| Studies on cryopreservation of fish sperm have been succeed in hundreds of fishes. But among tens thousands of fish species, they are just a few. The problem in fish sperm cryopreservation remains also in that most of the reported protocols are greatly different from each other, and making it very difficult to compare the reported results. At present, it is eagerly needed to set up one or some formal, general and practical protocol(s) for fish sperm cryopreservation.After a series of trying and improving, we constituted a quite easy protocol for cryopreservation of the spermatozoa for two commercially important flatfishes Paralichthys olivacelus and Verasper variegates (T & S). The protocol is greatly practical in conventional using, and resulting in good preservation with high sperm fertility. From checking sperm motility before and after cryopreservation, to testing fertility of the frozen-thawed sperm, and to reccording hatching rate after fertilization by frozen-thawed sperm, it forms a systematic series.After trying with many different extender preparations, we singled out the following ideal extenders: 12%glycerol (GLY) and 12%dimethyl sulphoxide (Me2SO) prepared in artificial sea water. Using the two extenders, we diluted semen to extender in the ratio 1 : 2, put the diluted semen in a 2-ml cryovial, 1 ml in each, and lowered down the cryovials in a canister to a liquid nitrogen container beginning from the top opening of it in stepwise, about 3 cm in a step and stay there for 2 minutes for each step. And then after suspension them 4 cm above the surface of the liquid nitrogen for 5 minutes, they were plunged into liquid nitrogen. By warming the cryovials in a 28 water bath after taking them out of the liquid nitrogen, just before using, we thawed the semen samples.After thawing, the percentage of motile sperm in Paralichthys olivacelus was 79.17 4.5% (GLY protected), and 60.5 ?3.6% (Me2SO protected). In the same sperm : egg ratio, we fertilized eggs with each frozen-thawed semen and fresh semen as control, and got 76.2 10% (GLY protected), and 67.06 ?15.1% (Me2SO protected) of eggsfertilized. After culture at about 16 for 60 hours, hatching rates of the embryos revealed to be 48.18 ?25.7% (GLY protected), and 37.4 ?8.3% (Me2SO protected). Comparing with those results from fresh semen, they are a little bit lower, but there is no significant statistical difference (P > 0.05) between treated semen and fresh control, except for motile rate of spermatozoa.The same protocol was used in cryopreservation of Verasper variegates sperm with an improvement of that 10% egg yolk (YK) was added to the extenders. The results showed that the percentage of motile sperm in frozen-thawed semen were 67.5 ?4.0% (for GLY+YK protected), and 65.0 ?4.0% (for Me2SO+YK protected). The percentages of eggs fertilized by frozen-thawed semen were 70.2 ?3.0% (for GLY+YK), and 69.0 ?3.5% (for Me2SO+YK). And eventually got the hatching rate at 18.0 ?2.6% (for GLY + YK), and 17.0 ?2.5% (Me2SO+YK) respectively. For the rates of fertilization and hatching, there is no significant difference (P > 0.05) compared to those rates obtain from fresh semen control. The lower hatching rate compared to other fishery fishes may due to the low quality of spermatozoa and eggs produced by the first domesticated generation.Ultrastructures of fresh and cryopreserved spermatozoa were studied under scanning and transmission electron microscope. Results showed that mitochondria swollen, lost or in losing in some cryopreserved spermatozoa, tail cut off totally or partly from some spermatozoa, and in some spermatozoa the cell membrane ruptured in some areas. This may attribute to cryoinjuries and results in lower motile sperm rate in the cryopreserved semen. As we studying the ultrastructure of Verasper variegates spermatozoon, we also found that there is a chromatin-free area in the anterior part of the nucleus, where a little bit deflect from the top of the nucleus. Such chromatin-free area is just correlated to the deep pit seen from scann...
|
PDF Full Text Request