Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores.
The objectives of this study were to determine the effect of cryoprotectants on sperm viability and develop a freezing protocol for long-term storage of P. monodon spermatophores. Spermatophores suspended for 30 min in calcium-free saline (Ca-F saline) containing the cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propylene glycol (PG), formamide, and methanol at concentrations of 5, 10, 15, or 20% were studied using a modified eosin-nigrosin staining technique. The smallest reductions in apparent sperm viability occurred with DMSO; therefore, a freezing protocol was developed using Ca-F saline containing 5% DMSO. Spermatophores were cryopreserved using three protocols; cooling to a final temperature of -30, -80 or -80 degrees C and immediately stored in liquid nitrogen (cooling rates of -2, -4, -6, -8, -10, -12, -14 or -16 degrees C/min). Frozen spermatophores were thawed (2 min) at 30, 60, 70, or 90 degrees C. Successful cryopreservation of spermatophores in liquid nitrogen was achieved by a one-step cooling rate of -2 degrees C/min between 25 and -80 degrees C before storing in liquid nitrogen. Optimal thawing was in a 30 degrees C water bath for 2 min; this yielded live sperm after storage in liquid nitrogen for 210 days. Average sperm viability for fresh (97.8+/-2.9%) and cryopreserved spermatophores held for less than 60 days (87.3+/-4.1%) did not differ (P>0.05); however, that for spermatophores stored in liquid nitrogen between 90 and 210 days were lower (P<0.05) and varied from 27.3+/-3.4 to 53.3+/-4.3%. Thawed spermatophores previously held in liquid nitrogen for less than 62 days fertilized eggs (fertilization and hatching rates of 71.6-72.2% and 63.6-64.1%, respectively) at rates comparable to fresh spermatophores (70.8-78.2% and 66.3-67.8%, respectively). In conclusion, sperm within cryopreserved spermatophores stored in liquid nitrogen retained their viability for up to 210 days.
Vuthiphandchai
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Department of Aquatic Science, Faculty of Science, Burapha University 20131, Thailand. verapong@vuu.ac.th
Nimrat
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Kotcharat
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Bart
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D000818:Animals; D015925:Cryopreservation / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000633:toxicity*; D004121:Dimethyl Sulfoxide / Q000633:toxicity; D019855:Ethylene Glycol / Q000633:toxicity; D005307:Fertilization in Vitro / Q000187:drug effects / Q000662:veterinary; D005559:Formamides / Q000633:toxicity; D008297:Male; D000432:Methanol / Q000633:toxicity; D033561:Penaeidae / Q000502:physiology*; D019946:Propylene Glycol / Q000633:toxicity; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D012965:Sodium Chloride; D013093:Spermatogonia / Q000187:drug effects*; D013997:Time Factors
D003451:Cryoprotective Agents; D005559:Formamides; D012965:Sodium Chloride; C031066:formamide; D019946:Propylene Glycol; D019855:Ethylene Glycol; D000432:Methanol; D004121:Dimethyl Sulfoxide