Introduction
Chub is a rheophilic species, preferring rapid rivers with gravelly bottoms. It is distributed in Europe and Asia Minor (Caffrey et al. 2008). Based on monitoring studies natural populations of chub have decreasing tendency due to overexploitation, loss of spawning habitat, water pollution, interspecific hybridization, remarkable angling demand and climate change (Cejko & Krejszef 2016). The improvement of applicable propagation technology, in which sperm cryopreservation plays a key role, is important for maintaining and stabilising the populations and for gene conservation. Cryopreserved sperm collected from farmed or natural populations is part of maintaining biological and genetic diversity. It can also contribute to increasing the natural abundance of fish species with high angling interest (Bernáth et al. 2021). The study aimed to compare the effects of two hormonal agents (Ovopel and carp pituitary extract) on chub sperm production, its cryopreservation and post-thaw storage time, moreover, the development of a novel large-scale cryopreservation procedure by testing various freezing methods.
Materials and methods
Experiment 1.
In the first experiment, 6-6 males were hormonally stimulated with two hormone preparations, carp pituitary extract and Ovopel. Sperm samples were frozen at a dilution ratio of 1:9 (sperm:diluent + protectant) in 0.5 mL straw in a Styrofoam box. Sperm motility assessment was carried out using Computer-Assisted Sperm Analysis (CASA) system. Sperm quality was determined by pMOT (progressive motility, %), VCL (curvilinear velocity, μm/s) and LIN (linearity, %). Motility parameters were recorded for fresh, cryopreserved and thawed chilled stored samples (at 0, 3, 6 h post-thawing).
Experiment 2.
In the second experiment, (N=5) sperm samples were cryopreserved in a Styrofoam box (dilution ratio: 1:9) using 5 mL straws and 4 mL cryotubes. Motility parameters of both fresh and thawed samples were recorded by CASA.
Experiment 3.
In our first propagation test, sperm samples (N=5) were pooled and filled into 4 mL cryotubes (dilution ratio: 1:9). The samples were frozen in a Styrofoam box and a Controlled-rate freezer (CRF). The kinetic parameters of fresh and frozen samples were recorded by CASA. In the experiment, 1-1 g of egg batches were fertilized with 10 µl of fresh and 100 µl of cryopreserved sperm. The control and cryopreserved groups were incubated separately at 19±0.8 °C using spawning nests. The hatching rate (hatched larvae per total egg number * 100) was determined for both groups (~100 larvae or eggs) at the moment of hatching (3 days post fertilization).
Experiment 4.
The second fertilization trial was carried out based on the results of the previous experiments (Ovopel, dilution ratio 1:1, 4 ml cryotube, Styrofoam box). Fertilization of 1-1 g egg batches was performed with fresh (10 µl) and frozen (20 µl) sperm. Kinetic parameters were recorded using CASA system in fresh samples, immediately before application (control, ~1 h post-collection) and after thawing. The hatching rate was determined for both fresh and frozen groups.
Results
Experiment 1.
No significant difference was measured between the carp pituitary extract and Ovopel treated groups for fresh and cryopreserved sperm samples (0, 3, 6 h post-thaw). Reduced pMOT values were also observed after 3 hours of chilled post-thaw storage in comparison to 0 h.
Experiment 2.
There was no significant difference in the motility parameters tested between the groups frozen in a Styrofoam box using 5 ml straws and using 4 ml cryotubes.
Experiment 3.
No significant difference was observed between the efficacy of the two cryopreservation methods either in terms of kinetic parameters or in terms of hatching rates (Styrofoam box (35±7%) and CRF (25±9%).
Experiment 4.
In the second fertilization trial, in pMOT and VCL parameters, a significant decrease was observed in the cryopreserved sperm in comparison to the fresh and control groups. Moreover, similar high (with no significant difference) hatching rates were observed in the control (72±19%) and cryopreserved (4 ml cryotube and Styrofoam box, 61±5%) groups.
Discussion and conclusion
Although there was no significant difference between the treatments. The chub sperm showed high sensitivity during 6 hours of chilled storage, the pMOT values were already significantly reduced after 3 hours. Before its implementation in common hatchery practices, it needs to be considered. Both the 5 mL straw and the 4 mL cryotube have been proven to be effective in freezing large-scale chub sperm. Both sperm frozen in a Styrofoam box and in a CRF seemed to be effective in propagation. For the first time, our study presents a novel and applicable method for the large-scale cryopreservation of chub sperm.
Acknowledgements
The research work was supported by the ÚNKP-22-3 new national excellence program of the Ministry for Culture and Innovation from the source of the National Research, Development and Innovation fund. The experiments were also funded by the GINOP-2.2.1-18-2020-00026, and by the Thematic Excellence Programme 2020 - National Challenges Subprogramme (TKP2020-NKA-16).
References
Bernáth G., Bokor Z., Horváth Á., Csorbai B. (2021): A természetes populációk megőrzésének lehetőségei. In: Urbányi B., Szabó T. & Horváth Á. (Eds.) Horgászati szempontból jelentős pontyfélék biológiája és tenyésztése. Gödöllő, Magyarország: Magyar Agrár- és Élettudományi Egyetem, Akvakultúra és Környezetbiztonsági Intézet 262 p. 169-186. pp. ISBN: 978-963-269-978-3
Caffrey, J. M., Acevedo, S., Gallagher, K., Britton, R. (2008): Chub (Leuciscus cephalus): a new potentially invasive fish species in Ireland, Aquatic Invasions, Reabic. 201-209. p.
Cejko, B. I., Krejszeff, S. (2016): Sperm characteristics of chub Leuciscus cephalus (L.) collected in artificial condition after Ovopel and Ovaprim treatment. Aquaculture Research 47, 847–856.