The objective of this study was to freeze sperm of sex-reversed females (neomales) of perch and to test their fertilization ability. Sperm used was testicular (TSN), collected from females that have been inverted by means of externally administered 17-alpha methyltestosterone. Sperm collected from intact males (SSNM) of the same origin were used as control. Prior to freezing, both TSN and SSNM were diluted into 300 mM M glucose solution at the ratio of 1 : 6 and DMSO was used as cryoprotectant (10% final concentration). Crypreservation was performed in 0.5 ml straws placed into a polystyrene box, three cm above the liquid nitrogen level for 10 min and thereafter transferred fully into liquid nitrogen. Samples were thawed in 40°C water bath for 8 s and used for the fertilization experiments. Spermatozoa concentration of fresh TSN and SSNM were estimated with 45.3 · 10 9 and 37.8 · 10 9 spermatozoa ml )1 , respectively. Both sperm velocity and motility showed significant decreases in the TSN (134.6 lm s )1 and 12.8%) compared to the SSNM (203.2 lm s )1 and 94.7%) at 10 s after sperm activation. However, no differences were observed in terms of hatching rates between fresh TSN and SSNM (42.5 vs 49.3%) at fertilization densities of 12 · 10 5 spermatozoa per egg. Frozen ⁄ thawed SSNM exhibited similar hatching rates at 12 · 10 5 and 2.4 · 10 5 spermatozoa per egg (37.2% vs 29.1%). Hatching rates for frozen ⁄ thawed TSN were about 7.3% with 12 · 10 5 spermatozoa per egg and did not show any difference at 2.4 · 10 5 spermatozoa per egg (6.6%). Stripped sperm of normal perch can be successfully frozen. Squeezing of the testes is not a good method for collection of testicular sperm resulting into low velocity, motility and hatching rate. To understand the influences of neomales on sperm quality on reproductive success further studies should be performed addressing a full assay of motility and fertility criteria when using stripped sperm from normal males and neomales. Additionally, the results indicate that many of sex reversed perch neomales are not able to release sperm and that for further studies some well spermiating neomales must to be selected.
Summary The objective of this study with common carp spermatozoa was to understand the fertilization potency of different males when different sperm quantities were applied per ova of a single female. The sperm of five males representing very good sperm motility and the ova from one female exhibiting the best apparent quality were used. The sperm of each male was collected at volumes of 5 (1000 spermatozoa), 10 (5000 spermatozoa), 20 (10 000 spermatozoa), 40 (20 000 spermatoza) and 400 μl (200 000 spermatozoa) and pre‐diluted with 995, 990, 980, 960 and 600 μl of Kurokura solution, respectively. Thereafter, 4000 eggs and pre‐diluted spermatozoa from each male, one by one, were simultaneously added to 1000, 5000, 10 000, 20 000 and 200 000 spermatozoa and activated with hatchery water. Initial sperm motility was in the range of 89.5–97.2% at 15 s, decreasing to 19.1–30.2% at 60 s post‐activation. At all times of evaluated post‐activation, the sperm motility did not differ significantly among the males. Sperm velocity decreased from 126.1 to 161.2 μm s−1 at 15 s to 11.9–35.2 μm s−1 at 60 s post‐activation. Sperm velocity was significantly different among males at 15 s post‐activation. Fertilization and hatching rates were similar in all males at a higher examined number of spermatozoa per ova (20 000 and 200 000). Similar fertilization and hatching rates were observed in four out of five males at 10 000 spermatozoa per ova. Lower spermatozoa per ova (5000) induced very different results, from 48 to 82% for fertilization rates and from 42 to 72% for hatching rates. At 1000 spermatozoa per ova a very high variability was observed: 10–50% for fertilization rates and 8–43% for hatching rates. These results did not correspond to sperm velocity among males. The 20 000 spermatozoa density was considered as providing a secure number of spermatozoa for reaching good fertilization in common carp. To avoid loss of genetic variability for future generations this recommendation is important to know for the management of hatcheries where these broodstocks will be generated.
Vitrification could provide a promising tool for the cryopreservation of fish embryos. However, in order to achieve cryopreservation using vitrification, chilling sensitivity and cryoprotectants toxicity were determined using tench embryos at four developmental stages (11, 17, 23 and 29-h). Embryos treated with alcalase (2mL 998ml 2mm at 22°C) were exposed to chilling with/without warming. Other embryos were exposed to methanol and glycerol at the concentration of 10% and 20% for periods of 20 min. Thereafter, embryos were incubated at special incubator to determin the hatching rates. Regarding chilling sensitivity, the hatching rates of embryos decreased significantly (P <0.001) after exposure to 0°C at all developmental stage except 29-h stage. The same results were reported after exposure to chilling followed by warming. It was found that the 11-h embryonic stage was most sensitive to chilling preservation. Whereas, the 29-h stage exhibited the least sensitivity, while 17-h and 23-h stages were intermediate in their sensitivity to chilling. The toxicity of methanol increased significantly (P <0.001) with developmental stage for 11-h, 17-h and 23-h stages. The largest change was a reduction in the toxicity of methanol in 29-h embryos. The highest hatching rates of tench embryos were obtained with 29-h embryos using various concentrations of methanol. The survival trends of different stages of tench embryos exposed to glycerol concentrations were approximately similar to those embryos exposed to methanol concentrations except for 11-h embryos which showed no hatching with both glycerol concentrations. Concerning the viability of embryos after vitrification, unfortunately, it could not obtained any viable embryos in any of conditions examined. In conclusion, as it was quite difficult to vitrify the tench embryos during this study using various vitrifying solutions and the method reported by Chen and Tian (2005) and so further studies using other developmental stages, basic, washing and vitrifying solutions are needed to achieve successful cryopreservation.
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