title: Effects of type III antifreeze protein on sperm and embryo cryopreservation in rabbit.
abstract: We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN2) and then preserved in the LN2. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN2. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos.
authors.affiliation: Division of Biological Resources and Development, Analytical Research Center for Experimental Sciences, Saga University, Japan. Electronic address: kanish@cc.saga-u.ac.jp.
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Nishijima
authors.affiliation: Division of Biological Resources and Development, Analytical Research Center for Experimental Sciences, Saga University, Japan.
authors.forename: Mai
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Department of Bio-resources, Division of Biotechnology, Frontier Science Research Center, University of Miyazaki, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Sakai
authors.affiliation: Department of Bio-resources, Division of Biotechnology, Frontier Science Research Center, University of Miyazaki, Japan.
authors.forename: Chihiro
authors.initials: C
authors.lastname: Koshimoto
authors.affiliation: Division of Biological Resources and Development, Analytical Research Center for Experimental Sciences, Saga University, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Morimoto
authors.affiliation: Division of Biological Resources and Development, Analytical Research Center for Experimental Sciences, Saga University, Japan.
authors.forename: Teruo
authors.initials: T
authors.lastname: Watanabe
authors.affiliation: Department of Molecular Pathology, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Japan.
authors.forename: Jianglin
authors.initials: J
authors.lastname: Fan
authors.affiliation: Division of Biological Resources and Development, Analytical Research Center for Experimental Sciences, Saga University, Japan.
authors.forename: Shuji
authors.initials: S
authors.lastname: Kitajima
mesh_terms: D000818:Animals; D021343:Antifreeze Proteins, Type III / Q000494:pharmacology*; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D004622:Embryo, Mammalian; D047108:Embryonic Development; D005260:Female; D006531:HEPES / Q000494:pharmacology; D008297:Male; D009028:Morula; D011817:Rabbits; D012662:Semen Preservation / Q000379:methods*; D013081:Sperm Motility / Q000502:physiology; D013094:Spermatozoa
chemical_list: D021343:Antifreeze Proteins, Type III; D003451:Cryoprotective Agents; D006531:HEPES
keywords: Antifreeze protein; Cryopreservation; Embryo; Rabbit; Sperm