ThesisItem Open AccessSTUDY ON PROLONGED OESTRUS IN CROSSBRED CATTLE(College of Veterinary Science Assam Agricultural University Khanapara, Guwahati-781022, 2017-07) Das, Nilotpal; Bhuyan, ManjyotiThe study was conducted in crossbred cows maintained in Instructional Livestock Farm (Cattle), College of Veterinary Science, Khanapara, Guwahati and in the private dairy farms located around Guwahati to record the incidence of prolonged oestrus, signs of oestrus, hormonal profiles and blood biochemical constituents, and to evolve a suitable treatment of prolonged oestrus. The incidence of prolonged oestrus in crossbred cows was 14.24 per cent comprising 7.60, 5.04 and 1.60 per cent of 2, 3 and 4 days durations respectively. The different behavioural signs of oestrus exhibited by prolonged oestrous crossbred cows were mounting, bellowing, decreased appetite, restlessness and sniffing of genitalia with a percentage frequency of 60.00, 66.66, 26.66, 100.00 and 20.00 respectively on 1st day of oestrus. The percentage frequency of different behavioural signs of oestrus decreased during subsequent days. The percentage of cows exhibiting mounting, decreased appetite and restlessness signs was 3.33, 5.00 and 10.00 respectively on 4th and 0.00, 1.66 and 3.33 respectively on 5th day. Out of different physical signs of oestrus, the most conspicuous signs were swelling of vulva, pink colour of vaginal mucous membrane, free flowing vaginal mucus, thin consistency of vaginal mucus and clear vaginal mucus. These signs were observed in 68.33 to 100.00 per cent of animals on 1st day of oestrus. Except clear colour of vaginal mucus, the percentage of animal showing other physical signs decrease on subsequent days and no animal showed free flowing and thin consistency of vaginal mucus on 4th day of oestrus. The frequency of occurrence of clinico-gynaecological changes in the genital organs viz., relaxed cervix, opened cervix, good uterine tone, palpable large follicle on right ovary, palpable large follicle on left ovary and tense follicular wall was 100.00, 100.00, 66.66 66.66, 33.33 and 100.00 per cent on 1st day of oestrus. The frequency of occurrence of these signs gradually decreased in subsequent days of oestrus upto 4th day. Statistical analysis showed that level of serum estradiol, progesterone, cortisol, calcium, phosphorus, zinc and cholesterol did not differ significantly between crossbred cows of 2, 3 and 4 days prolonged oestrus groups and between days within group. The mean levels of serum calcium and phosphorus in prolonged oestrous crossbred cows with 2, 3 and 4 days durations increased significantly (P<0.01) after treatment with mineral mixture supplementation and anti stress therapy. However, level of serum zinc increased significantly (P<0.01) in prolonged oestrous cow of 2 days duration and serum cholesterol in 3 and 4 days durations. The conception rate in prolonged oestrous crossbred cows was found to be 45.00, 55.00 and 40.00 per cent for mineral mixture supplementation and anti stress therapy, Buserelin acetate injection and for control group without treatment respectively. ThesisItem Open AccessMANAGEMENT OF POSTPARTUM ANOESTRUS USING HEATSYNCH PROTOCOL WITH NUTRITIONAL SUPPLEMENTATION IN CROSSBRED COWS(College of Veterinary Science Assam Agricultural University Khanapara, Guwahati-781022, 2017-07) KALITA, MRINAL; DEKA, K. C.The present study was conducted to find out the efficacy of Heatsynch protocol with nutritional supplementation on postpartum anoestrous crossbred cows. Forty postpartum anoestrous crossbred cows were selected based on the history and rectal palpation in Kamrup district of Assam. The animals were divided into four groups (n=10) viz., A, B and C and treated with Heatsynch + mineral mixture, Heatsynch + mineral mixture + bypass fat and Heatsynch respectively and AI was done at induced oestrus. Ten postpartum anoestrous crossbred cows were kept as untreated control (group D). Blood samples were collected from cows on day 0, 7, 8 and 9 of Heatsynch protocol for estimation of serum estrogen, progesterone, cortisol, cholesterol, total protein, calcium, phosphorus and zinc profile. The use of Heatsynch + mineral mixture, Heatsynch + mineral mixture + bypass fat and Heatsynch protocol resulted in 100.00, 100.00 and 90.00 per cent oestrus induction with mean interval of 34.65 ±1.56, 32.60 ± 1.54 and 45.65 ± 1.91 hours and conception rate at induced oestrus was 50.00, 60.00 and 44.44 per cent, respectively. The mean serum progesterone concentration on day 0, 7, 8 and 9 revealed that the values on 7 day (2.75 ± 0.01, 2.69 ± 0.01, 2.81 ± 0.02 ng/ml) were significantly higher (P<0.01) than the values obtained on day 0, 8 and 9 with the lowest concentration on day 9 (0.43 ± 0.01, 0.39 ± 0.01, 0.46 ± 0.02 ng/ml) in group A, B and C, respectively. Serum oestrogen concentration was significantly higher (P<0.01) on day 9 in all the three treatment groups than the values obtained on day 0, 7 and 8 of treatment. Serum cortisol level showed non-significant (P>0.05) difference between the treatment groups, however, varied significantly from the control group. The highest cortisol level was observed on day 9 of treatment (6.81 ± 0.02, 6.78 ± 0.07 and 6.84 ±0.06 ng/ml in group A, B and C, respectively). Serum cholesterol and total protein concentrations were the highest in group B and varied significantly (P<0.01) from the other groups. Serum calcium, phosphorus and zinc concentrations revealed that the values obtained in group A and B were significantly higher (P<0.01) than that in group C and D. It could be concluded that Heatsynch with nutritional supplementation resulted in effective induction of oestrus with satisfactory conception rate in postpartum anoestrous crossbred cows. ThesisItem Open AccessBIOCHEMICAL CHARACTERIZATION OF FRESH AND FROZEN SEMEN OF SWAMP BUFFALO(College of Veterinary Science Assam Agricultural University Khanapara, Guwahati-781022, 2017-07) BARUTI, MANNA; Deka, B. C.Four Swamp buffalo bulls aged 5 to 8 years were used to study the biochemical characteristics of fresh and frozen semen and the effect of freezing on biochemical characteristics. The semen was subjected to protein profiling through SDS-PAGE in both fresh and frozen semen and their correlations with fresh and frozen semen characteristics were analyzed. A total of 60 ejaculates comprising 15 from each bull and a total of 40 ejaculates comprising 10 from each bull were used for evaluation and biochemical characterization in fresh and frozen semen respectively. For studying the effect of freezing on biochemical constituents of semen the AST and ALT activities, total protein, total cholesterol and total lipid concentration were estimated in seminal plasma of fresh semen and extracellular fluid of frozen semen. The overall mean values of the AST and ALT activities and total protein, total cholesterol and total lipid concentration were 26.05±0.74 U/L, 49.14±1.72 U/L, 3.44±0.02 mg/ml, 23.12±0.65 mg/dl and 2.20±0.07 mg/ml respectively in seminal plasma of fresh semen. The corresponding values in extracellular fluid of frozen semen were 124.95±1.66 U/L, 87.80±2.29 U/L, 1.66±0.06 mg/ml, and 82.07±1.63 mg/dl and 32.70±0.74 mg/ml. The mean value of AST and ALT activities and total cholesterol and total lipid concentration increased significantly (P˂ 0.01) and the total protein concentration was decreased significantly (P˂ 0.01) in extracellular fluid of frozen semen comparison with that in seminal plasma of fresh semen. The proteins of Triton X-100-treated sperm membrane extract of fresh and frozen semen, seminal plasma of fresh semen and extracellular fluid of frozen semen were characterized by SDS-PAGE. Seven protein bands in seminal plasma and ten protein bands in extracellular fluid were identified. A total of eight and five protein bands were identified in sperm membrane extract of fresh and frozen semen respectively. The relative densities of sperm membrane proteins of different molecular weights as determined by Integrated Density Value were correlated with semen characteristics. The per cent initially motile sperm, live sperm percentage and intact acrosome of fresh and frozen semen were significantly correlated (P˂ 0.01) with sperm membrane proteins of different molecular weight in fresh and frozen semen. The study revealed that the 42, 55 and 104 kDa proteins of sperm membrane had significant correlation with semen characteristics and could be considered as potential marker in screening of swamp buffalo bulls. ThesisItem Open AccessTECHNOLOGY VALIDATION OF SEMEN PRESERVATION, SYNCHRONIZATION OF OESTRUS AND ARTIFICIAL INSEMINATION IN PIG(Assam Agricultural University, Khanapara,Guwahati, 2017-07) BANIA, BIRAJ KUMAR; Das, K.K.A total of 36 ejaculates from six boars (three Hampshire and three Rani) aged one and half to two years and maintained at National Research Centre on Pig, ICAR, Rani, Guwahati, collected twice in a week by gloved hand method, were used to study the efficacy of three extenders viz.: GEPS, Modena and Androheps for preservation of boar semen at liquid state. The ejaculates were split into three parts and extended in GEPS (Glucose-sodium salt of EDTA-Potassium sodium tartrate-Sodium citrate dehydrate), Modena and Androheps extenders for preservation upto 168 hours at 15⁰C and evaluated per cent sperm motility, live sperm and live intact acrosome at 24 hours of interval. The mean sperm motility live sperm and live intact acrosome differed significantly (P<0.01) between extenders and between preservation periods. The interaction between extender and preservation period was found to be significant (P<0.05) only for sperm motility. A total of 90 female pigs (thirty for each extender) were inseminated with a dose of 80 ml (20 ml semen + 60 ml extender) of semen preserved for 72 to 120 hours at 15⁰C with GEPS, Modena and Androheps extenders. The percentage of conception rates were 81.8 in gilts and 84.2 in sows, 64.2 in gilts and 68.76 in sows and 84.62 in gilts and 82.36 in sows for the three extenders respectively. GEPS and Androheps were found to be superior to Modena based on sperm motility, live sperm, live intact acrosome and conception rate (on non return basis). Thirty apparently normal and healthy cyclic Hampshire Crossbred gilts and sows (aged 10 months to 2 years) maintained at villages near to NRC on Pig Rani, Guwahati were used for the synchronization of oestrus by feeding 10 mg progesterone orally daily for 7 days in empty stomach. Animals at synchronized oestrus were artificially inseminated with semen extended in GEPS extender irrespective of preservation periods. The percentage of synchronized oestrus were 57.14 in gilts and 62.50 in sows. The mean interval from treatment to onset of synchronized oestrus was 7.36 ± 0.78 days in gilts and 8.40 ± 0.37 days in sows. The difference having significant (P<0.05).The mean duration of oestrus was 78 ± 2.98 and 85.20 ± 3.32 hours in synchronized gilts and sows respectively, the difference being not significant. Most frequent behavioural signs of synchronized oestrus were nervousness, frequent grunting, seeking male, stance reflex and inappetance; the frequencies were 100.00, 75.00, 75.00, 62.50 and 62.51 per cent respectively in gilts and 100.00, 80.00, 80.00, 70.00 and 60.00 per cent respectively in sows. Swelling of vulva and pinkish vulva were the most common physical signs of synchronized oestrus exhibited and the intensity of signs was more in gilts than in sows. The conception rate in gilts and sows was 87.50 and 80.00 per cent respectively. ThesisItem Open AccessMORPHOLOGICAL AND FUNCTIONAL CHARACTERISTICS OF CAPACITATED AND LIQUID BOAR SEMEN(Assam Agricultural University, Khanapara,Guwahati, 2017-07) HAQUE, AINUL; AHMED, K.A total of 24 ejaculates, collected twice in a week by simple fist method, six from each of the four trained and healthy crossbred Hampshire boars of one to two years of age maintained at ICAR-All India Coordinated Research Project (AICRP) on pig, College of Veterinary Science, AAU, Khanapara, Guwahati-22, were selected for the present study. The ejaculates were extended with Modena extender (1:3), hold at 22°C for 4 hours and preserved up to 120 hours at 15°C.The semen samples were evaluated at 0 (i.e. immediately after extension), 24, 48, 72, 96 and 120 hours of preservation. In the present study, the overall mean sperm motility showed a decline from 85.42% to 51.04%, live intact acrosome decreased from 95.32% to 76.09%, HOST reacted spermatozoa declined from 73.53% to 46.41%, sperm membrane protein decreased from 13.68 to 6.06 mg/109 spermatozoa, extracellular protein level in extender increased from 1.54 mg/ml to 1.84 mg/ml and sperm cholesterol level declined from 31.84 to 16.22 mg/108 spermatozoa. The overall mean values were found to be differed significantly (P ˂0.001) with increase in hours of preservation in the extender but no significant difference in overall mean extracellular protein level was observed between 24 hours and 48 hours as well as 72 hours and 96 hours of preservation. Sperms were suspended in TALP medium and incubated for 5 hours at 37°C for in vitro capacitation and evaluation was carried out at 0, 1, 2, 3, 4 and 5 hours of incubation. In the present study, the highest hyperactivation motility was observed at 4 hours of incubation from 18.96% at 0 hour to 71.86% at 4 hour, the hyperactivated motility of spermatozoa increased significantly up to 4 hours then it decreased significantly to 54.79% at 5 hours of incubation. The overall mean live intact acrosome declined from 94.76% to 39.06%, HOST reacted spermatozoa decreased from 76.32% to 60.59%, sperm membrane protein decreased from 24.90 to 11.40 mg/109 spermatozoa, extracellular protein in TALP medium increased from 0.49 to 1.07 mg/ml and sperm cholesterol level declined from 31.84 to 8.57 mg/108 spermatozoa. The overall mean values were found to be differed significantly (P ˂0.001) with increase in hours of incubation in the medium but no significant difference in overall mean extracellular protein level was observed from 1 hour to 3 hour of incubation. The aim of the present study was to determine the nature of capacitation-like changes during preservation by studying the morphological and biochemical characteristics, plasma membrane integrity of in-vitro capacitated and preserved boar spermatozoa. In the present study, the changes of the boar spermatozoa at 96 hours of preservation in respect of acrosomal status, plasma membrane integrity, membrane protein and cholesterol levels resembles with the changes of spermatozoa of in vitro capacitated for 3 hours of incubation at 37°C and maximum in vitro capacitation was observed at 4 hours of incubation at 37°C. ThesisItem Open AccessEFFECT OF EXTENDER AND DIFFERENT SPERM NUMBERS PER STRAW ON QUALITY OF FROZEN SEMEN IN BEETAL AND SIROHI BUCKS(Assam Agricultural University, Khanapara, Guwahati, 2016-01) KALITA, MANOJ KUMAR; Sinha, S.A total of 80 pooled ejaculates collected from two Beetal and one Sirohi bucks maintained at Goat Research Station, Burnihat were used to study the effect of tris extender containing 20% egg-yolk, 1% soy-lecithin and 1.5% soy-lecithin, and that of 37.5, 50 and 75 x106 sperm / straw on the quality of frozen semen, and also the effect of number of spermatozoa in frozen semen straws on fertility. The freezing of semen was done in French mini straw by rapid horizontal vapour freezing technique using liquid nitrogen. The overall mean post-thaw per cent sperm motility, live sperm, intact acrosome and HOST-reacted sperm in tris extender containing 20% egg yolk, 1% soy-lecithin and 1.5 % soy-lecithin was 61.20 ± 0.45, 57.77 ± 0.54 and 60.20 ± 0.45; 72.32 ± 0.47, 65.40 ± 0.56 and 67.07 ± 0.56; 68.42 ± 0.43, 61.30 ± 0.74 and 63.80 ± 0.58; and 64.35 ± 0.63, 57.35 ± 0.5 and 60.17 ± 0.46 respectively. The post thaw values of tris extender with 20% egg yolk were significantly (P<0.01) higher than that of tris extender containing 1 % and 1.5% soy-lecithin for live sperm, intact acrosome and HOST-reacted sperm, However, the difference was not significant between 20% egg yolk and 1.5% soy-lecithin for sperm motility. The post thaw values were significantly (P<0.01) higher for 1.5% than that for 1% soy-lecithin in all the parameters studied. The overall mean post-thaw per cent sperm motility, live sperm, intact acrosome and HOST-reacted sperm for straws containing 37.5, 50 and 75 x106 sperm/ straw was 56.02 ± 0.47, 57.50 ± 0.41 and 65.57 ± 0.58; 67.42 ± 0.62, 70.55 ± 0.55 and 73.45 ± 0.57; 61.12 ± 0.69, 64.37± 0.66 and 68.25 ± 0.66; and 59.00 ± 0.62, 62.77± 0.52 and 65.57 ± 058 respectively. The post-thaw values of semen with 75 x106 sperm/ straw were significantly (P<0.01) higher than that of 50 and 37.5 x106 sperm / straw for all the sperm parameters studied. The post thaw values with 50 x106 sperm/ straw was significantly (P<0.01) higher than that with 37.5 x106 sperm /straw for sperm motility, intact acrosome and HOST-reacted sperm but not for live sperm. The fertility rate based on non-return rate was the highest at 75 x106 sperm /straw in both Beetal and Sirohi goats. It was revealed from the present study that the quality of frozen semen was superior in tris extender containing 20% egg yolk to that containing 1% or 1.5 % soy-lecithin. The post thaw quality and fertility of goat semen was significantly superior for 75 x106 sperm / straw to 50 x106 and 37.5 x106 sperm / straw. ThesisItem Open AccessINFLUENCE OF ADDITIVES ON IN-VITRO MATURATION OF BOVINE OOCYTE(Assam Agricultural University, Khanapara, Guwahati, 2016-07) BHAJONI, MADHURIMA; Bhuyan, D.A study was conducted to find an effective in-vitro culture system based on in-vitro maturation of bovine oocytes. Ovaries from slaughter house were utilized to study ovarian biometry, follicular biometry and performance of in-vitro maturation of oocyte. Significantly higher weight, length, width and thickness were recorded in ovary with CL than that without CL. The number of large, medium and small follicles was more in ovary without CL than with CL group. The mean number of medium size follicles was significantly (P<0.01) higher in ovary without CL (6.32±0.75) than with CL (3.33±0.18). The recovery rates of grade A (47.58%) and B (37.42%) oocytes were higher than that of grade C (8.82%) and D (6.12%) by aspiration method. In the present study in-vitro maturation of oocytes was done at 38.5 0C in humidified atmosphere of 5% CO2 for 24 hours and matured in-vitro in medium-I or control (TCM-199+10% FBS+L-glutamine+sodium pyruvate+Gentamicin+pFSH+ Estradiol 17-β), medium-II (control+5% ECS), medium-III (control+100µM/ml cysteamine), medium-IV (control+10ng/ml EGF) and medium-V (control+ 5% ECS+ 100µM/ml cysteamine+10ng/ml EGF). The mean diameter of oocytes with cumulus cells for grade A oocytes varied significantly (P<0.01) after in-vitro maturation (IVM) in different media. The medium having either epidermal growth factor or cysteamine as additives showed higher diameter of oocytes after IVM as compared to medium with estrous cow serum or foetal bovine serum or combination of all three additives. The mean diameter of oocyte without cumulus cells before and after IVM did not differ significantly between different media. The increase in diameter of oocytes with cumulus cell for grade A was significantly (P˂0.05) higher in medium-III than that of I, II and V and in medium-IV than that of I, II and V. There was no significant difference in increase in diameter of oocyte without cumulus cells for grade A and oocyte with and without cumulus cells for grade B between different media. The rates of maturation based on cumulus cell expansion and nuclear maturation were the highest in the medium-IV (86.92% and 62.36%) containing epidermal growth factor (EGF) followed by medium-III (82.24% and 56.82%) containing cysteamine among all the media, the difference between media III and IV being non-significant. ThesisItem Open AccessEFFECT OF ADDITIVES IN MEDIUM ON IN-VITRO MATURATION AND FERTILIZATION OF GOAT OOCYTES(Assam Agricultural University, Khanapara, Guwahati, 2016-07) BORAH, DHARITRI; Biswas, R. K.A total of 2539 oocytes were recovered from 712 goat ovaries obtained from slaughter house soon after sacrifice and the mean recovery rate of oocytes per ovary was 3.43 ± 0.06, 4.21 ± 0.08 and 3.24 ± 0.78 by aspiration, slicing and puncture techniques respectively, being significantly higher (P<0.01) in slicing as compared to other two techniques. The recovery of good quality oocytes with two or more cumulus cells layers around the oocytes was significantly higher (P<0.01) in puncture (73.92 ± 0.92%) than that in aspiration (66.27 ± 0.68%) and slicing (64.76 ± 0.92%) techniques. The effect of addition of 10ng/ml EGF + 50 ng/ml IGF-1, 10ng/ml EGF + 600µM/ml cysteine and 10ng/ml EGF + 0.2mM/ml sodium pyruvate in TCM-199 + 100µl/ml foetal bovine serum + 100µM/ml cysteamine + 1µg/ml 17β-Oestradiol + 5µg/ml pFSH + 5µg/ml oLH + 10 per cent follicular fluid and 10 per cent estrous goat serum (control medium) was studied for in-vitro maturation (IVM) of goat oocytes on incubation at 38.50C for 24 hours in a CO2 incubator maintaining 5 per cent CO2 under humidified condition. The IVM rate of oocytes on the basis of cumulus cell expansion and polar body extrusion was found to be significantly higher (P<0.01) with EGF + IGF-I (88.74± 1.85% and 61.71 ± 1.61%) than that with EGF + sodium pyruvate (82.86 ± 0.97% and 54.62 ± 1.88%), EGF + cysteine (78.58 ± 1.45% and 49.02 ± 1.52%) and control medium (75.27 ± 1.58% and 43.03 ± 1.48%). The oocytes matured in the IVM media used were fertilized in-vitro in Fert-TALP using swimmed-up sperm capacitated in sperm TALP. The incidences of in-vitro fertilization of oocytes on the basis of two polar bodies and 2-cell stage were also higher when oocytes were matured in-vitro using EGF + IGF-I (44.67 ± 8.86 and 15.39 ± 4.48%) than that with EGF + sodium pyruvate (25.51 ± 7.31 and 11.56 ± 4.72%), EGF + cysteine (22.46 ± 8.37 and 11.56 ± 4.72%) and control medium (20.48 ± 4.27 and 8.10 ± 3.84%) although the differences were not found to be significant. ThesisItem Open AccessA STUDY ON REPRODUCTIVE DISORDERS IN CROSSBRED CATTLE WITH SPECIAL REFERENCE TO REPEAT BREEDING(Assam Agricultural University, Khanapara, Guwahati, 2016-07) ACHARYA, CHIRANJEEVI; Deka, B. C.