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20131
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Subject: Animal Reproduction and Gynecology × Author: BAISHYA, SANTOSH KUMAR × Thesis Type: Ph.D ×
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    ThesisItemOpen 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.