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Establishment of Freezing Conditions for Improving Cryosurvival in Miniature Pig Spermatozoa  

Lee, Yong-Seung (College of Animal Life Sciences, Kangwon National University)
Yoo, Han-Jun (College of Animal Life Sciences, Kangwon National University)
Cheong, Hee-Tae (School of Veterinary Medicine, Kangwon National University)
Yang, Boo-Keun (College of Animal Life Sciences, Kangwon National University)
Woo, Jea-Seok (National Institute of Animal Science, Hanwoo Ex)
Park, Choon-Keun (College of Animal Life Sciences, Kangwon National University)
Publication Information
Reproductive and Developmental Biology / v.35, no.3, 2011 , pp. 329-334 More about this Journal
Abstract
This study was conducted to establish a freezing method of miniature pig spermatozoa. The semen 더aculated from PWG M-type miniature pig was collected by gloved-hand method. The semen was diluted with same volume extender (m-Modena B). The frozen solution used frozen solution of four different (LEY, TCG, BF-5 and m-Modena+egg yolk) for find optimal frozen solution in miniature pig sperm. The diluted semen for frozen rate assay was added to LEY solution (solution I: 11% lactose+egg yolk; solution II: solution I+glycerol+OEP), and frozen depending on freezing rate by the three different freezing methods (A: until $5^{\circ}C$ for 1 hrs, holding at $-102^{\circ}C$ for 10 min; B: until $5^{\circ}C$ for 2 hrs, holding at $-102^{\circ}C$ for 10 min; C: until $5^{\circ}C$ for 3 hrs, holding at -80 and $-102^{\circ}C$ for 10 min). Semen cooled until $5^{\circ}C$ was added with glycerol 1, 3 and 5%, and take a equilibrium time for 0, 10 and 30min. Frozen-thawed sperm were evaluated for viability, acrosomal status and morphological abnormality. The results of frozen-thawed sperm ability by frozen solution, viability was higher in LEY solution compared to other three different frozen solution. AR pattern of LEY solution were lower than other three different frozen solution. The results of freezing rate, viability was higher in B method compared to other methods (p<0.05). Acrosomal statute was intacted in A and B methods than C method. The experiment for glycerol condition was showed that sperm viability was higher in extender with 1% and 3% glycerol and equilibrium time of 0 min. The acrosome damage was lower in extender with 1% glycerol and equilibrium time of 10 min than other conditions. In conclusion, the optimal conditions for cryopreservation of miniature pig spermatozoa obtained in LEY frozen solution, cooling rate of 1~2 hrs, 1~3% glycerol concentrations and glycerol equilibrium time of 0~10 min.
Keywords
Miniature pig; Freezing rate; Glycerol equilibrium; Freezing method; Spermatozoa;
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Times Cited By KSCI : 3  (Citation Analysis)
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1 Hernandez M, Roca J, Gil MA, Vazquez JM, Martinez EA (2007): Adjustments on the cryopreservation conditions reduce the incidence of boar ejaculates with poor sperm freezability. Theriogenology 67:1436- 1445.   DOI   ScienceOn
2 Holt WV (2000): Basic aspects of frozen storage of semen. Anim Reprod Sci 62:3-22.   DOI   ScienceOn
3 Huang SY, Kuo YH, Lee WC, Tsou HL, Lee YP, Chang HL, Yang JJ (1999): Substantial decrease of heatshock protein 90 precedes the decline of sperm motility during cooling of boar spermatozoa. Theriogenology 51:1007-1016.   DOI   ScienceOn
4 Johnson LA (1998): Current developments in swine semen: preservation, artificial insemination and sperm sexing. In: Proceedings of 15th IPVS Congress, pp 225-229.
5 Lee SH, Cheong HT, Yang BK, Park CK (2005): Development of semen extenders by assessment of sperm viability in miniature pig semen. Reprod Dev Biol 29(4):247-252.
6 Lin DS, Connor WE, Wolf DP, Neuringer M, Hachey DL (1993): Unique lipids of primate spermatozoa- desmosterol and docosahexaenoic acid. J Lipid Res 34:491-499.
7 Maxwell WMC, Johnson LA (1997): Membrane status of boar spermatozoa after cooling or cryopreservation. Theriogenology 48:209-219.   DOI   ScienceOn
8 Mazur P, Leibo SP, Farrant J, Chu EHY, Hanna MG Jr, Smith LH (1970): Interactions of cooling rate, warming rate and protective additive on the survuval of frozen mammalian cells. In: Wolstenholme GEW, O'Connor M (Eds), The Frozen Cell, Churchill, London, pp 69-88.
9 Mazur P (1984): Freezing of living cells: mechanisms and implications. Am J Physiol 247:C125-C142.
10 Molinia FC, Evans G, Casares PI, Maxwell WM (1994): Effectof monosaccharides and disaccharides in Tris based diluentson motility, acrosome integrity and fertility of pellet frozen ram spermatozoa. Anim Reprod Sci 36:113-122.   DOI   ScienceOn
11 Parks JE, Arion JW, Foore RH (1987): Lipids of plasma membrane and outer acrosomal membrane from bovine spermatozoa. Biol Reprod 1249-1258.
12 Plummer JM, Watson PF (1985): Ultrastructural localization of calcium ions in ram spermatozoa before and after cold shock as demonstrated by a pyroantimonate technique. J Rprod Fertil 75:255-263.   DOI
13 Polge C, Smith AU, Parkes AS (1949): Revival of spermatozoa after vitrification and dehydration at low temperature. Nature 164:666-676.   DOI
14 Wilmut I, Salamon S, Polge C (1973): Deep freezing of boar semen II. Effect of method of dilution, glycerol concentration, and time of semen-glycerol contact on survival of spermatozoa. Aust J Biol Sci 26:231.
15 Purdy PH (2006): A review on goat sperm cryopreservation. Small Rum Res 6:215-225.
16 Wang WH, Abeydeera LR, Fraser LR, Niwa K (1995): Functional analysis using chlortetracycline fluorescence and in vitro fertilization of frozen thawed ejaculated boar spermatozoa incubated in protein-free chemically defined medium. J Reprod Fertil 104: 305-313.   DOI
17 Watson PF (1990): Artificial insemination and the preservation of semen. In: Lamming G (Ed), Marshall's Physiology of Reproduction vol. 2 Churchill Livingstone, Edinburgh, London, pp 747-869.
18 김성곤, 장현용, 박동헌, 박춘근, 정희태, 김정익, 양부근 (2006): 돼지 정액의 간편 동결 방법 확립과 동결정액의 융해 후 생존성 평가. 한국동물번식학회지 30: 59-64.
19 이용승, 최원철, 이승형, 정희태, 이상영, 양부근, 박춘근 (2006): Miniature Pig와 Duroc 종간의 동결-융해후 정액 성상 비교. 한국수정란이식학회지 21:263-271.
20 Aisen E, Medina V, Venturino A (2002): Cryopreservation and post thawed fertility of ram semen frozen in different trehalose concentrations. Theriogenology 57:1801-1808.   DOI   ScienceOn
21 Berger B, Fischerleitner F (1992): On deep freezing of boar semen: investigations on the effects of different straws volumes, methods of freezing and thawing extenders. Reprod Dom Anim 27:266-270.
22 Bwanga CO (1991): Cryopreservation of boar semen: 1. A literature-review. Acra Vet Scand 32:431-453.
23 Byrne GP, Lonergan P, Wade M, Duffy P, Donavan A, Hanrahan JP (2000): Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro. Anim Reprod Sci 62:265-275.   DOI   ScienceOn
24 Eriksson BM, Rodriguez-Martinez H (2000): Effect of freezing and thawing rates on post-thaw viability of boar spermatozoa. Anim Reprod Sci 63:205-220.   DOI   ScienceOn
25 Evans G, Maxwell W (1987): Salamon's Artificial Insemination of Sheep and Goats. Butterworth, Sydney, p 194.
26 Fiser PS, Fairfull RW (1990): Combined effect of glycerol concentration and cooling velocity on motility and acrosomal integrity of boar spermatozoa frozen in 0.5 ml straw. Mol Reprod Dev 25:123-129.   DOI   ScienceOn