ThesisItem Open AccessOPTIMIZATION OF CULTURE MEDIA FOR IN-VITRO BOVINE EMBRYO DEVELOPMENT: GROWTH FACTORS AND SERUM INFLUENCES(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2019-07) BAISHYA, DIPANNITA; Bora, ArundhatiThe present experiment was conducted to study the effect of certain growth factors (EGF, IGF-1and their combination) and serum influences on possible potentialization of culture media for in vitro cattle embryo development. In experiment I, 224 nos. of cattle ovaries were collected. The mean number ovarian follicles recovered per type-I ovary were 5.30 which was significantly higher (P≤0.001) than the corresponding values 3.27 of the type-II ovaries. The mean recovery of cumulus oocyte complexes per type-I ovary was 3.41 and the corresponding value was 1.67 in type-II ovaries. Two different types of maturation and culture media viz: SBMM (Serum Basic Maturation Media) containing modifiedTCM-199+ serum (10%,Fetal Bovine Serum)+ Sodium pyruvate + glutamine + gentamicin + pFSH + hMG inj+ E2 (estradiol), SFBMM(Serum Free Basic Maturation Media) containing modified TCM-199 + PVP + BSA + Sodium pyruvate+ L-glutamine+ p FSH+ gentamicin + hMG inj+ E2 (estradiol), SBCM (Serum Basic culture media): mCR2aa stock +10%FBS+ Gentamicin, SFBCM (Serum Free Basic Culture Media) containing mCR2aa stock+ BSA-V+ PVP+ Gentamicin were used for in vitro maturation and in vitro culture of the oocytes. EGF (30ng) and IGF-1 (100ng) were added in maturation media as well as embryo culture media singly and in combination in both the groups of media. Frozen bull semen straws of proven fertility were used and prepared for in vitro capacitation by density gradient method using B.O. media. The results revealed a significant (P<0.05) increase in maturation rate in serum supplemented media than serum free media (75.43± 3.25 vs 64.20 ±3.77) based on cumulus cells expansion. The cleavage percentages of serum supplemented culture media was found to be significantly higher (P<0.05) than serum free culture media (70.33±3.21 vs 55.81±4.33). In experiment -2: A total of 318 nos. of ovaries were collected with a recovery of 65 per cent culturable oocytes, representing 6.5 COCs per ovary. Growth factors EGF (30ng/ml), IGF-1 (100ng/ml) and their combination (EGF+IGF-1) were used in serum basic and serum free basic maturation and culture media for the study. There was no significant difference in respect of maturation, fertilization and embryonic development between EGF supplemented, IGF-1 supplemented and their combination (EGF+IGF-1) in serum and serum free basic culture media. However, when compared with the results of serum free basic maturation media supplemented with 30ng EGF and serum basic maturation media without EGF, the mean in vitro maturation percentage based on extrusion of polar body were found to be significantly higher (P<0.05) in supplemented media than the serum basic maturation media (70.00±14.49 vs 54.17±7.19). Similarly, when comparison was made with IGF-1 supplemented serum free basic maturation media with serum basic maturation media without IGF-1, the in vitro maturation percentage based on cumulus cells was found to be significantly higher (P<0.05) than serum basic maturation media (67.27±6.33 vs 75.43±3.25). However, in case of serum free basic maturation media supplemented with 30ng EGF+100ng IGF-1, the mean in vitro maturation percentage based on extrusion of polar body was significantly higher (P<0.05) than serum basic maturation media (80.00±10.33vs 54.17±7.19). The efficacy of EGF supplemented serum free basic culture media and serum basic culture media in respect of cleavage and early embryonic development was comparable at cleavage (2-cell) and blastocyst stage, while significantly higher (P<0.05) values were observed in 4 cell (57.14±4.83 vs 47.25±4.86), 8 cell (45.71± 4.83vs 31.87± 4.99) 16 cell (37.14±4.72 vs 20.88±3.21) and morula stage (27.62±4.36 vs 7.69±4.32) in EGF supplemented serum free culture media than serum basic culture media. Similarly, when the efficacy of IGF-1 supplemented serum free basic culture media were observed, no significant difference was obtained in 2-cell, 4-cell and blastocyst stages. On the contrary, the serum supplemented media showed significantly higher (P<0.05) 8-cell (45.71±4.86 vs 31.87±4.99), 16-cell (37.14± 4.72 vs 20.88±3.21) and morula stage (27.62±4.36 vs7.69±4.32) than serum basic culture media. EGF+IGF-1 supplemented serum free basic culture media when compared with serum basic culture media, significantly higher (P<0.05) values were found in respect to 2-cell (79.63± 2.35 vs 70.33±3.21), 4-cell (65.00±4.83 vs 47.25±3.23), 8 cell (56.00±3.42vs 31.87±4.99) 16 cell (48.00±4.72 vs 20.88±3.21) morula (37.00±2.13 vs 7.69±4.32) and blastocyst stage (10.23±2.08 vs 4.40±3.11). From the above findings, it can be concluded that addition of EGF and IGF-1 in combination in serum free basic maturation media has better stimulatory effect on nuclear maturation of oocytes in comparison to EGF and IGF- 1 supplementation individually. EGF and IGF-1 in combination in Serum Free culture media significantly increased blastocyste rates when compared with serum based culture media. ThesisItem Open AccessPHYSIOLOGICAL, BEHAVIOURAL AND MOLECULAR CHANGES IN PIGLETS IN RESPONSE TO WEANING STRESS(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2018-07) TIGGA, GLORIA; Dutta, ArupThe present experiment was conducted to study the effect of weaning stress on various physiological, biochemical, hormonal, hematological response including behavioural changes and pattern of gene expression profile of HSP27, HSP70 and HSP 90 in piglets weaned at different age. The present investigation was carried out in Hampshire x Ghungroo cross-bred piglets of instructional farm of NRC on pig, Rani. Three litters of six piglets were taken for the study and were weaned at different age of 28 days (Gr-I), 35 days (Gr-II) and at 42 days (Gr-III). Blood collection was scheduled from the day of weaning (0d) , 7th day, 15day followed by subsequent collections at 15 days interval up to six months of age. Physiological and behavioural studies were done at the piggery farm and molecular studies were done in the laboratory of NRC on pig, Rani whereas biochemical, hormonal and hematological studies were conducted in the laboratory of the department of Biochemistry, Nuclear research laboratory of the department of Physiology and in the laboratory of Teaching Veterinary Clinical Complex of C.V.Sc., Khanapara, Guwahati respectively. No significant difference was found in average growth rate and body weight at maturity among the groups. There was significant difference in rectal temperature (day 30, 45 and 60) and respiration rate (day 0 and 15) found between Gr-I and Gr-II. The mean heart rate (beats/ minute) was found towards decreasing trend in all the groups. The level of biochemical indices were found to be varied within normal range on day 15 post-weaning and the variations in the level in later part were found to be associated with hormonal level and age related. Higher level of phosphorus was found in piglets of Gr-III as compared to Gr-I and Gr-II. During the initial phase of experimental period significantly lower level of iron was found in piglets of Gr-I as compared to Gr-II (day 15 and 30) and Gr-III (day 0 and 15). Significantly higher level (P<0.001) of zinc was found in Gr-III as compared to Gr-I and Gr-II from day 75 onwards. Significantly lower level of copper was found in Gr-II as compared to Gr-I and Gr-III on day 15 and 30 which may be the cause of subnormal hemoglobin level (%) in this group during this period. The range of cortisol level was comparatively higher throughout the experimental period. Significant differences (p<0.001) were found in the level of hematological parameters viz TEC, Hb% and PCV between younger and older pigs on day 0 to 45. Significantly higher level of neutrophils and cortisol were observed in younger piglets. Initially, there was increase in neutrophil (%) and decrease in lymphocyte (%) indicating a stress response on day 15 post-weaning in all the groups, with significantly higher level in Gr-I. The mean values of DLC viz. neutrophil (%) (day 15, 30, 45, 60, 75, 90) and lymphocyte (%) (day 30, 45, 60, 75, 90) were found to be significantly higher and lower respectively in Gr-I as compared to Gr-II. Significantly higher number of feeding bouts (P<0.05) and higher level of aggression (P<0.05) on d+2 and d+7 was observed in Gr-III compared to Gr-I and Gr- II The level of mRNA expression of HSPs viz; HSP27, HSP70 and HSP90 were found to be increased significantly (P<0.001) on day 7 in piglets of Gr-I. Significantly higher level of mRNA of HSP90 was expressed in Gr-I on day 15 as compared to piglets of Gr-II and Gr-III` indicating that adaptive process were slower in younger piglets. ThesisItem Open AccessULTRASONOGRAPHIC MONITORING OF OVARIAN FOLLICULAR AND LUTEAL DYNAMICS IN COW(College of Veterinary Science, Assam Agricultural University, Khanapara, Guwahati, 2022-03) RALTE, VANLALNGILNEII; Dutta, DevojyotiThe ovarian follicular and luteal dynamics, blood flow pattern in the corpus luteum (CL) including the pattern of follicular wave and luteal development were studied using portable ultrasound device with 5-10 MHz linear array transrectal probe in female Crossbred and Lakhimi, indigenous cattle breed of Assam. In the experiment, twelve each cyclic Crossbred (Jersey X L) and Lakhimi (L) cows were subdivided into two groups viz. Group-I/Natural or Spontaneous oestrus and Group-II/Induced or Synchronized oestrus for each breed comprising of six cows in each group. They were evaluated in the subsequent cycle of natural luteolysis (Group-I) and induced luteolysis by prostaglandin F2α (Group-II) and circulating steroids were estimated on alternate days of the cycle. From scanning the ovaries for two inter-ovulatory cycle the experimental cows exhibited two and three follicular waves per cycle. Most of the cows 30(62.50 %) exhibited 3-wave cycle and 18(37.50 %) had 2-wave cycle. The diameter of 12.4 mm and 10.5 mm were the threshold diameter for ovulation according to follicle diameter in crossbred and Lakhimi cows, respectively. The inter-ovulatory interval (IOI) was found to be significantly longer (P<0.05) in Crossbred cows experiencing 3-wave (21-22 day) than 2-wave (20-21 day) per cycle. The first wave emerged on day 0 (day of ovulation) to day 1 of cycle with mean day of 0.13 ± 0.12 to 0.83 ± 0.38 in both the breed and group. The second wave onset was significantly (P < 0.05) earlier (9.00 ± 0.19, 8.50 ± 0.22) in 3-wave cycle than the 2-wave cycle (10.50 ± 0.29, 10.67 ± 0.21) in both Crossbred and Lakhimi cows. The average number of follicles (≥ 2 mm) observed on wave onset was 10-13 in Crossbred and 9-13 in Lakhimi cows. The first wave dominant follicle (DF) became deviated in between day 3 to 4 in both the breeds, while the second wave DF deviated on day 11-12 in Crossbred and day 10-11 in Lakhimi cows exhibiting 2-wave cycle. Deviation of the third DF occurred on day 17-18 in both the breeds. Maximum mean diameter of DF in the second waves recorded as 8.74 ± 0.52 and 7.70 ± 0.27 mm in Crossbred and Lakhimi cows respectively in 3-wave cycles was significantly smaller (P < 0.05) than the second wave DF diameter (12.83 ± 0.65 and 10.53 ± 0.76 mm) in 2-wave cycles in both crossbred and Lakhimi cows. The DF maximum diameter was attained significantly (P < 0.05) earlier in 3-wave cycle in both the first and second wave than in 2-wave cycle. The average growth rate of ovulatory DF in 3-wave cycle was 1.19 and 1.37 mm/day in Group-I and II respectively in Crossbred while, 0.88 and 1.18 mm/day in Group-I and II respectively in Lakhimi. The dominant follicles of the first wave (non-ovulatory) began atresia on day 8.0 ± 0.41 at the rate of 1.36 to 1.53 mm/day in Crossbred cow and on day 7.75 ± 0.25 at the rate of 0.97 mm/day in Lakhimi cows. The developing CL was detected on day 0 to day 1 in both Crossbred and Lakhimi cows. Maximum diameter (mm) of the CL for the two and three follicular wave cycle in Lakhimi (15.29 ± 0.64 mm and 15.08 ± 0.45 mm) were significantly smaller (P <0.05) than in Crossbred cows (22.98 ± 0.87 mm and 21.94 ± 0.66 mm). Onset of luteal regression of the 2-wave cycles (day 12 to 13 ) was significantly earlier (P < 0.05) than in the 3-wave cycle (day 15 to 16 ). In early luteal phase (day 0 to 5) the Doppler signal increased to 65.73 ± 5.02 mm2 in Crossbred and 57.30 ± 9.83 mm2 in Lakhimi. During the mid-luteal phase (day 7 to 13) the area gradually increased to 119.82 ± 5.54 mm2 in Crossbred and 93.82 ± 4.12 mm2 in Lakhimi cows then rapidly declined in the late luteal phase (day 15 to 18) to 10.66 ± 2.02 mm2 in Crossbred and 11.99 ± 5.38 mm2 in Lakhimi. Similar pattern was observed in 2-wave cycle. On the day of oestrus, the mean serum Oestradiol-17β level ranged from 35.75 ± 0.64 to 51.01 ± 2.13 pg/ml in Crossbred and 32.43 ± 0.74 to 35.74 ± 0.97 pg/ml in Lakhimi while the serum progesterone level ranged from 0.58 ± 0.71 to 1.27 ± 0.33 ng/ml in crossbred and 0.43 ± 0.77 to 0.47 ± 0.76 ng/ml in Lakhimi cows. ii There was a positive correlation between LBF area and progesterone level in 2-wave crossbred (r = 0.92), 2-wave Lakhimi (r= 0.82), 3-wave crossbred (r = 0.81) and 3-wave Lakhimi (r = 0.90) during the cycle. There was positive correlation between DF size and oestradiol level in 2-wave (r = 0.40) also in 3-wave crossbred and Lakhimi cows (r = 0.41). Negative correlation was observed between DF size and progesterone level in 2-wave (r = - 0.47) and in 3-wave (r = - 0.40) in crossbred and Lakhimi cows. ThesisItem Open AccessBIOMOLECULAR EXPRESSION ON MELATONIN AND VITAMIN E SUPPLEMENTATION DURING SUMMER AND WINTER IN PIG(Assam Agricultural University, Khanapara, Guwahati, 2016-01) CHAKRABORTY, ARINDAM; Baruah, AnubhaThe 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.