Reproductive and Developmental Biology

The Korean Society of Animal Reproduction (한국동물번식학회)
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Effect of Tris, Sodium Bicarbonate and Caffeine in Fertilization Medium on In Vitro Fertilizability of Boar Spermatozoa Frozen in Straws

  • Lee, Eun-Song (School of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University)
  • Published : 2008.12.31
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Abstract

The objective of this study was to examine the effect of caffeine and sodium bicarbonate in a fertilization medium on the fertilizability of boar spermatozoa that were frozen in straws. Boar spermatozoa were extended with Beltsville F5 extender and frozen in 0.25-ml straws. In vitro matured porcine oocytes were fertilized in vitro (IVF) with frozen-thawed boar spermatozoa for 6h in a modified tris-buffered medium (mTBM) or in its modified medium by substituting the tris with 25mM sodium bicarbonate (modified bicarbonate-buffered medium; mBBM). Some of inseminated oocytes were fixed and stained for examination of sperm penetration. IVF embryos were cultured in a North Carolina State University-23 medium for embryo development. The percentage of live sperm was $47{\pm}4%$ and morphological abnormality of acrosome was found in $14{\pm}3%$ of spermatozoa. Optimal sperm concentration for IVF was $0.75{\sim}1.0{\times}1.0{\times}10^6$ sperms/ml when mTBM containing 5mM caffeine was used as the fertilization medium. Sperm penetration was significantly (p<0.05) stimulated by increasing caffeine concentration in the IVF medium. In addition, mBBM significantly (p<0.05) increased sperm penetration (92%) compared to mTBM (65%). More (p<0.05) blastocysts (22% vs. 32%) developed from the oocytes that were fertilized in mBBM containing 1mM caffeine than from those fertilized in mTBM with 5mM caffeine. Our results indicate that boar spermatozoa can be frozen successfully in straws with holding their normal fertilizability and that caffeine and sodium bicarbonate stimulates sperm penetration in vitro.

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References

  1. Abeydeera LR, Day BN (1997): In vitro penetration of pig oocytes in a modified Tris-buffered medium: Effect of BSA, caffeine and calcium. Theriogenology 48:537-544 https://doi.org/10.1016/S0093-691X(97)00270-7
  2. Bavister BD, Leibfried ML, Lieberman G (1983): Development of preimplantation embryos of the golden hamster in a defined culture medium. Biol Reprod 28:235-247 https://doi.org/10.1095/biolreprod28.1.235
  3. Bhattacharyya A, Yanagimachi R (1988): Synthetic organic pH buffers can support fertilization of guinea pig eggs, but not as efficiently as bicarbonate buffer. Gamete Res 19:123-129 https://doi.org/10.1002/mrd.1120190203
  4. Coy P, Grullon L, Canovas S, Romar R, Matas C, Aviles M (2008): Hardening of the zona pellucida of unfertilized eggs can reduce polyspermic fertilization in the pig and cow. Reproduction 135:19-27 https://doi.org/10.1530/REP-07-0280
  5. Coy P, Romar R (2002): In vitro production of pig embryos: A point of view. Reprod Fertil Dev 14:275-286 https://doi.org/10.1071/RD01102
  6. Farrel PS, Bavister BD (1984): Short-term exposure of two-cell hamster embryos to collection media is detrimental to viability. Biol Reprod 31:109-114 https://doi.org/10.1095/biolreprod31.1.109
  7. Hao Y, Mathialagan N, Walters E, Mao J, Lai L, Becker D, Li W, Critser J, Prather RS (2006): Osteopontin reduces polyspermy during in vitro fertilization of porcine oocytes. Biol Reprod 75:726-733 https://doi.org/10.1095/biolreprod.106.052589
  8. Hao Y, Murphy CN, Spate L, Wax D, Zhong Z, Samuel M, Mathialagan N, Schatten H, Prather RS (2008): Osteopontin improves in vitro development of porcine embryos and decreases apoptosis. Mol Reprod Dev 75:291-298 https://doi.org/10.1002/mrd.20794
  9. Hong JY, Yong HY, Lee BC, Hwang WS, Lim JM, Lee ES (2004): Effects of amino acids on maturation, fertilization and embryo development of pig follicular oocytes in two IVM media. Theriogenology 62:1473-1482 https://doi.org/10.1016/j.theriogenology.2004.02.013
  10. Hunter RHF (1991): Oviduct function in pigs, with particular reference to the pathological condition of polyspermy. Mol Reprod Dev 29:385-391 https://doi.org/10.1002/mrd.1080290411
  11. Kim JS, Cho YS, Song BS, Wee G, Park JS, Choo YK, Yu K, Lee KK, Han YM, Koo DB (2008): Exogenous dibutyryl cAMP affects meiotic maturation via protein kinase A activation: It stimulates further embryonic development including blastocyst quality in pigs. Theriogenology 69:290-301 https://doi.org/10.1016/j.theriogenology.2007.09.024
  12. Koo DB, Kim YJ, Yu I, Kim HN, Lee KK, Han YM (2005): Effects of in vitro fertilization conditions on preimplantation development and quality of pig embryos. Anim Reprod Sci 90:101-110 https://doi.org/10.1016/j.anireprosci.2005.01.005
  13. Palasz AT, Beltran Brena P, De la Fuente J, Gutierrez-Adan A (2008): The effect of different zwitterionic buffers and PBS used for out-of-incubator procedures during standard in vitro embryo production on development, morphology and gene expression of bovine embryos. Theriogenology 70:1461-1470 https://doi.org/10.1016/j.theriogenology.2008.06.092
  14. Park Y, Hong J, Yong H, Lim J, Lee E (2005): Effect of exogenous carbohydrates in a serum-free culture medium on the development of in vitro matured and fertilized porcine embryos. Zygote 13: 269-275 https://doi.org/10.1017/S0967199405003369
  15. Pelaez J, Breininger E, Alegre B, Pena1 FJ, Dominguez JC (2006): In vitro evaluation of the quality and fertilizing capacity of boar semen frozen in 0.25 ml straws. Reprod Dom Anim 41:153-161 https://doi.org/10.1111/j.1439-0531.2006.00659.x
  16. Petters RM, Wells KD (1993): Culture of pig embryos. J Reprod Fertil Suppl 48:61-73
  17. Piasecka-Serafin M (1972): The effect of the sediment accumulated in containers under experimental conditions on the infection of semen stored directly in liquid nitrogen (-$196^{\circ}C$). Bull Acad Pol Sci Biol 20:263-267
  18. Pursel VG, Johnson LA (1975): Freezing of boar spermatozoa: Fertilizing capacity with concentrated semen and new thawing procedure. J Anim Sci 40: 99-102 https://doi.org/10.2527/jas1975.40199x
  19. Richter L, Romeny E, Weitze KF, Zimmermann F (1975): Deep freezing of boar semen. VII. Communication: Laboratory and Field experiments using extender Hülsenberg VIII. Dt Tierarztl Wschr 82:155-162
  20. Suzuki K, Ebihara M, Nagai T, Clarke NG, Harrison RA (1994): Importance of bicarbonate/$CO_2$ for fertilization of pig oocytes in vitro, and synergism with caffeine. Reprod Fertil Dev 6:221-227 https://doi.org/10.1071/RD9940221
  21. Suzuki C, Yoshioka K, Itoh S, Kawarasaki T, Kikuchi K (2005): In vitro fertilization and subsequent development of porcine oocytes using cryopreserved and liquid-stored spermatozoa from various boars. Theriogenology 64:1287-1296 https://doi.org/10.1016/j.theriogenology.2005.03.009
  22. Wang WH, Abeydeera LR, Okuda K, Niwa K (1994): Penetration of porcine oocytes during maturation in vitro by cryopreserved ejaculated spermatozoa. Biol Reprod 50:510-515 https://doi.org/10.1095/biolreprod50.3.510
  23. Wang WH, Abeydeera LR, Prather RS, Day BN (1998): Morphologic comparison of ovulated and in vitro-matured porcine oocytes, with particular reference to polyspermy after in vitro fertilization. Mol Reprod Dev 49:308-316 https://doi.org/10.1002/(SICI)1098-2795(199803)49:3<308::AID-MRD11>3.0.CO;2-S
  24. Wongsrikeao P, Kaneshige Y, Ooki R, Taniguchi M, Agung B, Nii M, Otoi T (2005): Effect of the removal of cumulus cells on the nuclear maturation, fertilization and development of porcine oocytes. Reprod Dom Anim 40:166-170 https://doi.org/10.1111/j.1439-0531.2005.00576.x
  25. Yi YJ, Manandhar G, Sutovsky M, Li R, Jonakova V, Oko R, Park CS, Prather RS, Sutovsky P (2007): Ubiquitin C-terminal hydrolase-activity is involved in sperm acrosomal function and anti-polyspermy defense during porcine fertilization. Biol Reprod 77: 780-793 https://doi.org/10.1095/biolreprod.107.061275
  26. Yoon KW, Shin TY, Park JI, Roh S, Lim JM, Lee BC, Hwang WS, Lee ES (2000): Development of porcine oocytes from preovulatory follicles of different sizes after maturation in media supplemented with follicular fluids. Reprod Fertil Dev 12:133-139 https://doi.org/10.1071/RD00027
  27. Zou CX, Yang ZM (2000): Evaluation on sperm quality of freshly ejaculated boar semen during in vitro storage under different temperatures. Theriogenology 53:1477-1488 https://doi.org/10.1016/S0093-691X(00)00290-9