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2016 - 201912020 - 20223
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Subject: Veterinary Physiology × Thesis Type: M.V.Sc. ×
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    ThesisItemOpen Access
    Strategy for development of stem cell like embryonic fibroblast cells
    (College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-09) Das, Prerana; Nath, Nikhil Ch
    Fibroblast cells are the type of cells that play an important role in the formation of connective tissue. The use of fibroblast cell is versatile, for e.g., demonstration of avian viruses, feeder cells, production of vaccines, preservation of genetic resources etc. In this present study, duck embryonic fibroblast cells were isolated, cultured, and sub-cultured up to six passages. The cells were grown in four culture media i.e., Medium 1(MEM), Medium 2(MEM+IGF-1), Medium 3 (MEM+10% FBS), Medium 4 (MEM+10% FBS+IGF-1). In serum and serum-free media the time required for the cells to attain 70% confluence in primary culture was 84.667±.0.152 hours and 111.867±0.161 hours respectively. The cells grown in medium containing serum showed better results than cells grown in serum-free medium. The time taken to reach 70% confluence in 6th passage in Medium 2 and Medium 4 which are IGF-1 supplemented are 94.583±0.217 hours and 62.167±0.096 hours respectively whereas time taken in Medium 1 and Medium 3 which are IGF-1 free media are 95.350±0.039 hours and 62.667±0.152 hours respectively. Therefore, the cells grown in IGF-1 supplemented media showed significant difference compared than the rest of the culture media (p≤0.01). Morphologically, the cells showed characteristic spindle shape, turgor vitalis cytoplasm, centrally located nuclei and flame-like pattern up to the sixth passage. The viability assessment was carried out in first, second, third, fourth, fifth and sixth sub-culture and the viability percentage of the cells in six different sub-cultures were 89.843±0.108, 91.427±0.082, 91.228±0.081, 91.867±0.079, 92.231±0.073, 93.431±0.069 in the case of Medium 1, 90.425± 0.085, 92.358± 0.124, 93.692±0.084, 93.982 ±0.282, 94.625 ±0.089, 94.892 ±0.096 in the case of Medium 2, 89.145 ±0.263, 90.482±0.09, 91.643±0.143, 92.713±0.186, 93.460±0.079, 94.543±0.074 in Medium 3, and 88.597±0.132, 89.387±0.143, 90.552±0.101, 91.423±0.078, 93.077±0.140, 93.077±0.140 in Medium 4. The viability percentage between the passages was significantly different (p≤0.01). However, the viability of the cells was better from the second subculture compared to primary cultures. The pluripotency of the cells was observed by immunostaining using NANOG antibody, a pluripotent marker that is expressed in embryonic stem cells. It was observed that cells showed positive for NANOG at every subculture depicting their pluripotent nature.
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    ThesisItemOpen Access
    Physiological effect of cryoprotectants in freezing of embryonic fibroblast cells
    (College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-09) Toufiki, Faijun; Bora, Arundhati
    Fibroblast cells are the most common cells of connective tissue and form structural framework. In the present study duck embryonic fibroblast cells were developed up to third subcultures and were cryopreserved in three freezing media consisting of freezing medium 1 (10% DMSO), freezing medium 2 (0.9 M Trehalose) and freezing medium 3 (10% DMSO+ 0.09 M Trehalose in 1:1). The cells conforming the morphologically characteristics of fibroblast like typical fusiform shape, turgor vitalis cytoplasm, centrally located nuclei and flame like migration pattern were used for the experiment. The effect of cryoprotectant at equilibration and at different time of post thaw was assessed by their viability and post thaw characteristics. Trypan blue is an azo-based hydrophilic, tetra sulfonated blue acid dye which is used to determine the number of viable cells present in a cell suspension. A viable cell will have clear cytoplasm and non-viable cell will have a blue cytoplasm. The viability percentage before cryopreservation the viability of the duck fibroblast was 90.75±0.047. For freezing medium 1 (10% DMSO), the viability percentage at equilibration was found to be 89.75±0.047 and subsequently at 7 days 89.61±0.064, at 14 days 89.30±0.035, at 21 days 89.06 ±0.011, at 28 days 89.69±0.14. For freezing medium 2 (0.9 M trehalose), the viability percentage at equilibration was found to be 87.69±0.82 subsequently at 7 days 86.73±0.14, at 21 days 86.42±0.04, and at 28 days 86.00±0.06. The viability percentage was significantly higher (p<0.05) in freezing medium 3 (10% DMSO+0.9 M Trehalose in 1:1) followed by freezing medium 1 (10% DMSO) and freezing medium 2 (0.9 M trehalose). For freezing medium 3 (10% DMSO + 0.9 M Trehalose in 1:1), the viability percentage was found to be at equilibration 90.39±0.084 and subsequently at 7 days 89.78±0.068, at 14 days 89.78± 0.068, at 21 days 89.71 ± 0.13, at 28 days 89.68±0.021 respectively. The revival of freezing media 3 (10% DMSO + 0.9 M Trehalose in 1:1) was found to be at 24 hours 14.12±1.65, at 48 hours 26.44±1.93, at 72 hours 40.44±2.27, at 84 hours 50.64±2.89, at 96 hours 59.32 ±0.23. For freezing media 1 (10% DMSO) the confluency was found to be at 24 hours 17.68±0.97, at 48 hours 32.32±0.99, at 72 hours 43.12±1.12, at 84 hours 49.56±0.18 and at 96 hours 59.28±0.14. For freezing media 2 (0.9 M trehalose) the revivability was found to be 24 hours 9.72±0.08, at 48 hours 14.84±1.14, at 72 hours 25.20±1.20, at 84 hours 44.56±0.30, at 96 hours 53.76±0.10. The confluency of freezing medium 3 was significantly higher (p<0.05) found better than freezing medium 1 and freezing medium 2. Found that both intracellular and extracellular cryoprotectant which may favor the normal physiological process at equilibration and at thawing. NANOG, a noble pluripotent marker was found to be present in the developed fibroblast cells as well as after cryopreservation.
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    ThesisItemOpen Access
    Monitoring certain physiobiochemical parameters of post weaned crossbred kids raised under three different climate resilient housing system
    (College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-09) Patgiri, Dhiman; Dutta, Arup
    The present experiment was conducted to study the growth and physio-biochemical performances of post-weaned (90 days) female crossbred kids (Beetal × Assam Hill Goat) kept under three different climate resilient housing system (i.e., Shed-A, Shed-B and Shed-C) from weaning (90 days) till the attainment of puberty. A total of 21 numbers of post-weaned (90 days) female crossbred kids (Beetal × Assam Hill Goat), maintained in intensive care of management and fed a uniform ration in accordance to ICAR, 2013 feeding standard were randomly divided into three groups of average equal body weight having 7 animals in each group and housed in three different housing system which differed in terms of their ventilation system, materials used, colour, ground clearance etc. Temperature, relative humidity and THI of the ambient surrounding and inside the three sheds were recorded thrice daily viz., morning, afternoon and evening hours during the experimental period and it was found that temperature and THI varied significantly among the ambient surrounding and the three Sheds with lowest temperature and THI recorded in the Shed-B i.e., having side ventilation with walls and floor made of bamboo material. The physiological parameters related to thermal stress viz., respiratory rate, pulse rate and rectal temperature were recorded at alternate days in the morning hours during the experimental period and statistical analysis revealed significant differences (P<0.01) in those parameters with the lowest value recorded in the Shed having the lowest THI. The body weight measurement was done at fifteen days (Fortnightly) interval and statistical analysis revealed significant differences (P<0.01) in the body weight of the animals in the different housing system and the housing system having lowest THI showed highest body weight gain (9.75±0.24 Kg) compared to the animals of other housing systems. Similarly, the age of attainment of puberty was attained by close monitoring of the animals for signs of puberty and statistical analysis revealed significant differences (P<0.01) in the age of attainment of puberty with the lowest age (200.43±5.96 Days) of attainment of puberty was found in animals reared in the system having the lowest THI value. Blood samples were collected at fifteen days (Fortnightly) interval for the analysis of certain haemato-biochemical parameters. Statistical analysis revealed non-significant differences in the Hb, RBC, Glucose and SOD concentrations. Although, there was an apparent decrease in the SOD (1.25±0.03 u/g) concentration in the housing system having lowest THI value. The PCV concentration showed significant differences (P<0.01) among the various housing system with the minimum PCV (18.86 ±0.16 %) value was recorded in the animals housed in the Shed having lowest THI. Analysis of variance revealed that the Cortisol, T3 and T4 concentration showed significant differences (P<0.01) among the animals housed in the different housing system. Thereby it can be seen that housing system having strong implication on various physiobiochemical parameters of an animal and also has the potential to ameliorate the effect of thermal stress on animal.
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    ThesisItemOpen Access
    BIOMOLECULAR EXPRESSION ON MELATONIN AND VITAMIN E SUPPLEMENTATION DURING SUMMER AND WINTER IN PIG
    (Assam Agricultural University, Khanapara, Guwahati, 2016-01) CHAKRABORTY, ARINDAM; Baruah, Anubha
    The present experiment was conducted to study the changes of various physiological, haematological and hormonal parameters including expression of HSP70 gene in the crossbred pigs (Hampshire × Local) under the agroclimatic condition of Assam. The experiment included a total of 36 numbers of crossbred weaned female pigs. Eighteen (18) animals were subjected to treatment separately during summer and winter. The selected animals were divided into three groups with six pigs in each group consisting of the control group (Treatment 1), one group was fed melatonin @3 mg/animal (Treatment 2) and the other group was fed Vitamin E @100 mg (Treatment 3) for both the seasons. The animals were maintained at AICRP on Pig, College of Veterinary Science, AAU, Khanapara, Guwahati-22. The physiological parameters such as body temperature, pulse rate and respiration rate were recorded following standard methods. Temperature-Humidity Index was calculated out from the data of ambient temperature and relative humidity by using standard formula. About 5 ml of blood was collected from each experimental animal aseptically at 15 days interval for the whole experimental period. The haematological parameters viz. Haemoglobin (Hb), Packed cell volume (PCV), total erythrocyte count (TEC) and total leucocyte count (TLC) were estimated from fresh blood by using MS4 Automated Haematological Cell Counter. The enzyme superoxide dismutase (SOD) and Lactate dehydrogenase (LDH) were estimated by using SOD and LDH assay kit manufactured by Cayman Chemical Company, USA as per manufactures protocol. Growth hormone and Progesterone were estimated by ELISA technique using Elisa kits procured from LDN Immunoassays and services. Melatonin was estimated by ELISA technique using Elisa kits procured from, Genway, Biotech Inc. The level of thermal stress related blood hormones such as triiodothyronine (T3), thyroxine (T4) and cortisol hormones were estimated by Radioimmunoassay (RIA) technique. The relative expression of HSP 70 gene was done by Real time PCR. The Temperature Humidity Index (THI) during the study period was indicative of thermal stress to the experimental animals in the summer as compared to winter season. Physiological parameters viz., body temperature, respiration rate and pulse rate were found to be positively correlated with THI. All the physiological parameters showed significant difference (P<0.01) between summer and winter seasons irrespective of treatments. Haematological parameters viz. Hb, PCV, TEC was significantly lower during summer while TLC concentration was significantly higher during summer season as compared to winter in all the treatment groups. The mean body weight in the experimental pigs was significantly higher (P<0.01) in winter compared to summer. Serum T3 concentrations was significantly (P<0.01) lower during summer as compared to winter in all the treatment groups. Serum T4 concentration showed significant difference between treatment, between season and also between treatment and season. Serum cortisol concentration showed significant difference between treatment, between season and also between treatment and season. The serum cortisol concentration was found lowest in the melatonin and vitamin E supplemented group in both the seasons as compared to the control group. Significant difference (P<0.01) was found in the mean GH values between season with significantly higher values in the winter season. Statistical analysis revealed significant difference (P<0.01) in the mean progesterone concentration between treatment and between season. Significant difference (P<0.01) was found in the mean age at puberty between treatment with lower age at puberty in the melatonin supplemented group followed by vitamin E supplemented group and control group with highest age at puberty. There was also significant difference (P<0.01) in the mean age at puberty between season with lower age at puberty in the winter compared to summer. There was also significant difference (P<0.01) between day and season. Serum LDH activity was significantly higher (P<0.01) during summer as compared to winter season. The serum SOD activity was found to differ significantly (P<0.01) higher between treatment and between season and also between treatment and season. The serum progesterone concentration showed significant difference (P<0.01) between treatment and between season. There was also significant difference (P<0.01) between day and season. The mean melatonin concentration showed significant difference (p<0.01) between groups with significantly higher melatonin concentration in the melatonin supplemented group in both the season. Similarly serum vitamin E concentration was significantly higher (p<0.01) in the vitamin E supplemented group than the other two treatment groups in both the seasons. The normalized expression for HSP70 during summer shows that the animals with Melatonin treatment had 1.98 fold lower expression than the animals of control group. Likewise, animals with Vitamin E treatment showed 0.56 fold lower expression than control animals during summer season. During winter, the animals with Melatonin treatment showed 0.70 fold higher expression compared to control animals. Similarly, animals with Vitamin E treatment showed 1.28 fold higher expressions than control animals.