The investigation of post-thaw chilled storage and the applicability of large-scale cryopreservation in chub (Squalius cephalus) sperm.

Chub (reophillic cyprinids) is one of the most sensitive bioindicator fish of environmental changes following anthropogenic activities. The improvement of different biotechnological procedures could help support its conservation and strengthen the natural populations. The aim of this study was to compare the effects of two different hormonal agents (carp pituitary extract and Ovopel™) on various motility parameters (pMOT-%, DAP-µm, VCL µm s-1, VSL-µm s-1, LIN-%, ALH-µm, BCF-Hz) of fresh and cryopreserved/thawed sperm (stored at 4 °C for 6 h). Additionally, we sought to develop a novel, large-scale cryopreservation method for chub sperm, assessing freezing methods (Styrofoam box and a controlled-rate freezer) and different containers (0.5, 5 mL straw and 4 mL cryotube) for sperm cryopreservation. The results of this study indicated no difference between the carp pituitary extract and Ovopel treated groups in either the fresh or frozen/thawed sperm (at 0, 3, 6, hour post thawing, P = 0.4351). In contrast, the quality of the thawed chub sperm was negatively affected after 3 h chilled storage in both hormonal treatments (P = 0.0036, P < 0.0001). When assessing the motility parameters of the sperm between the 5 mL straw and 4 mL cryotube groups cryopreserved in a Styrofoam Box, no difference was observed (P = 0.103). Additionally, sperm loaded in 4 mL cryotubes showed no difference in motility when cryopreserved with either the Styrofoam box or controlled-rate freezer methods (P = 0.109). A similar hatching rate was observed in sperm preserved using the Styrofoam box (35 ± 7 %) and controlled rate freezer (25 ± 9 %) methods (P = 0.300). In a second fertilization trial, hatching rate was similar between control (72 ± 19 %) and cryopreserved (4 mL cryotube and Styrofoam box, 61 ± 5 %) groups. (P = 0.257). Based on our findings and its standard features (less species specific, precise dose calculation), Ovopel can be a good candidate for the stimulation of spermiation in chub sperm prior to cryopreservation. Furthermore, our study presents a novel and applicable method for the large-scale cryopreservation of chub sperm.

The systematic development and optimization of large-scale sperm cryopreservation in northern pike (Esox lucius).

In our study, a systematic development of a new large-scale sperm cryopreservation protocol was carried out in northern pike (Esox lucius). The effect of 2 sugar based (glucose and trehalose) extenders, 3 dilution ratios (1:3, 1:9 and 1:19) 2 vol straws (0.5 and 5 mL) and a 10 mL cryotube, 2 different cryopreservation methods (Polystyrene box-P. box and Controlled Rate Freezer-CRF), as well as 3 different thawing periods (3, 3.5 and 4 min) were investigated on the motility of thawed sperm. The glucose based extender showed significantly higher pMOT (1:3-18 ± 16%, 1:9-20 ± 13%, 1:19-16 ± 12%) at all dilution ratios than in the trehalose based extender (1:3-0.3 ± 1%, 1:9-1±1%, 1:19-4±2%). A similar tendency was recorded in VCL and STR at a ratio 1:3 and 1:9. No significant difference was measured in sperm movement between the P. box and CRF using the 0.5 mL straw. Similarly no significant difference was observed in all motility parameters with 10 mL cryotube frozen in CRF at a ratio 1:3-1:19. An effective and short thawing period (3 min) was experimentally specified for the 10 mL cryotube cryopreserved in the CRF. In all large-scale cryopreservation methods, high pMOT (straw CRF: 57 ± 10%, straw P. box: 50 ± 9%, cryotube CRF: 41 ± 10%), and STR were measured, and no significant difference was recorded in all motility parameters. Our results demonstrate the effectiveness of our newly developed extender and the applicability of 3 different large-scale cryopreservation methods in pike sperm. Our protocols could be new prospective candidates for future exploitation in hatchery practice.