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ThesisItem Open Access EFFECT 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 Access INFLUENCE 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 Access EFFECT 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 Access A 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.ThesisItem Open Access EFFECT OF MEDIA ON IN-VITRO MATURATION OF BOVINE FOLLICULAR OOCYTES(Assam Agricultural University, Khanapara, Guwahati, 2016-07) DAS, ARUNIMA; BARUA, P. M.The present research was planned to study the effect of different concentrations of Vitamin E and Oestrous cow serum and their combination on in-vitro maturation of bovine follicular oocytes incubated in TCM-199 medium containing Follicular fluid, Oestradiol-17β, p-FSH, Gentamicin, Sodium Pyruvate, Cysteamine and Fetal Bovine Serum at 38.5 0C with 5% CO2 for 24 hours in a CO2 incubator. A total of 825 oocytes were recovered from slaughterhouse ovaries of cattle by aspiration cum slicing technique and graded as A, B, C and D with the recovery rates of 77.64 ± 0.02, 11.60 ± 0.01, 6.84 ± 0.01 and 3.91 ± 0.01 per cent, respectively. A total of 704 oocytes of grade A and B were used in the study. Maturation of oocytes was determined on the basis of rate of cumulus cell expansion and polar body formation. Vitamin E concentrations at 0, 100 and 150 µM levels in the maturation medium resulted in cumulus cell expansion rate of 74.46 ± 2.68, 74.28 ± 1.66 and 73.59 ± 0.78 per cent, respectively, while polar body formation rates were found to be 44.02 ± 4.37, 50.06 ± 2.40 and 45.43 ± 4.20 per cent, respectively. Oocytes maturation rates both in terms of cumulus cell expansion and polar body formation were not affected by levels of Vitamin E supplementation in the in-vitro maturation medium. Oestrous Cow Serum supplementation at 0, 20 and 30 per cent levels to the in-vitro maturation medium resulted in the cumulus cell expansion rate of 74.46 ± 2.68, 68.41 ± 2.40 and 63.90 ± 3.49 per cent, respectively; corresponding figures for polar body formation rate were 44.02 ± 4.37, 32.10 ± 2.84 and 29.50 ± 0.67 per cent. Addition of either 20 or 30 per cent Oestrous Cow Serum in the maturation medium did not improve the oocytes maturation rate both in terms of cumulus cell expansion as well as polar body formation. Cumulus cell expansion and polar body formation rates were recorded as 67.20 ± 3.45 and 38.26 ± 2.28 per cent, respectively when 100 µM Vitamin E and 20 per cent Oestrous cow serum combination was used in the in-vitro maturation medium. The corresponding rates for the control medium (without supplementation) were 74.46 ± 2.68 and 44.02 ± 4.37 per cent.ThesisItem Open Access CRYOPRESERVATION OF BOAR SEMEN WITH SPECIAL EMPHASIS ON CRYODAMAGE AND ITS MITIGATION(Assam Agricultural University, Khanapara, Guwahati, 2013-07) BAISHYA, SANTOSH KUMAR; Biswas, R. K.A total of 92 sperm-rich fraction of ejaculates were collected from two boars each of Hampshire (HS), Hampshire x Khasi Local (HS x KL) with 87.5 per cent exotic inheritance and HS x KL with 75 per cent exotic inheritance maintained at Livestock Research Farm, Division of Livestock Production, ICAR Research Complex for NEH Region, Umiam, Meghalaya by gloved/bared hand technique once or twice in a week. The seminal ejaculates thus obtained were frozen in 0.5 ml straws in liquid nitrogen using 3 per cent glycerol in BTSLEYG extender allowing 3 hours holding time and 1 hour equilibration period to study the quality of semen before and after freezing to find out the extent of cryodamage which was sought to be mitigated by resorting to different freezing methods, addition of different antioxidants, and incorporation of cholesterol-loaded Methyl-β-cyclodextrin (CLC) either alone or in combination with antioxidant in the extender. Sixty ejaculates were used to study the effect of different freezing methods. Semen was frozen either by conventional method or by controlled freezing method adopting cooling @ 3˚C/min from 5 to −6˚C, 1 minute holding at −6˚C and then freezing either @ 20˚C, 40˚C or 60˚C/min from -6˚C to -140˚C before plunging into liquid nitrogen. Equal number of ejaculates were used in different freezing methods. Sixteen ejaculates were subjected to study the effect of supplementation of Glutathione (GSH, 1 mM), Vitamin E (Trolox, 0.2 mM) and Butylated hydroxytoluene (BHT, 0.2 mM) as antioxidants in the first fraction of the extender as compared to without supplementation. Another sixteen ejaculates were used to find out effect of addition of CLC either alone or in combination with BHT in the extender. In both the experiments split sample technique of the ejaculates was followed. Quality of semen was estimated by evaluating the following sperm parameters: motile sperm by subjective method, live sperm by Eosin- Nigrosin staining, live intact acrosome by Nigrosin-Eosin-Giemsa staining, plasma membrane intact sperm by Carboxyfluorescein Diacetate and Propidium Iodide staining, sperm hypo-osmotic swelling test in 100 mOsm, live sperm with high mitochondrial membrane potential (MMP) by JC-1 plus Propidium Iodide staining, lipid peroxidised sperm by BODIPY C-11 staining, DNA-damaged sperm by Acridine Orange staining, and sperm plasma membrane protein profile by SDS-PAGE. The mean values of all the sperm parameters assessed declined significantly (P<0.05) after freezing as compared to that after equilibration irrespective of freezing method adopted. The mean per cent sperm motility after freezing was significantly (P< 0.05) higher in controlled freezing methods as compared to that in conventional method. The mean per cent post thaw live sperm, live intact acrosome, plasma membrane intact sperm, HOST-reacted sperm and live sperm with MMP were higher, although non-significantly, in controlled freezing than in conventional freezing method. Loss in number of protein bands in sperm plasma membrane after freezing was lower in controlled freezing than in conventional freezing method. Out of the three methods employed, controlled freezing @ 40˚C/min yielded relatively higher mean per cent post-thaw motile sperm, live sperm, HOST-reacted sperm and live sperm with high MMP revealing its superiority. The mean percentages of all sperm parameters decreased significantly (P < 0.05) after freezing than that after equilibration irrespective of with or without antioxidant supplementation after adopting controlled freezing @ 40˚C/min that was found to be superior. The supplementation of antioxidants resulted in significant (P < 0.05) increase in mean per cent plasma membrane intact sperm and significant (P < 0.05) decrease in mean per cent lipid peroxidised sperm after freezing as compared to no supplementation. The mean percentages of motile sperm, live sperm, live intact acrosome, HOST-reacted sperm and live sperm with MMP after freezing were higher, and per cent DNA-damaged sperm after freezing was lower non-significantly in antioxidant supplemented groups than in control group. Out of the three antioxidants used, BHT yielded a relatively higher mean post thaw percentages of motile sperm, live sperm and live intact acrosome and lower percentage of lipid peroxidised sperm that indicated its superiority. To study the effect of addition of CLC on sperm quality, each ejaculate was split into four equal parts. BHT, that was found to be superior among the antioxidants, was added in one part. CLC @ 5 mg/ 200 – 240 x 106 sperm was added at 18˚C in the second part and diluted with BTS during holding and incubated for 30 to 60 minutes. CLC @ 5 mg/ 200 – 240 x 106 sperm was added at 18˚C in the third part and diluted with BTS during holding and incubated for 30 to 60 minutes and first fraction of LEYG extender containing 0.2 mM BHT was added to it. No antioxidant and no CLC were added in the extender for the fourth part which served as control. The mean values recorded in all sperm parameters diminished significantly (P <0.05) after freezing as compared to that after equilibration with or without the additives. The mean per cent post thaw motile sperm, live sperm, plasma membrane intact sperm and HOST-reacted sperm increased significantly (P < 0.05) and the mean percentage of lipid peroxidised sperm decreased significantly (P < 0.05) as compared to that of control when semen was frozen with supplementation of BHT and CLC either alone or in combination in the freezing medium. The mean percentage of motile sperm after freezing was significantly (P < 0.05) higher in BHT plus CLC supplemented group as compared to that in BHT and CLC alone and control group; however, the difference with supplementation of BHT was narrow(2.19 %). The mean values recorded in respect of other sperm parameters after freezing did not differ significantly between BHT plus CLC, and BHT supplemented group. The overall mean percentage of DNA-damaged sperm irrespective of stage was significantly (P < 0.05) lower with supplementation of BHT alone or in combination with CLC as compared to CLC alone and control group. In view of high cost of CLC and cumbersome process involved in preparation of CLC and comparable efficacy of BHT, and BHT plus CLC, frozen semen produced with the supplementation of BHT alone adopting freezing @ 40˚C/min was used for insemination. Twenty five sows were inseminated artificially carrying out double cervical insemination by Golden AI Pig Catheter at 30 and 42 hours following onset of oestrus using 4-5x109 spermatozoa per dose. The percentage of farrowing was 44.00 and the mean litter size at birth was 5.91 ± 0.69.ThesisItem Open Access EFFICACY OF IMMUNOMODULATORS IN ADDRESSING UTERINE INFECTION IN CATTLE(Assam Agricultural University, Khanapara, Guwahati, 2013-07) BHUYAN, MANJYOTI; Nath, K.C.A study was conducted on 72 crossbred cows comprising 18 normal cyclic and 54 metritic cows with a view to comparing efficacy of intrauterine treatment of E. coli LPS and Oyster glycogen in inducing uterine immunity, controlling bacterial contamination of the uterus and obtaining conception in the metritic cows. Metritis was diagnosed on the basis of clinical signs, pH of uterine discharge and white side test. E. coli LPS and Oyster glycogen was used at the dose of 100µg and 500 mg respectively each in 20 ml of phosphate buffer saline solution (pH 7.2). The study revealed that all metritic cows showed mucopurulent discharge, thick uterine wall, alkaline pH (≥8) and positive reaction to white side test while none of the normal cows showed these diagnostic characteristics of metritis. Level of uterine defence mechanism as measured by per cent neutrophil, total leucocyte count (TLC), concentration of total protein and IgG of the uterine lavage was higher in metritic cows as compared to that in the normal. In normal and metritic cows the respective mean values were 57.18 ± 0.60 and 69.62 ± 1.71 per cent for neutrophil, 0.07 ± 0.01 and 0.22 ± 0.02×106/ml for TLC, 0.104 ± 0.00002 and 0.144 ± 0.0003 g/dl for total protein and 0.006 ± 0.0001 and 0.013 ± 0.0003 mg/ml for IgG concentration. E. coli LPS and Oyster glycogen when administered intrauterine at the dose of 100 g and 500 mg respectively stimulated uterine defence mechanism in terms of increased TLC and concentration of total protein and IgG in both normal and metritic cows. In E. coli LPS and Oyster glycogen treated normal cows the respective mean values were 3.31 ± 0.22 and 1.43 ± 0.53 × 106/ml for TLC, 0.149 ± 0.00002 and 0.135 ± 0.00002 g/dl for total protein concentration and 0.011 ± 0.0001 and 0.008 ± 0.0001 mg/ml for IgG content of the uterine lavage. In metritic cows for the two treatment groups the respective mean value were 13.97 ± 0.47 and 7.88 ± 0.34 × 106/ml for TLC, 0.250 ± 0.0001 and 0.227 ± 0.0002 g/dl for total protein and 0.025 ± 0.0001 and 0.020 ± 0.0001 mg/ml for IgG content of the uterine lavage. In both normal and metritic cows mean values recorded following E. coli LPS treatment were higher than that observed in Oyster glycogen treatment, indicating superior immunomodulatory effect of E. coli LPS over Oyster glycogen. Bacteriological study revealed that 83.33 per cent metritic and 16.67 per cent normal cow uteri were positive for bacterial isolates and the bacteria were Staphylococcus sp., E. coli, Streptococcus sp. and Brucella sp. in metritic and only Staphylococcus sp. in normal cows. Both E. coli LPS and Oyster glycogen were effective in controlling bacterial infection in metritic cows. Following intrauterine treatment with either E. coli LPS or Oyster glycogen only 16.67 per cent were found positive for bacterial isolates against 83.33 per cent in the control (non-treated) cows. Inseminations carried out in metritic cows after 24 hours of intrauterine treatment with E. coli LPS resulted in only 16.66 per cent conception rate against none following insemination with Oyster glycogen and Ciprofloxacin with Tinidazole .When inseminations were carried out in the subsequent oestrus period following treatment conception rate was 83.33, 50.00 and 50.00 per cent in cows treated with E. coli LPS, Oyster glycogen and Ciprofloxacin with Tinidazole respectively.