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.

Chilled and post-thaw storage of sperm in different goldfish types.

The effective storage time of sperm after stripping (for 48 hr in 6-hr intervals) and after thawing (for 6 hr in 2-hr intervals) in Black moor, Oranda and Calico goldfish types was investigated. Variations in sperm density were also measured in all lines. The efficiency of a sperm cryopreservation method formerly developed for common carp was recorded in all three goldfish lines. Motility parameters ((pMOT, %), curvilinear velocity (VCL, µm/s) and straightness (STR, %)) of Black moor sperm did not decrease significantly during 48 hr of storage. A significant reduction in the Oranda type compared to the fresh control was observed in pMOT after 42 (23 ± 2%) and VCL after 36 (94 ± 12 µm/s) hours (pMOT 84 ± 5%, VCL 150 ± 11 µm/s). In the Calico type, pMOT decreased significantly already after 18 (42 ± 26%) and VCL after 6 (105 ± 8 µm/s) hours (fresh: pMOT 92 ± 5%, VCL 151 ± 6 µm/s). A high pMOT immediately following thawing was measured in Oranda (46 ± 12%) and Calico (55 ± 15%) types, whereas a reduced pMOT was recorded in Black moor (24 ± 19%). In Calico, pMOT showed a significant reduction after 6 hr (19 ± 11%) in comparison with the initial value, with no changes observed in VCL and STR. None of the parameters changed in the Black moor and Oranda types. Evidence was found that different goldfish lines have different sperm quality and characteristics. Further studies can investigate the possible effects of chilled and post-thaw storage on the fertilizing capacity of sperm in the Black moor, Oranda and Calico goldfish types.

Commercial-scale out-of-season cryopreservation of Eurasian perch (Perca fluviatilis) sperm and its application for fertilization.

The quality and fertilizing capacity of perch (Perca fluviatilis) sperm collected outside of the spawning season (off-season) and cryopreserved at a commercial scale, were tested. Basic parameters (equilibration time, dilution ratio, sperm concentration, post-thaw motility duration) which can have a significant effect on cryopreservation success were systematically investigated for effects on sperm quality using computer assisted sperm analysis (CASA). No significant decrease in progressive motility (pMOT) and straightness (STR) of fresh-diluted sperm was recorded among groups equilibrated for 0, 30 or 60min in an extender with cryoprotectants. Curvilinear velocity (VCL) was reduced significantly after 30min (30min: 146±15µm/s, 60min: 124±18µm/s) of equilibration compared to the control (174±9µm/s). After thawing, no decrease in pMOT or VCL was observed at different equilibration times in any of the analyzed groups. No correlation was observed among progressive motility, dilution ratios (p=0.7) and cell concentrations (p=0.1). The use of different activating solutions resulted in similar pMOT and VCL in the first 120s post-thaw. Nevertheless, post-thaw sperm motility was reduced after 30s using all activators. Motility parameters with low variation were recorded after thawing of 57 straws (pMOT: 37±7%, VCL: 92±10µm/s, STR: 89±3%). Ten randomly selected straws from commercial-scale cryopreservation resulted in a high fertilization rate (cryopreserved sperm: 72±14%, fresh control: 94±2%). An optimized commercial-scale cryopreservation protocol was successfully developed for Eurasian perch. The applicability of the off-season collected perch sperm for cryopreservation and fertilization was demonstrated.

Comparison of two different methods in the cryopreservation of Eurasian perch (Perca fluviatilis) sperm.

Two different cryopreservation methods were compared and an optimal dilution ratio for the use of controlled-rate freezer (CRF) was established for Eurasian perch (Perca fluviatilis) sperm. Progressive motility (72 ± 15%) and curvilinear velocity (VCL, 146 ± 11 µm/s) of sperm cryopreserved with CRF did not reduce significantly compared to fresh sperm [progressive motility (90 ± 4%), VCL (173 ± 24 µm/s)]. On the other hand, progressive motility (62 ± 15%) and VCL (120 ± 21 µm/s) of sperm cryopreserved with the conventional floating frame technique were significantly lower when compared to the fresh control. Sperm in both cryopreserved groups showed significantly higher straightness [STR, CRF (84 ± 4%), frame (84 ± 2%)] than in the fresh control group (68 ± 4%). Perch sperm cryopreserved with CRF at a dilution ratio of 1:20 showed significantly higher progressive motility (49 ± 6%) than at a ratio of 1:5 (39 ± 6%) and showed significantly higher VCL (129 ± 11 µm/s) than at dilution ratios of 1:10 (112 ± 17 µm/s) and 1:5 (115 ± 9 µm/s).

Cryopreservation of sperm of two European percid species, the pikeperch (Sander lucioperca) and the Volga pikeperch (S. volgensis).

Experiments were carried out on sperm cryopreservation of two European percid fish species, the pikeperch (Sander lucioperca) and the Volga pikeperch (S. volgensis). Two experiments were conducted on pikeperch sperm. In the first, the effects of three extenders (Glucose, KCl, Sucrose) and two cryoprotectants (dimethyl-sulfoxide: DMSO, methanol: MeOH) were tested on motility and fertilization. In the second, the effects of two dilution ratios (1 : 1, 1: 9) and two cryoprotectants (DMSO, MeOH) on hatching were investigated. In the experiment on Volga pikeperch the suitability of using cryopreservation for fertilization was investigated. In the first experiment on pikeperch the highest post-thaw motility (28 +/- 21%) and fertilization rate (43 +/- 12%) was found with DMSO as cryoprotectant in combination with Glucose extender. In the second, the highest hatch rate (41 +/- 22%) was observed with MeOH as cryoprotectant and 1 : 1 sperm dilution ratio, however no significant difference was found among the results. In the experiment on Volga pikeperch hatch rates with cryopreserved sperm (60 +/- 2%) did not significantly differ from the control (60 +/- 6%). Contamination of sperm with urine seems to be a key problem in the success of sperm cryopreservation of these species.