Cryopreservation of tench, Tinca tinca, sperm: Sperm motility and hatching success of embryos

• Published in: Theriogenology Vol. 67; no. 5; pp. 931 - 940
• Main Authors: Rodina, M., Gela, D., Kocour, M., Alavi, S.M. Hadi, Hulak, M., Linhart, O.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15.03.2007
• Subjects: Animals; Cryopreservation; Cryopreservation - methods; Cryopreservation - veterinary; cryoprotectants; Cryoprotective Agents; Cyprinidae - physiology; Dimethyl Sulfoxide; embryo (animal); Embryonic Development - physiology; Female; fertilization (reproduction); Fertilization in Vitro - veterinary; fish eggs; fish larvae; hatching; Hatching rate; Male; propanediols; Propylene Glycols; Semen Preservation - methods; Semen Preservation - veterinary; Sperm Count - veterinary; sperm cryopreservation; Sperm motility; Sperm Motility - physiology; sperm volume; spermatozoa; storage time; Tench Tinca tinca; Tinca tinca; Sperm motility; Hatching rate; Cryopreservation; Tench Tinca tinca
• DOI: 10.1016/j.theriogenology.2006.11.007
• ISSN: 0093-691X

The aim of the present study was to elaborate cryopreservation methods for ex situ conservation of tench. Success of cryopreservation was tested during two series of experiments. The first set of experiments studied the effects of two types of cryoprotectants (DMSO and a combination of DMSO with propanediol at ratio 1:1) at concentrations of 8 and 10% and three different equilibration times in two different immobilization solutions (IS) (Kurokura 180 and Kurokura) before freezing (0.0, 2.0 and 4.0 h after T 0). The K4 cooling programme was used to freeze 1 ml of cryoextended sperm using 1.8 ml cryotubes. Main monitored parameter was hatching rate after using of cryopreserved sperm. The second set of experiments studied the volume effect of 0.5, 1 and 5 ml straws and compared these with 1.8 ml cryotubes as well as the effect of the cooling programme (K4 and L1). Following the results of the first study, a combination of DMSO and propanediol (ratio 1:1) at concentration of 10% was added to extended sperm in Kurokura 180 IS. Main monitored parameter was hatching rate after using cryopreserved sperm, supplementary parameters were sperm velocity and motility percentage assessed at 10 s post-activation. Sperm was collected directly into IS and stored at 4 °C for 2.5 h. Thereafter were sperm samples pooled, equlibred in IS (first set of experiments) or directly mixed with cryoprotectants (DMSO or a mixture of DMSO with propanediol at ratio 1:1) and transferred to 1.8 ml cryotubes or straws (0.5, 1 and 5 ml). Then the cryotubes/straws were directly transferred to pre-programmed PLANER Kryo 10 series III and cooled using two different cooling programmes including a slow cooling programme (a) named K4 (from +4 to −9 °C at a rate of 4 °C min −1 and then from −9 to −80 °C at a rate of 11 °C min −1) and a rapid cooling programme (b) named L1 (directly from +4 to −80 °C at a rate of 20 °C min −1). Both slow (K4) and rapid (L1) cooled samples were held 6 min at −80 °C. Finally, samples were transferred into liquid N 2. The frozen spermatozoa were thawed in a water bath (40 °C) according to the frozen volume and checked for fertilization and hatching rates. Percentage of sperm motility and sperm velocity were measured using video recorded frames. ANOVA showed a significant influence of frozen and fresh sperm in all treatments. The hatching rates of 33.8% were obtained when sperm was equilibrated for 0 h before freezing in IS of Kurokura 180 and frozen with a 10% of mixture 1:1 of DMSO and propanediol into straws of 5 ml and cooled using program L1. The velocity of frozen-thawed spermatozoa ranged from 31 to 46 μm s −1 and in post-thawed sperm was not significantly different according to frozen sperm volume, but a higher velocity was obtained when sperm was fast frozen using programme L1. A large volume of frozen sperm could reveal the best procedure for freezing, but also for simulating methods of artificial propagation for future practical use of frozen tench sperm at a large scale.