Study on physico-morphology, biochemical constituents and effect of different levels of egg yolk on cryopreservation of Pantja buck semen
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Date
2021-09
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G.B. Pant University of Agriculture and Technology, Pantnagar - 263145 (Uttarakhand)
Abstract
Present study was conducted to assess the cryopreservation of extended Pantja buck semen in Tris-citric acid-fructose-egg yolk-glycerol (TCF-EY) extender containing 4 different levels (5, 10, 15 and 20%) of egg-yolk and to compare the neat, post diluted, post equilibrated and post-thawed quality of semen. Artificial Vagina (AV) method was used to collect semen from four reproductively mature, 2.5 to 4 years old, healthy breeding Pantja bucks. A total of thirty-two ejaculates were collected twice a week. After semen collection from all bucks, neat semen was examined for pH, volume, concentration, mass motility, live spermatozoa count, HOST, acrosome and abnormal spermatozoa percent. Thereafter semen samples were pooled and evaluated for some biochemical parameters viz; Malondialdehyde (MDA), Glutamic Oxaloacetic Transaminase (GOT), Glutamic Pyruvate Transaminase (GPT) and Glutathione Peroxidase (GSH-Px). Pooled semen sample was divided and diluted into four groups as D1[Neat semen + tris-fructosecitric acid-egg yolk (@5%)-glycerol], D2[Neat semen + tris-fructose-citric acid-egg yolk (@10%)-glycerol], D3[Neat semen + tris-fructose-citric acid-egg yolk (@15%)-glycerol] and D4[Neat semen + tris-fructose-citric acid-egg yolk (@20%)-glycerol]. Pooled semen samples were processed and evaluated without washing after dilution, post equilibration and after thawing for progressive motility, live and dead sperm percentage, sperm abnormalities, plasma membrane integrity (HOST) and acrosome integrity. Evaluation of MDA, GOT, GPT and GSH-Px was also done at post thawing stage. Biochemical parameters were evaluated in pooled semen before dilution and at post-thawing stage only. Dilutions were made according to the SOP and equilibrated at 5°C for 3 h and then the French mini (0.25 ml) straws were filled with semen and prefrozen in liquid nitrogen (LN2) vapours followed by stocking in LN2 container for seven days. After 7 days frozen semen straws were thawed in a water bath at 37˚C for 30 seconds and post thaw evaluation was done for above mention parameters. The mean value for neat seminal parameters of Pantja buck semen were recorded as volume 0.51±0.090 ml, pH 6.83±0.004, concentration 3.94±0.228, mass motility 4.49±0.078, live spermatozoa 84.75±1.75%, head abnormality (%) 2.13±0.057 %, mid piece abnormality 0.46±0.021 %, tail abnormality 4.85±0.29%, total abnormality (%) 7.48±0.34 %, plasma membrane integrity (%) 83.53±1.696 %, Intact acrosome (%) 82.63±1.760 %, MDA 2.22±0.16 (nmol/ml), GOT 127.36±0.556 Units/l, GPT 16.99±0.348 Units/l and GSH-Px 7.94±0.56 Units/ml. Significant difference (P< 0.05) was observed for all the parameters in different stages of freezing viz; post dilution, post equilibration and post thawing and in various diluters viz; D1, D2, D3 and D4. All seminal parameters except total abnormality showed higher value in D2 (10% EY) group at post dilution, post equilibration and post thawing stages compared to other groups. Likewise, significantly lower value for MDA, GOT, GPT and GSH-Px was observed in 10% egg yolk at post thawing stage. It was therefore concluded that the Pantja buck semen can be cryopreserved successfully at 10% egg yolk level (V/V) in Tris-citric acid-fructose-egg yolk -glycerol extender.