60 ± 0.06 to 0.80 ± 0.02 in 0 to 90 days of experiment but in T1 and T2 groups, blood MDA value decreased significantly (P<0.01) from 0 to 90 days of experiment. Analysis of variance revealed no significant differences during different days of experiment and also amongst the experimental groups of the animal in respect of blood super oxide dismutase (SOD) and glutathione peroxidase (GPX) concentration. The levels of vitamin C (μg/ml) were apparently higher from 0 to 90 days of experiment but analysis of variance revealed no significant differences. From this study, it can be concluded that three medicinal plants viz., A. heterophyllus, C. papaya, T. bellerica are rich source of phytochemicals with good antioxidant properties and do not have any harmful effect on normal functioning of the rumen, liver and kidney. Thus, the plant leaves can be incorporated in the diet up to 10% of the total mixed ration.ThesisItem Open Access THERAPEUTIC POTENTIAL OF BONE-MARROW DERIVED MESENCHYMAL STEM CELLS IN WOUND HEALING(Assam Agricultural University, Khanapara, Guwahati, 2017-07) HAZORIKA, MOUSUMI; Kalita, Dhruba JyotiThe present experiment was carried out to explore the therapeutic potential of rabbit bone-marrow derived mesenchymal stem cells (rBM-MSCs) in wound healing in New Zealand White rabbits. The experiment was conducted in the Department of Veterinary Biochemistry, College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati-781022. Eighteen numbers of clinically healthy male New Zealand white rabbit of 3-4 months with mean body weight of 1.52 kg were randomly divided into three different groups of six animals each viz. Gr-I (Control group), Gr-II (Test group-1) and Gr-III (Test group-2). A total of thirty-six surgical wounds were created on the skin of eighteen rabbits with two in each. The Gr-I rabbits received no treatment and was considered as Control group. Gr-II rabbits were treated with a standard drug i.e. Povidine iodine ointment (Betadine®, USP 10 % w/w) while Gr–III rabbits were treated with cultured BM-MSCs topically. They were raised under standard laboratory conditions and veterinary supervision throughout the experimental period and were given standard pellet feed. The experiment was conducted in a 2x2 factorial design for a period of 30 days. Bone marrow was collected from rabbits to culture and characterize the BM-MSCs. Blood was collected from all the animals of each group on days 0, 3, 7, 15 and 30 to estimate haemato-biochemical constituents viz. haemoglobin, PCV, ESR, TEC, TLC and DLC; serum glucose, total protein, triglycerides, SGOT, SGPT, alkaline phosphatase, uric acid, Ca2+, P, Mg2+, Na+, K+, Cl-, iron, copper, zinc, cytokine proteins: TNF-α, IL-1β, IL-4, IL-6, IL-10 and to study cytokine gene expression pattern during wound healing. Wound area was measured on days 1, 3, 7, 15, 21 and 30 of each animal, of different experimental groups. Tissue was collected on day 10 and day 20 of surgery from each animal of different experimental groups to estimate certain granulation tissue markers viz. LPO, NO; SOD, CAT, GSH, vitamin A, vitamin C, vitamin E; MPO; protein and DNA; and connective tissue markers viz. HYP, HXA, HUA, collagen and elastin. The tissue was also collected on days 1, 10 and 20 of surgery to observe the histopathological changes during wound healing. The rBM-MSCs was isolated, cultured and characterized as per the standards set by ISCT. The rBM-MSC treated group showed complete healing of wound by day 21 whereas complete healing of wound was observed on day 30 in both Gr-I and Gr-II. All the haemato-biochemical constituents were found within the normal range throughout the experimental period. However, the levels of certain parameters significantly increase or decrease amongst the groups with the advancement of time. The rBM-MSCs significantly increased the antioxidants’ levels and reduced the generation of free radicals’ and acute inflammatory marker on day 10 and day 20 of surgery. Significant increase observed in the content of granulation tissue biochemical markers (protein and DNA) and connective tissue markers (HYP, HXA, HUA, collagen and elastin). Histopathologically, the BM-MSCs treated group showed early tissue changes compared to other groups and approximately resembles the adjacent normal skin on day 20. On the basis of macroscopic, biochemical and histopathological evaluation of wounds, it can be concluded that the rBM-MSCs can be used successfully for the treatment of surgical wounds without any significant adverse effects on the animal.ThesisItem Open Access EXPLORATION OF CYTOKINE PROFILES FOR PREDICTION OF SUCCESSFUL PREGNANCY AND LACTATION AND MANIPULATION OF CYTOKINE LEVEL USING CERTAIN STIMULANT IN CROSS BRED COWS OF ASSAM(Assam Agricultural University, Khanapara, Guwahati, 2017-07) BARKAKATI, JURI; Kalita, Dhruba JyotiThe present study was conducted to explore the cytokine profile of crossbred cattle of Assam for the prediction of successful pregnancy and lactation and to manipulate the level of cytokine by using certain cytokine stimulants/ regulators/plant leaves. Selected animals was divided into five groups keeping six in each in accordance with the feeding of different cytokine stimulator/regulator. All cows included for the experiment was in similar managemental practices and were offered same type of ration throughout the study. Group I was control without any treatment. Group II was treated with neem leaves (Azadirachta indica) at the rate of 3gm per kg body weight, Group III was fed with bogori leaves (Zizyphus mauritiana) at the rate of 300 mg per kg body weight orally. Group IV and group V were fed Restobal, a herbal immunomodulator at the dose rate of 50 ml orally and bamboo leaves (Bambusa bambos) at the rate of 3gm per kg body weight daily from the last three months of pregnancy to three months after parturition. Inflammatory indices, liver specific biomarkers, energy metabolism indices, protein metabolism index, oxidative stress response, different serum minerals etc. and different hematological parameters were estimated. Estimation of different cytokine profiles was carried out namely TNF-α, IL-1β. IL-6, IL-10 in serum. And record of daily milk yield and growth performance of calves with different cytokine stimulator/regulator/plant leaves. A non significant rise in the level of RBC count was seen in group IV (Restobal treated) in the late gestation than that of the control group. All the groups showed a similar trend of increase in RBC count till late gestation and then a decline on the day of calving and thereafter. Overall mean showed that there was a higher level of haemoglobin in group II followed by group IV, group III and II. All the groups showed an apparent rise in the level of haemoglobin than that of control group. Overall mean of WBC count in Group II and III were found to be higher than that of the control group but the increase was not significant. No significant difference was observed between the control and treated groups. A higher level of protein was found in group II and group V as compared to the control group which was non significant. A significantly higher (P < 0.0001) level of protein was recorded 30 days after parturition than that of the day of calving. Group II (Neem fed) and group IV (Restobal treated) had a significantly higher (P <0.001) level of globulin as compared to that of the control group. A non significant reduction in the level of creatinine in group IV after parturition . Almost all the groups showed a higher level of albumin during the late gestation which showed a slight decline around calving and on the day of calving and then there was gradual increase in the level of albumin after around one month post parturition. Lower level of total bilirubin was recorded in the experimental groups as compared to the control group. The pre partum values of the paraoxonase enzyme was found to be higher than the level at calving and at postpartum .No significant difference in the overall mean between the experimental groups and that of the control group was recorded. The experimental groups showed a higher level of GGT enzyme activity upto the day of calving and then thereafter showed a reduced level at postpartum . There was a non significant decrease in the level of ALP in the experimental groups as compared to that of the control group. Significant difference (P<0.01) was found in between group I and III and in between group I and V. (P<0.05).The groups showed a higher level of SGOT at parturition which declined after calving. There was non significant decrease in the level of SGOT in the neem treated group.Lower level of NEFA was found in group III and IV as compared to the control group. The groups showed a higher level of NEFA during early lactation. Group I, II and III showed a lower level of beta hydroxyl butyric acid as compared to the control group. However, an increase in the level of BHBA was recorded in group V than that of the control group. Group II (neem leaves fed) and III (Bogori leaves fed) showed a higher level of SOD as compared to the control group. Antioxidant enzyme activity was found to be higher at prepartum, then showed a reduction in the level at calving and thereafter there was gradual increase in the activity of enzyme at early lactation. Enhanced antioxidant activity was observed in the treated groups as compared to the control group. Dietary Neem leaf meal did not significantly (P>0.05) affect the calcium, sodium and potassium levels but significantly (P<0.05) decreased the phosphate level while significantly (P<0.05) and steadily, increasing the chloride level as its level increased. Group II that was fed with neem leaves showed a high preparum level of TNF -α and the peak level (1248.33±226.53 pg/ml) on the day of parturition. The increased level of tumor necrosis factor alpha (TNF-alpha) might be due to the cytokine and immune system stimulating property of neem plants. Significantly lower level of IL-6 was recorded in group III (Bogori leaves fed) and group V (bamboo leaves fed) than that of control. IL-6 level increased non significantly in the Restobal treated and Neem treated group. The level of IL-10 showed an increased level up to the day calving and then a decline was recorded approximately 15 days after calving. Significantly higher level of IL- 1β was recorded in group II (neem treated) (P<0.001) and in group V (P<0.01) than that of the control group. The level of (IL-1β) showed a high level during the advanced stage of pregnancy.Overall mean level of milk yield (litres) was found to be 9.25± 0.115 in group 1, 9.7 ± 0.137 in group II, 9.3±0.139 in group III, 9.6± 0.133 and 8.63±0.085 in group IV and V respectively.All the experimental animals differed significantly from that of the control group, except group IV .The highest average milk yield was recorded in group II (neem fed ) followed by group IV (Restobal treated) and then group III (Bogori leaves ). A lower level of milk yield was found in group V (bamboo leaves fed). The highest body weight was recorded in group IV ( Restobal treated ) followed by group II (neem fed) and then group III and V. supplementation of neem leaves , Restobal, Bogori leaves has a positive effect on the performance of the animals. The cytokine regulators/plant leaves used in the study can be given to the animals during pregnancy which might play an important role in enhancing immunity, maintenance of pregnancy and a successful lactation period.ThesisItem Open Access EXPLORING SEASON RELATED BIOCHEMICAL INDICES IN CROSS-BRED AND LOCAL CATTLE(Assam Agricultural University, Khanapara, Guwahati, 2016-07) Chetia, Mayuri; Sarma, SatyaThe present experiment was conducted to study the significant changes of various physiological, biochemical, hormonal and HSP gene expression profiles in Cross-bred (HF X Jersey) cattle under the agro-climatic condition of Assam and Local cattle of Assam were included in the experiment as reference experimental animals. The experiment was aimed at determining the status of some of the important season related thermal stress biomarkers with an idea to get some clues about the heat tolerance and the adaptability status of the Cross-bred cattles compared to the Local breeds of Assam. The experiment comprised of a total twelve numbers of healthy cattle of ages between 2-3 years, which were divided into two groups each containing six numbers of Cross-bred and six numbers of Assam Local cattle being reared under the semi-intensive system in Instructional Livestock Farm (Cattle) of College of Veterinary Science Khanapara, Assam Agricultural University and Experimental Animal Shed, Department of Veterinary Physiology, College of Veterinary Science Khanapara, Assam Agricultural University. The animals were kept under standard feeding and management. The monthly ambient temperature (°C), relative humidity (%) were recorded from the automatic weather station and temperature humidity index (THI) were calculated during the summer and winter season using standard formula. The physiological parameters such as the rectal temperature, respiration rate and pulse rate were recorded daily twice for the two seasons. The different biochemical parameters such as serum total protein, serum glucose, serum electrolytes - Na+, K+, Cl-, Ca2+, P and Mg2+, erythrocytic oxidative enzymes - superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), serum Lactate Dehydrogenase (LDH), serum hormonal parameters namely, Cortisol, T3 and T4 and mRNA expression profile of HSP 40, HSP 60, HSP 70, HSP 90 genes were studied in both the breeds of cattle. The Temperature Humidity Index (THI) during the study period was found to be indicative of thermal stress to the experimental animals during the summer season compared to winter season. The different physiological parameters viz. rectal temperature, respiration rate and pulse rate were found to increase in both the breeds during summer compared to winter season. All the physiological parameters were increased in the evening hours in both the cattle types compared to the morning hours. The serum total protein and serum glucose concentrations were significantly lower (P<0.001) during the summer season as compared to the winter season in both cross-bred and Assam local cattle. Similarly, the serum electrolytes, such as Na+, K+, Cl-, Ca2+, P and Mg2+ concentrations were significantly (P<0.001) lower in the summer as compared to winter season in both cross-bred and local cattle types of Assam. A significant difference was observed in serum total protein, serum glucose, serum Na+, K+, Cl-, Ca2+ and Mg2+ concentration between the breeds.The activity of erythrocytes SOD, GSH-Px, CAT and serum LDH was found to be significantly (P<0.001) higher in the summer season in both the breeds compared to the winter season. In addition, a significant (P<0.001) difference on the concentration of different erythrocytic and serum enzymes was observed between the two breeds. The concentrations of Cortisol, T3 and T4 were found to be significantly (P<0.001) different between the seasons in both cross-bred and local cattle of Assam. The cortisol level significantly increased during the summer season in both the breeds compared to the winter season while T3 and T4 level significantly decreased in the summer season in both the cattle types. A significant (P<0.001) difference in the hormonal concentration of Cortisol, T3 and T4 was found between the breeds. The present experiment shows a wide variation in the expression of different HSP genes during summer and winter season. The mRNA expression of HSP 40, 70 and 90 genes were significantly higher (P<0.01) during summer season as compared to winter season. The mRNA expression of HSP40, HSP 60, HSP70 and HSP90 was significantly differed between the two breeds of cattle types. HSP 70 has been found to be the potential biomarkers of heat stress in both the breeds of cattle and based on the relative expression, the ranking of genes from higher to lower abundance in cross-bred cattle were in the following order: HSP70>HSP90>HSP40>HSP60 and in local cattle, it is HSP70>HSP60>HSP90>HSP40. All the physiological and biochemical alterations were observed in local and cross-bred animals in the different seasons. However, these alterations were quite more prominent in case of cross-bred cattle in comparison to the local cattle. Hence, the local cattle have better heat tolerance than those of the cross-bred cattle under the agro-climatic condition of Assam.