Sperm motility and fertilizing ability of frozen spermatozoa of males (XY) and neomales (XX) of perch (Perca fluviatilis)

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 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 × 109 and 37.8 × 109 spermatozoa ml−1, respectively. Both sperm velocity and motility showed significant decreases in the TSN (134.6 μm s−1 and 12.8%) compared to the SSNM (203.2 μm 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 × 105 spermatozoa per egg. Frozen/thawed SSNM exhibited similar hatching rates at 12 × 105 and 2.4 × 105 spermatozoa per egg (37.2% vs 29.1%). Hatching rates for frozen/thawed TSN were about 7.3% with 12 × 105 spermatozoa per egg and did not show any difference at 2.4 × 105 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.