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.

The effects of different preservation methods on ide (Leuciscus idus) sperm and the longevity of sperm movement.

The present study investigated the effects of chilled storage and cryopreservation on ide sperm motility and fertilizing capacity alongside the longevity of sperm movement. The parameters of motility (progressive motility-pMOT, curvilinear velocity-VCL and straightness-STR) have been recorded during 48 h of chilled storage (4 °C) at 24-h intervals. The longevity of sperm movement was measured following activation for up to 120 s (in a range at 10-120 s) in freshly stripped and thawed sperm. A formerly established cryopreservation method was tested on ide sperm where motility parameters, hatching rate and larval malformation (according to 7 category groups) were investigated. Significant decrement of pMOT has already been observed after 24 h (6 ±â€¯5%) compared to the freshly stripped sperm (49 ±â€¯22%). pMOT and STR showed no significant changes for up to 120 s following activation in fresh sperm, whereas VCL showed significant difference between 10 (51 ±â€¯11 µm/s), 90 (33 ±â€¯3 µm/s) and 120 (31 ±â€¯4 µm/s) seconds as well as between 20 (48 ±â€¯12 µm/s), and 120 s. No negative effect of cryopreservation was recorded on pMOT (fresh: 49 ±â€¯19%, cryopreserved: 22 ±â€¯22%), VCL (fresh: 45 ±â€¯9 µm/s and cryopreserved: 57 ±â€¯5 µm/s), STR (fresh: 81 ±â€¯3% and cryopreserved: 92 ±â€¯1%) hatching rate (fresh: 22 ±â€¯15%, cryopreserved: 33 ±â€¯18%) or larval malformation (fresh: 12 ±â€¯4%, cryopreserved: 12 ±â€¯4%). No significant correlation was found between the three motility parameters and hatching rate. Cryopreservation had no effect on hatching and the prevalence of larval deformity. Furthermore craniofacial and eye deformities were characteristic in the group originating from fertilization with cryopreserved sperm, while edemas (pericardial, yolk) occurred more frequently in the control. The formerly developed cryopreservation protocol (method for cyprinids) was applicable to ide sperm.