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ThesisItem Open Access EFFECT OF PARENTERAL MICRONUTRIENTS SUPPLEMENTATION ON THE IMMUNE FUNCTIONS OF PERIPARTUM COWS AND THEIR CALVES(ICAR-NDRI, KARNAL, 2022) Yallappa Mallappa Somagond; AJAY KUMAR DANGPeriparturient dairy cows undergo great stress due to major physiological and metabolic changes as well as immunosuppression occurs during this period. This period is linked to a decrease in the plasma concentrations of various minerals and vitamins. The present study was conducted on peripartum cows to investigate the effect of repeated injections of vitamins and trace elements on the functions and population of blood immune cells, the expression profile of the first cellular line of defence (i.e., neutrophils), and the concentration of pro and anti-inflammatory cytokines in cows and their calves. Twenty-four peripartum crossbred cows were randomly grouped into four (n=6): Control, Multi-mineral (MM), Multi-vitamin (MV), and Multi-minerals and Multi-vitamin (MMMV) groups. Five ml of MM (Zinc 40 mg/ml, Manganese 10 mg/ml, Copper 15 mg/ml, Selenium 5 mg/ml) and five ml of MV (Vitamin E 5 mg/ml, Vitamin A 1000 IU/ml, B-Complex 5 mg/ml, and Vitamin D3 500 IU/ml) were injected intramuscularly (IM) to the MM and MV groups. Animals of the MMMV group were injected with both. In all the treatment groups, injections and blood sampling were carried out on the 30th, 15th, and 7th days before and after the expected date of calving and also on the day of calving. In calves, blood samples were collected at birth i.e., day 0 and on days 2, 3, 4, 7, 8, 15, 30, and 45 post-calving. Each cow's colostrum/milk was collected on the day of calving (day 0) and 2, 4, and 8 post-calving days. A lower percentage of total neutrophils, immature neutrophils, and a higher percentage of lymphocytes along with a significant (P<0.05) neutrophil phagocytic activity and lymphocyte proliferation were noticed in the micronutrient-injected groups (Cows and their calves). Lower expression of TLRs and CXCRs and higher expression of GR-α, CD62L, CD11b, CD25, and CD44 was seen in micronutrient (MMMV) injected groups. In both cows and calves, pro-inflammatory cytokines significantly (P<0.05) decreased and antiinflammatory cytokines increased in MMMV injected groups. Total antioxidant capacity was higher, activity of antioxidant enzymes (SOD and CAT), TBARS levels were lower in the blood plasma of treated cows/calves. In addition, a lower incidence of diseases was observed in micronutrient (MMMV) injected cows and their calves. Our results indicate that repeated injections of trace elements and vitamins to peripartum dairy cows could be a major strategy to improve the blood immune cell functions, decrease oxidative stress and in regulating the inflammatory response in periparturient dairy cows and their calves.ThesisItem Open Access EFFECT OF CHLOROPHYTUM BORIVILIANUM SUPPLEMENTATION ON BIOCHEMICAL PROFILE, HORMONES AND PRODUCTIVE PERFORMANCE OF CATTLE(ICAR-NDRI, KARNAL, 2020) DEVI, POOJA; SINGH, MAHENDRAThe present investigation was carried out on Chlorophytum borivilianum (CB) supplementation effect as heat stress ameliorator and immunomodulator in Tharparkar (TP) and Karan Fries (KF) cows. CB was supplemented at low (40 mg/kg bwt/day) and high dose (80 mg/kg bwt/day) during hot-humid (HH) and hot-dry (HD) season. The parameters like physiological responses, milk yield, DMI and body weight were recorded and milk composition, gene expression, hormones and energy metabolites were estimated. Second experiment was conducted during thermoneutral condition to isolate the effect of reduced DMI during HH season or high ambient temperature effect. The data on milk production performance, dry matter intake and physiological responses were recorded. THI was calculated to assess the level of heat stress on animals. Body weight of KF and TP cows during HD and HH season did not varied. CB supplementation in higher dose (80 mg/kg bwt) increased DMI (per 100 kg bwt) (p<0.01) in KF and TP cows during both the seasons, however, milk yield did not declined in HD season. The supplementation of CB in high dose increased the milk yield, milk fat, protein, plasminogen and decreased SCC in comparison to low dose. Further, ratio of SFAs:UFAs was significantly reduced (p<0.01) due to CB in both the seasons. However, higher SFAs and lower UFAs (p<0.01) were found in milk of KF cows in comparison to TP. Physiological responses viz. RR, PR, RT and skin temperatures (Forehead, neck, rear body and udder) were significantly lower (p<0.01) in TP cows in comparison to KF cows and decreased by CB treatment in both the seasons. Thermal imaging analysis of skin temperature at various sites was higher (p<0.01) in the afternoon in comparison to morning in KF and TP cows. Plasma Glucose was lower and NEFA, Urea and creatinine level was higher during both the seasons and declined at high dose than low dose. Plasma cortisol and prolactin level were higher (p<0.01) in HH than HD season in KF and TP cows. CB at higher dose reduced (p<0.01) plasma cortisol in both the breeds without affecting insulin and aldosterone levels. Plasma SOD, catalase, TBARS were higher in both the seasons in KF and TP cows and CB in high dose reduced (p<0.01) their levels and improved TAC. The CB supplementation downregulated (p<0.01) mRNA expression of pro-inflammatory cytokines (TNF-α and IL1-β) and anti-inflammatory cytokine (IL10) in both breeds during HD and HH season. However, effect of higher dose was more (p<0.01) than lower dose. Pairfed experiment revealed no effect of adjusted DMI on milk yield in thermoneutral conditions in comparison to HH season in which milk yield and DMI declined (p<0.01) indicating adverse effect of high temperature on milk yield. It can be concluded that higher dose (80mg/kg bwt) significantly reduced plasma energy metabolites, circulatory levels of stress hormones, and augmented the immunity of crossbred and Indigenous cows by downregulating the proinflammatory and antiinflammatory cytokines in comparison to lower dose (40mg/kg bwt) without any adverse effect. Chlorophytum borivilianum @ 80mg/kg bwt could be used as effective heat stress ameliorator and immunomodulator in dairy animals.ThesisItem Open Access ROLE OF AQUAPORINS IN THERMOREGULATION OFBUFFALOES DURING DIFFERENT SEASONS(ICAR-NDRI, KARNAL, 2019) DEBBARMA, SUSHANTA; ASHUTOSHThe study was conducted on buffaloes (n=12) to study the role of aquaporins in thermoregulation of buffaloes during different seasons. The relative gene expression of aquaporins in skin and upper respiratory tract were studied. For study of gene expression in upper respiratory tract, tissue samples were collected from slaughter house. In skin, AQP1, AQP3 and AQP5 expression were studied during winter, spring and summer seasons. The relative mRNA expression of AQP1, AQP3, AQP4 and AQP5 in nasal turbinate and upper tracheal mucosa during winter, spring and summer seasons were studied. The results of the study confirmed the presence of aquaporins in the studied tissue by gene expression studies and immunolocalization in the skin, nasal turbinate and tracheal mucosa. Further physiological responses, hematological parameters, electrolyte, biochemical and hormonal concentrations of the animals were evaluated to ascertain their role in the thermoregulation of buffaloes. The physiological responses viz. respiratory rate, heart rate, rectal temperature and skin temperature of buffaloes increased with increase in ambient temperature. There was a significant increase in evaporation rate from skin and pulmonary surface of buffaloes with the concomitant increase in ambient temperature during summer. The hematological components varied seasonally with PCV being lowest during summer, Hb and TEC count being highest during spring, and TLC being highest during winter. The high concentration of plasma anti diuretic hormone accompanied by low packed cell volume during summer is indicative of its role to conserve body water content in buffaloes. The higher relative expression of AQP1, AQP3 and AQP5 during summer season in skin of buffaloes along with increasing sweating rate and evaporative loss through skin is indicative of their role in thermoregulation of buffaloes. The up regulation of AQP1 in nasal turbinate during summer and the concomitant rise in pulmonary evaporation rate is suggestive of its role in water diffusion through nasal mucosa during hot ambience. The down regulation of AQP1, AQP3, AQP4 and AQP5 in tracheal mucosa during summer indicates their minor role during evaporative water loss from upper respiratory tract during summer. Different aquaporins are regulated at different levels in skin, nasal turbinate and tracheal mucosa of buffaloes during different seasons.ThesisItem Open Access DEVELOPMENT OF ELISA FOR ESTIMATION OF BOVINE ANTI-MULLERIAN HORMONE USING EPITOPE SPECIFIC ANTIBODY(ICAR-NDRI, KARNAL, 2022) PRASANNA PAL; ANJALI AGGARWALAnti-Mullerian Hormone (AMH) is a dimeric glycoprotein and a member of the transforming growth factor β (TGF-β) family of growth and differentiation factors. AMH secretion is not dependent on other hormones and it can be used potentially to predict fertility in cattle. So, assessment of AMH levels in animals can be beneficial for the selection of animals. Unfortunately, generating polyclonal and monoclonal antibodies used in immunological assays have several disadvantages. The problem can be solved through selection of epitopes from the AMH protein and generating antibody against those. Therefore, we aimed to generate antibodies against selected epitope specific peptide sequence of bovine AMH; to develop and validate a suitable bovine AMH assay using raised antibodies and to estimate the basal level of AMH in indigenous cows in different age groups. For this purpose, we predicted three linear B cell epitope and synthesized three peptides. The peptides were conjugated to Keyhole limpet hemocyanin (KLH) and emulsified with FCA (Freund’s Complete Adjuvant) or FIA (Freund’s Incomplete Adjuvant). Nine female rabbits of three months of age were selected and 250 μg of peptides were administered in the rabbits (n=3) through subcutaneous route with a volume of 0.5 ml (day 0, 14, 28, 42). After final dose, we collected sera, determined antibody titer, purified it and conjugated with HRP. A good titer was observed for AMH 1 and AMH 3 peptides but not in AMH 2. The purified Ig G antibody concentration in serum was found to be 8.005 mg/ml and 10.7725 mg/ml for peptide 1 and 3 group respectively. The antibodies were used for development of direct, competitive and sandwich ELISA. Among three ELISA, sandwich was found to be the best and selected for further study. The assay was highly specific as it did not bind with AMH-related members and with non-related members. The sensitivity of direct and competitive ELISA was 50 pg/ml and for the sandwich ELISA was 5.0 pg/ml. The intra-assay CV was <6% and the inter-assay CV was <9%. The average recovery percentages were found to be within 88-100%. LLOQ was found to be 5 pg/ml and ULOQ at 50 μg/ml (CV< 20%). Blood samples (5 ml) were collected from Sahiwal and Tharparkar cattle of 0-10 years of age for standardizing the level of AMH. The concentration of AMH changes with progression of age in both the breeds (p<0.001) but no difference was observed between the breeds (p>0.05). The minimum AMH level was found in 0 to six months of age for both the breeds. Afterwards it increased significantly (p<0.05) and reaches a peak value between 1.5-2.5 years. After attaining the peak value, the level of AMH remained nearly same up to about eight years of age (p>0.05) with slight fluctuations. Thereafter it decreased progressively and reached lowest value at ten years of age. In conclusion, we developed a new highly sensitive ELISA against bAMH using epitope specific antibodies and performed age specific characterization of bAMH in Sahiwal and Tharparkar breeds for the first time.ThesisItem Open Access EFFECT OF MONENSIN SUPPLEMENTATION ON ATTAINMENT OF SEXUAL MATURITY AND SEMEN QUALITY IN BUCKS(ICAR-NDRI, KARNAL, 2021) RAVI KUMAR SONI; ROY, A.K.The present study was conducted to study the effect of monensin supplementation on the attainment of sexual maturity and semen quality in bucks. Eighteen weaned kids aged 3 months were divided into three groups. The control animals were fed according to the requirement of ICAR (2013) standards, with 40:60 ratios of concentrate and green roughage. The treatment group was fed similar to control with addition of 10 mg/head/d and 20 mg/head/d of monensin in T1 and T2 group respectively. Kids were selected according to similar body weight and one month period of adaptation allowed before starting the experiment. The supplementation experiment was started at the age of four months for sixty days. Blood samples were collected to evaluate the hormonal profile and metabolic profile of the animals during supplementation at every fortnight. Body conformation attributes and scrotal biometry were performed at fortnightly intervals. Age of puberty was accessed on the basis of semen parameters. Semen samples were collected for two months after 15 days of onset of puberty. At the end of sixty days supplementation the four animals from each group were selected to find out the responsiveness of control and treated animal to GnRH. The testosterone and LH were estimated at -2, -1, 0, 1, 2, 3, 4, 6, 8, and 24 hours after GnRH shot (1μg/kg of BW). Selection of most appropriate dose (20 mg/head/d) of monensin was done on the basis of supplementation results and used to see the effect of monensin on semen quality and antioxidants status in seminal plasma. The final body weights of animals (kg) did not differ (P>0.05) significantly; i.e. 19.32±0.54, 20.44±0.78 and 21.42±0.36 kg in control, treatment I and treatment II respectively. The ADG was higher (P<0.01) in treatment groups (119.3±3.9 and 127.3±6.09 respectively in T1 and T2) as compare to control (91.00±2.33). There was no significant effect on DMI in treatment groups. Mean value of heart girth differed (P<0.05) in T2 (64±1.03) as compare to control 60.67±0.42 and T1 61.33±0.80 groups respectively. Body conformation attributes viz. body length, height at hip and height at wither did not differ (P>0.05). Scrotal circumference was higher (P<0.05) in T2 (15.03±0.44) as compare to control (14.02±0.35) and T1 (14.36±0.32) whereas testis length and width did not differ (P>0.05). The puberty was achieved earlier (P<0.05) in T1 (226.7±2.48) and T2 (219.7±2.27) group as compared to control (241.7±2.48) groups. Semen attributes like sperm concentration, motility, volume, percentage of live, mass motility and abnormality did not differ significantly (P>0.05). There was no significant difference in the basal and peak LH as well as testosterone concentration in different groups and group time interaction before and after GnRH injection. The concentration of LH and testosterone was different (P<0.0001) with respect to time in all three groups. The selected dose of monensin (20 mg/head/d) had no significant effect on semen quality parameters and antioxidant status of seminal plasma. However, the supplementation of monensin significantly improved the growth and early onset of puberty/sexual maturity without affecting the semen quality.ThesisItem Open Access EFFECT OF THERMAL AND NUTRITIONAL STRESSES ON GROWTH AND METABOLIC GENE EXPRESSION IN SAHIWAL AND KARAN FRIES CALVES DURING SUMMER(ICAR-NDRI, KARNAL, 2021) SHASHANK C. G. GOWDA; PARVEEN, KUMARThe present study evaluated the impact of Thermal, Nutrition stresses on growth and metabolic gene expression in calves. The study was conducted from May to September on twenty Sahiwal (SW) and Karan Fries (KF) calves. Calves were divided into Control (C) (n=5), Heat stress (HS) (n=5), Nutrition Stress (NS) (n=5), and Combined stress (CS) (n=5) in both breeds. The experiment was divided into two periods; Period 1: Exposing calves to Heat stress, Nutrition stress, and Combined Stress for 93 days, followed by Period 2: ad libitum access to feed for 60 days. During Period 1, KF HS group had significantly (P<0.05) higher RR and PR during both morning and afternoon. During afternoon, SW HS group had significantly (P<0.05) higher RR and PR. KF and SW, HS and CS had significantly (P<0.05) higher RT during the afternoon. In KF, the HS group had significantly higher (P<0.05) Body Weight. Whereas in SW, HS and C groups had greater Body Weight (P<0.05). DMI in C and HS groups were significantly (P<0.05) higher than NS and CS groups in both breeds. Plasma IGF-1, T3 and T4 in C and Cortisol in CS were significantly higher (P<0.05) in both breeds. On mRNA sequencing ACSM4, AK5 genes were significantly (FDR<0.05) down regulated in the KF CS group whereas LIPG, APOA1, and CPS1 genes were significantly (FDR<0.05) up regulated in SW NS and CS. During Period 2, C and HS groups significantly increased body weight (P<0.05) in both the breeds. IGF-1 in HS, T3 and T4 in C and Cortisol in CS groups were significantly (P<0.05) higher in KF whereas, in SW IGF-1, T3 and T4 were significantly (P<0.05) higher in the C group and Cortisol in CS group. Findings suggest that when both stressors were combined, it had serious consequences on the growth performance in both breeds. On a comparative basis, the SW breed performed better than the KF breed for the target variables which could be attributed to the extreme climate resilience potential of the SW breed.ThesisItem Open Access EFFECT OF MICRONUTRIENTS SUPPLEMENTATION ON IMMUNOMODULATORY COMPONENTS OF COLOSTRUM IN PERIPARTURIENT CROSSBRED COWS(ICAR-NDRI, KARNAL, 2021) YOGESH PANDEY; DANG, A.K.The present research work was undertaken to investigate the effect of micronutrients supplementation on immunomodulatory functions of Karan Fries cows during the periparturient period. A total of 35 periparturient cows were selected and divided five groups, T1, T2, T3 and T0, acted as control. Each groups was consist of seven animals. Group T0 (Control) was not supplemented with any micronutrient whereas, group T1, T2, T3 and T4 were supplemented with, vitamin A (100000 IU/animal/day), Zinc (60 ppm/animal/day), Vitamin E (2500 IU/animal/day), and their combination respectively. Colostrum and calves blood samples were collected from each animal and evaluated for the DLC, viability, immunoglobulins and growth factor. Among the treatment groups, macrophage was significantly (P˂0.05) higher in T4 group colostrum, however there was no significant difference between T3 and T2 treatment group, similarly there was no significant difference between T0 and T1 supplemented group. Maximum percentage of lymphocyte were recorded in the colostrum of T4 group cows, followed by Group T3, T2, T1, and T0 group respectively. Highest mean percentage of neutrophils in colostrum were recorded in the control group of cows, followed by T1, T2, T3, and finally T4 group. Somatic cell (SC) viability was recorded significantly higher in the T4 group of cows, followed by T3, T2, T1, and finally control group. Maximum mean value (mg/ml) of immunoglobulin IgG and IgA were observed on Day 0 in the colostrum of T4 group of cows, followed by T3, T2, T1, and finally in the T0 group. Highest mean IgG was reported in T4 group of calf followed by T2, T3, T1 finally in the control group. Highest mean value of IGF1 concentrations were observed in the colostrum of T4 group of cows, compared to cows of other groups. Whereas, IGF1 concentration did not differ between the treated and control group of calves. In the study carried out on colostrum exosomes revealed a total of, 299 and 246 differentially proteins (P˂0.05) in control and treatment group respectively. Exosomes were found to be rich in protein related to immune defense, signaling, cell development and proliferation, differentiation, repair, and growth. Dietary supplementation of micronutrient decreased significantly (P<0.05) summed concentration of SFAs and increased the significantly (P<0.05) USFAs compared with the control group. Micronutrient supplementation showed highest depression in saturated fatty acid in T4 group and simultaneously USFA, increased significantly (P<0.05) particularly MUFA. Apart from this we were also synthesis macrophage activation factor (MAF) from colostrum. MAF was used to evaluate their effect on in vitro phagocytic activity of mice peritoneum and cow milk macrophages, which was significantly (P<0.05) increased in the treatment group as compared to the control group. Supplementation of micronutrient to periparturient dairy cows ameliorate their immune-physiology, which improves the quality of colostrum and thus calf wellbeing. Therefore, micronutrients should be fed to the pregnant cows at least 30 days before calving.ThesisItem Open Access EXPRESSION OF AQUAPORINS IN MAMMARY GLAND AND SKIN OF MURRAH BUFFALOES DURING DIFFERENT SEASONS(ICAR-NDRI, KARNAL, 2019) AJITHAKUMAR H M; SINGH, MAHENDRAPresent study experiment was conducted to determine the relative gene expression of aquaporins in different stages of buffalo mammary gland and skin tissue with heat stress. Mammary gland samples were obtained from New Delhi slaughter house during summer season. The results of the present study confirmed presence of AQP1, AQP3 and AQP5 in the mammary gland by immunolocalization and RT-PCR and AQP3 in BuMEC under in vitro studies. The relative expression of AQP3 in BuMEC was significantly higher (P<0.01) due to hyperosmotic stress 7% lactose over the 5% lactose, heat (42 oC) and NaCl treatment. Experiment 2 on restricted (twice a day) and ad lib (5 times a day) water intake on AQP gene expression (AQP3 and AQP5) in Murrah buffaloes during summer season revealed water Intake, plasma Na, ADH and ANG II was significantly (p<0.01) higher in ad lib feeding group as compared to restricted water intake. Experiment 3 on lactating Murrah buffaloes during hot dry and hot humid season indicated increase in DMI, body weight, milk production and fat content (p<0.01) by using with mist and fan cooling in hot dry season and fan cooling in hot humid season due to treatment by decreasing values of physiological responses. Plasma sodium concentration was significantly (p<0.01) higher cooling group as compared to control group buffaloes. Mist and fan downregulated (p<0.01) AQP3 and AQP5 gene expressions during hot dry season, however expression was upregulated in fan provided group in hot humid season. Plasma ADH, ANG II was lower (p<0.01) and ALD was higher in mist and fan group. It was concluded that higher expression of AQPs ( AQP1, AQP3 and AQP5) in lactating mammary gland tissue regulate milk secretion further hyperosmotic stress of lactose was more potent to induce AQP3 expression in BuMEC. Increasing THI upregulates the relative mRNA expression of AQP3 and AQP5. The relative lower expression of AQP3 and AQP5 mRNA in the mist and fan treatment group as compared to control during hot dry season indicated stress on animals and there by high expression occurred to cope up with the heat stress. The lower expression of AQP3 and AQP5 indicated lower evaporative loss at high humidity in control as compared to fan treatment group.ThesisItem Open Access IDENTIFICATION AND CONFIRMATION OF EARLY PREGNANCY ASSOCIATED URINARY METABOLITES IN MURRAH BUFFALO(ICAR-NDRI, KARNAL, 2019) SARANGI, ARCHANA; BALHARA, A. K.Evaluation of urinary metabolite dynamics pertaining to day 0 and day 42 post-insemination in healthy Murrah buffaloes (Bubalus bubalis). Metabolite analysis was performed on urine samples of pregnant, non-pregnant, nonpregnant inseminated heifers derived from three groups of artificially inseminated, thirty (n=30) healthy cyclic Murrah buffalo heifers. Urinary metabolites were analyzed in these samples corresponding to Day 0, 10, 18, 35 and day 42 of pregnancy by nuclear magnetic resonance (NMR) and the results were further validated by gas chromatography-mass spectrometry (GC-MS) analysis. Current experiments revealed and confirmed 32 metabolites in urine of both pregnant, non-pregnant buffaloes urine with global changes during early pregnancy by multivariate statistical analysis such as principal components analysis (PCA) model and heatmap. Significant increase in urinary concentration of some 2- amino-3-(1H-indol-2-yl)propanoic acid derived metabolite was observed on day 42 as compared to day 0 of pregnancy. Similarly, ethylene glycol, melatonin and fumarate appeared on specific days of early pregnancy. Contrary to this, decrease in concentration of 1-methyl-(2S)-2-amino-3-(1H-imidazol-5-yl)propanoic acid and 3-indoxysulfate were observed with progression of pregnancy from day 0 to 42. Multivariate analysis of the analysed metabolites generated distinct differential pattern with progression of pregnancy at early stage. Pathway analysis of the corresponding metabolites suggested high impact correlation with establishment and succession of pregnancy in buffaloes. Urinary metabolites measured during early pregnancy in buffaloheifers depicted close association of the metabolites with physiological processes involved in successful conception and progression of pregnancy. This study opens new avenues for use of urinary metabolites in pregnancy diagnosis in bovine especially buffaloes. Key words: Murrah buffalo; Urinary metabolites; Early pregnancy; NMR; GC-MS
