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Subject: Animal Reproduction, Gynaecology and Obstetrics × Author: Das, Arunima × End date: 2023 × Start date: 2020 × Thesis Type: Ph.D ×
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    ThesisItemOpen Access
    Morphological and functional characterization of boar spermatozoa on incubation in capacitating media and preservation
    (College of Veterinary Science, Assam Agricultural University, Khanapara Campus, 2022-03) Das, Arunima; Barua, P M
    A total of 24 ejaculates comprising 6 ejaculates from each of four HD-K75 boars of 10-12 months age maintained at ICAR - All India Coordinated Research Project (AICRP) on Pig C.V.Sc, A.A.U., Khanapara, Guwahati are being selected for the present study. The semen was collected by simple fist method twice weekly to study the morphological and functional characterization of in-vitro capacitated and preserved boar spermatozoa. After initial evaluation (volume, concentration and initial motility), the fresh semen was split into three parts. One part of the semen was used for fresh semen evaluation, second for capacitation and the other for preservation. For capacitation, the semen was incubated in TALP and m-KRB media at 37oC for 5 hours. For preservation semen was extended (1:4) in BTS and GEPS extenders and held at 22ºC for 4 hours. The extended semen was then preserved at 15oC in BOD incubator upto 120 hours. The overall mean of strained volume of semen, initial motility, hyperactivated spermatozoa, sperm concentration, live spermatozoa, live acrosome reacted spermatozoa and per cent hypo-osmotic swelling test (HOST) was 220.65±5.34 ml, 83.29±0.92 per cent, 92.21±0.54 per cent, 270.87±2.94 million per ml, 90.82±0.83 per cent , 82.76±0.36 per cent and 65.06±0.27 per cent and the overall range being 150-265 ml, 78 to 95 per cent, 88 to 95 per cent, 245- 298 million per ml, 86 to 95, 79-86 and 62 to 78 per cent respectively. Sperms were suspended in TALP media and m-KRB media and incubated for 5 hours at 370C for in-vitro capacitation and evaluation was carried out at 0, 3 and 5 hours of incubation. In the present study, the highest hyperactivated motility was observed at 3 hours of incubation, from 18.51% at 0 hour to 57.32% in TALP and 17.96% at 0 hour to 43.25% at 3 hour in m-KRB, the hyperactivated motility of spermatozoa increased significantly upto 3 hours then it decreased upto 44.72 in TALP and 43.25 at 5 hours of incubation. The overall mean live acrosome reacted spermatozoa per cent declined from 85.31% to 35.52 % in TALP and 84.95% to 34.04% in m-KRB media, HOST reacted spermatozoa decreased from 68.49% to 53.37% in TALP and 67.54% to 51.41% in m- KRB, FITC-PSA(-ve) spermatozoa percentage increased from 0.17% to 13.92% in TALP and 0.17 % to 12.58% in m-KRB, total protein increased from 0.41mg/ml to 1.21 mg/ml in TALP and 0.44 mg/ml to 1.25 mg/ml in m-KRB, total cholesterol decreased from 31.58 mg/dL to 11.28 mg/dL in TALP and 32.24 mg/dL to 10.61 mg/dL in m-KRB, total phospholipid 61.90 mg/dL to 59.24 mg/dL in TALP and 62.15 mg/dL to 59.40 mg/dL in m-KRB. The overall mean values were found to be differed significantly (P<0.01) between periods, while between media no significant difference was observed except in live acrosome reacted spermatozoa (P<0.05) and HOST (P<0.01). In preserved group, Semen was extended with BTS and GEPS extender (1:4), held at 22oC for 4 hours and preserved upto 120 hours at 15oC. The semen samples were evaluated at 0 (i.e. immediately after extension), 24, 48, 72, 96 and 120 hours of preservation. In the present investigation, the overall mean sperm motility showed a decline from 82.63% to 30.21% in BTS and 83.04% to 31.75% in GEPS, hyperactivated motility percentage decreased from 84.64% to 32.70% in BTS and 83.95% to 34.24% in GEPS and live spermatozoa decreased from 84.92% to 45.08% in BTS and 85.75% to 46.92 % in GEPS, live acrosome reacted decreased from 80.79% to 44.79% in BTS and 82.29% to 44.79% in GEPS, host reacted spermatozoa decreased from 61.64% to 36.09% in BTS and 60.62% to 34.20% in GEPS, FITC-PSA(-ve) spermatozoa increased from 0 to 13.17% in BTS and 0 to 12.58% in GEPS, total protein (g/dL) level increased from 1.39% to 2.01% in BTS and 1.32% to 2.02% in GEPS, total cholesterol (mg/dL) level decreased from 32.82 to 15.54 in BTS and 32.82 to 15.21 in GEPS and total phospholipid (mg/dL) level decreased from 62.03 to 60.90 in BTS and 62.31 to 60.24 in GEPS. The overall mean values were found to be differed significantly (P<0.01) between periods, while between media significant difference (P<0.05) was observed in sperm motility and HOST while, hyperactivated motility, live spermatozoa and live acrosome reacted spermatozoa were differed significantly higher (P<0.05). The aim of the present study was to determine the nature of capacitation like changes during preservation by studying the morphological and functional characteristics of in-vitro capacitated and preserved boar spermatozoa. In the present study, the maximum in-vitro capacitation was observed at 3 hours of incubation at 37oC. While, changes of the boar spermatozoa after 72 hours of preservation in respect of acrosomal status, plasma membrane integrity, total protein, total cholesterol and FITC-PSA (-ve ) spermatozoa resembled with the changes of spermatozoa of in-vitro capacitated for 3 hours of incubation.