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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.ThesisItem Open Access UPSCALING OF BOVINE INFERTILITY COUNTERING TECHNOLOGIES(Assam Agricultural University, Khanapara, Guwahati, 2017-07) DUTTA, LAKSHYA JYOTI; Nath, K.C.A study was conducted on a total of 909 crossbred cows maintained in 60 private farms of Kamrup, Darrang and Lakhimpur districts of Assam with the primary objectives of identification and characterization of common reproductive disorders and studying fortification needs of commonly practised therapeutic techniques for the treatment of infertility. Incidence of reproductive disorder was determined based on breeding history provided by the animal owners and clinico-gynaecological examination of 133 problem breeder cows. The study revealed that overall incidence of infertility was 14.63 per cent comprising 5.28 per cent for repeat breeding with uterine infection, 3.41 per cent for repeat breeding without uterine infection, 2.97 per cent for true anoestrus, 2.53 per cent for silent oestrus and 0.11 per cent for each of pyometra, ovarian cyst, infantile genital organ and ovario-bursal adhesion. Out of the total number of infertile cows 59.39 per cent suffered from repeat breeding and 37.59 per cent anoestrus. Poor management system in respect of flooring, drainage, sunlight exposure, ventilation and roofing of the cattle shed was associated with higher incidence of infertility with the frequency of occurrence of 58.69, 28.10, 17.11, 51.28 and 21.47 per cent respectively against 12.28, 11.0, 12.60, 12.98 and 9.38 per cent for good flooring, good drainage, good sunlight exposure, good ventilation and good roofing system respectively. Feeding condition was normal in 48.33 per cent and poor in 51.66 per cent cattle farms. Incidence of infertility was 13.36 and 15.78 per cent under normal and poor feeding conditions respectively. Of the infertile cows 53.38 per cent had body condition score between 2.5 and 3.5 and 46.61 per cent had the score less than 2.5. Out of repeat breeder cows with uterine infection 43.66 per cent had good body condition and 27.41 per cent poor body condition. Level of serum calcium, zinc, leptin, ghrelin and IGF-1didnot vary significantly between types of infertility. Level of phosphorus and iron was low in cows affected with repeat breeding due to infection. Serum oestrogen level remained significantly low in cows affected with silent oestrus and true anoestrus while level of progesterone was significantly higher in cows affected with silent oestrus. Treatment of silent oestrus in crossbred cows with double injection of PGF2α 11 days apart resulted in 100.00 per cent oestrus response and 66.66 per cent conception rate. Fortification of PGF2α with supportive treatment comprising oral bypass fat and mineral mixture and injectable phosphorus and vitamins did not improve conception rate. Supportive treatment alone when used in true anoestrus cows resulted in 83.33 per cent oestrus response and 50.00 per cent conception rate. Fortification of GnRH with supportive treatment did not improve conception rate in treating true anoestrus in cows. Fortification of intrauterine Lugol’s iodine with supportive treatment used for treating repeat breeding due to uterine infection resulted in higher post treatment conception rate of 83.33 per cent against 66.66 per cent obtained with Lugol’s iodine alone. The hormone hCG was the choice of drug for treatment of repeat breeding without uterine infection resulting in post treatment conception rate of 83.33 per cent which increased to 100.00 per cent when fortified with supportive treatment.ThesisItem Open Access VITRIFICATION OF FOLLICULAR OOCYTES AND IN VITRO FERTILIZATION IN PIG(Assam Agricultural University, Khanapara, Guwahati, 2017-01) KALITA, KRISHNA; DEKA, B. C.