The initial attempts at cryopreserving sperm involved the use of vials, with freezing performed in nitrogen vapor (Moczarski and Kołdras, 1982). For most of cryopreservation trials in the 90-ties, sperm was cryopreserved mainly by pelleting on dry ice (-79°C). In the first decade of the 21^st century, the method of pelleting on dry ice was gradually abandoned and 0.25 ml and 0.5 ml plastic straws (IMV Technologies, L’Agile, France) were introduced (Glogowski et al., 2002). These straws were frozen 3 cm above liquid nitrogen (in the vapor of liquid nitrogen) for 5 min in a Styrofoam box with an isolating Neopor block (Minitübe GmbH, Tiefenbach, Germany). In 2014, an efficient glucose-methanol extender was developed (Ciereszko et al., 2014) which was subsequently refined to optimize the cryopreservation of fish semen (primarily salmonids). This optimization involved controlling the final concentration of spermatozoa and cryoprotectants (Nynca et al., 2017; Judycka et al., 2018a; Judycka et al., 2019b, c, d, 2020, 2021).

During the first decades, sperm motility of fresh and cryopreserved semen was subjectively evaluated. However, in the first decade of the 21st century, Computer-Assisted Sperm Analysis (CASA) was introduced for objective and advanced sperm motility analysis. The first system introduced was the Hobson Sperm Cell Tracker, developed by Hobson Vision Ltd., Bakewell, UK (Ciereszko et al., 2008). Subsequently, other systems followed, such as the Sperm Class Analyzer v. 4.0.0 developed by Microptic S.L, Barcelona, Spain (Dziewulska et al., 2011), the CRISMAS computer-assisted sperm analysis software (Image House Company, Copenhagen, Denmark; Judycka et al., 2015; Sarosiek et al., 2016a); and the CEROS II System (Hamilton Thorne, Beverely MA, USA; Judycka et al., 2019c).

Fish spermatozoa are small, therefore counting under the microscope is time-consuming. Fast methods for sperm concentration measurements are prerequisite, especially for the quick and successful execution of standardized cryopreservation procedures, which rely on controlling the number of spermatozoa in straws. Three methods are commonly used for this purpose: i) spectrophotometric method based on estimation of sperm concentration (Ciereszko and Dabrowski, 1993), ii) computer-aided fluorescence microscopy using NucleoCounter SP-100 (Chemometec, Allerød, Denmark; Nynca and Ciereszko, 2009), and iii) flow cytometry employing a portable cytometer, Muse Count and Viability Assay Reagent (Millipore, Burlington, MA, USA) (Nynca et al., 2016b). The latter two methods can also assess sperm viability.

The fertilizing ability represents the most crucial and ultimate parameter of cryopreserved semen and has been measured in several studies, both at the stage of eyed eggs and hatched larvae, preferably employing a controlled sperm-to-egg ratio (for example, (Babiak et al., 1998b, Babiak et al., 1997; Glogowski et al., 2002; Ciereszko et al., 2008; Dziewulska et al., 2011; Dziewulska and Domagała, 2013; Ciereszko et al., 2014; Dietrich et al., 2014; Nynca et al., 2014; Judycka et al., 2015; Nynca et al., 2015b; Dietrich et al., 2016; Sarosiek et al., 2016b; Judycka et al., 2018b; Judycka et al., 2019c; Judycka et al., 2019d).

Semen cryopreservation has been utilized to establish a semen bank housing valuable carp genetic lines at the Institute of Ichthyobiology and Aquaculture of the Polish Academy of Sciences in Gołysz. A leader in carp breeding since the 1950s, the Institute has curated a unique collection of 17 breeding lines, forming a “living gene bank”. This living gene bank plays a pivotal role in national and ex situ programs for the protection and reproduction of aquatic animals, aligning with Poland’s commitment to the Convention on Biological Diversity. Semen cryopreservation activities for the carp breeding lines maintained in Gołysz were conducted in accordance with “National programs for maintaining the genetic reserve of farmed fish and the conservation of genetic resources of farm animals” (2003 to 2008) founded by Ministry of Agriculture and Rural Development, operational program “Sustainable development of the fisheries sector and coastal fishing areas”, 2.2. project title – “Protection of genetic resources of original pure lines of farmed carp” (2013–2016) and operational program “Fisheries and Sea” (PO RYBY 2014–2020), actions within Priority 2 - Supporting environmentally sustainable, resource-efficient, innovative, competitive, and knowledge-based aquaculture, the operation titled “Protection of the possessed genetic resources of fish implemented in accordance with national environmental protection programs and biodiversity restoration” (2017–2023). The primary goal of these programs is to preserve genetic variability and selection material for future breeding by safeguarding the gene pool of males across various breeding lines.

Cryopreservation activities were carried out in two phases, from 2003 to 2008 and 2013 to 2023, involving the freezing of semen from carp breeding lines: Polish lines (Landek line (2), Ochaby line (3), Gołysz line (6) and Knyszyn line (K)) and foreign lines such as Hungarian (8, 7, 0, W, T), Lithuanian (B, BVP), Ukrainian (U, Up), French (F), Israeli (D), German (N), Yugoslavian (J) as detailed in Table 1 . The lines designated for freezing were selected by the staff of Institute in Gołysz. The selection process focused on endangered lines, particularly those whose breeding stock had diminished in recent years. The lines with the fewest breeders were prioritized for preservation, as were lines experiencing significant declines. This allowed us to protect the lines in the weakest state and those facing the greatest threats. Semen was collected from hormonally stimulated carp and samples showed a minimum of 70% motility were selected for cryopreservation. From 2003 to 2005, an effective pellet freezing method was used, involving the dilution of semen in Kurokura’s extender (with 10% DMSO and 10% egg yolk, Kurokura et al., 1984) before being dripped into solidified carbon dioxide (dry ice, -79°C) wells. The frozen semen took the form of pellets, which were then transferred into labeled plastic boxes and stored in liquid nitrogen (-196°C). This method ensured a high fertilization and hatching rate, typically ranging from 80% to 90%. Subsequently, in 2006, to align with modern cryopreservation standards, the method transitioned to plastic straw (volume 0.5 mL) freezing. The great advantage of straws was possibility to print the information concerning individual fish directly on straws with the use of straw printer. The developed method involved diluting semen in the Kopeika, Lahnsteiner or “Pike” extender (at 1:6 or 1:9 ratio) (Kopeika, 1986; Lahnsteiner et al., 2000; Bernáth et al., 2017). Semen was immediately packed into 0.5-mL straws and held in liquid nitrogen vapor 3 cm over the surface of the liquid nitrogen for 5 min. Afterward, the straws were placed into liquid nitrogen for storage. From 2013, each male’s semen was evaluated by thawing one straw in a 40°C water bath for 13 s, and post-thaw sperm motility showed a range of 42 to 66%. The cryopreserved semen, stored in both pellets and straws in Cryobank at the Cryogene company in Poland, is maintained under the supervision of the Institute in Gołysz. Detailed records of cryopreservation protocols, including extender compositions, dilution factors, and post-thaw motility, are documented, contributing significantly to the field of aquaculture and genetic preservation research.

In 2023, as part of the project “Protection of genetic resources of farmed and environmentally valuable freshwater and dual-environment fish”, we performed cryopreservation of semen from four breeding lines of carp (Izraelian: DOR-70, Bubianski: B, Zatorski: Z and Starzawski: SK) maintained at the Experimental Fish Farm in Zator, National Inland Fisheries Research Institute. Semen was collected from 60 non-stimulated male carps, and concurrently, a fin sample was taken from each individual for genetic analysis to facilitate future crossbreeding. The cryopreservation process involved diluting the semen at a 1:9 ratio with a ‘Pike’ extender, resulting in a post-cryopreservation motility of 43–51%, varying by lines ( Table 2 ). Semen from 60 males were cryopreserved and is being stored in Fish Sperm Bank in Olsztyn. It has been emphasized, that these works started in 2022 when dr Kowalski’s team cryopreserved semen in 180 straws from 45 individuals.

Summing up, currently, semen from carp lines maintained in two Polish breeding stations are under a cryopreservation program. This should secure the variability of carp in Poland, allowing the constant development of aquaculture for this species.

The autochthonous population of common whitefish in Lake Łebsko, located within the Słowiński National Park, was first recognized in the 19th century and documented at the beginning of the 20th century (Ciereszko et al., 2008). This population is currently classified as endangered and is supported by stocking programs.

In 2006–2007, as part of the grant project “Protection of the autochthonous population of common whitefish in Lake Łebsko,” funded by EKOFUNDUSZ, Warsaw, we conducted initial research focusing on semen characteristics and the development of a semen cryopreservation procedure. Fish were collected by fishermen in 2006 (testicular and stripped, n = 17; Table 3 ) and 2007 (stripped, n = 19). Obtaining good-quality semen from captured fish posed a significant challenge, partially resolved through close cooperation with the fishermen. Semen characteristics, including volume, sperm concentration, motility parameters, and seminal plasma parameters, were established (Ciereszko et al., 2008). The efficacy of four cryoprotectants—methanol, glycerol, DMSO, and DMA—was evaluated. Methanol emerged as superior, ensuring both post-thaw sperm motility and fertilizing ability. Consequently, the cryopreservation procedure was refined, involving the use of an extender consisting of 0.3 M glucose and 10 or 15% methanol, with a semen-extender ratio of 1:3. Extended semen was then filled into 0.25 ml plastic straws and frozen in nitrogen vapor, positioned 3 cm above the surface of the liquid nitrogen. This procedure resulted in a decrease in sperm motility to half of the values observed in fresh semen, while maintaining similar fertilizing ability. Gonadal semen from six males (collection in 2006) was cryopreserved and currently stored in the Fish Sperm Bank in Olsztyn (Ciereszko et al., 2008).

The cryopreservation efforts continued under the grant project “Ichthyological Biodiversity of Lakes – Elaborating a Model for Problem Solution: A Case Study of Natural Resources of Autochthonous Common Whitefish in Lake Łebsko (Łebsko Lake Whitefish),” funded by EEA grants. In 2009, semen from 41 males was collected using an improved procedure based on careful fish collection and limited semen specimen handling (5–6 per hour), characterized, and subsequently cryopreserved using the glucose (0.3 M)-methanol (10%) extender. Previously, methanol concentration at 10% resulted in obtaining similar fertilization ability to 15% methanol, therefore we decided to use in further experiments 10% methanol. Post-thaw sperm viability and motility were measured at 67% and 35%, respectively. In 2011, cryopreservation was performed for an additional 30 males, followed by cryopreservation of semen from 12 males in 2013. All these samples are currently stored in the Fish Sperm Bank in Olsztyn.

We have developed a cryopreservation procedure for Salmonid fish semen based on the glucose-methanol extender, whose effectiveness was further confirmed by high fertilization rates (Nynca et al., 2017; Judycka et al., 2018b; Judycka et al., 2019b; Judycka et al., 2019d). This formed the basis for establishing cooperation with the “Dąbie” Fish Hatchery and joint implementation of the NCRD project (Innovative Hatchery Project – Implementation of Semen Cryopreservation in the Development of Breeding Programs of Salmonid Fish, CRYOHATCH TANGO 1/266953/NCBR/2015), where a standardized cryopreservation procedure (in terms of sperm concentration and cryoprotectant concentrations) for sex-reversed female rainbow and brook trout semen was developed. The optimal conditions for cryopreservation of semen were 3.0×10⁹ spermatozoa ml⁻¹, 7.5% methanol and 0.15 M glucose for sex-reversed females rainbow trout, or 0.19 M glucose for sex-reversed females brook trout. In this elaborated procedure, semen mixed with cryoprotectants (glucose and methanol) was loaded into 0.5 ml plastic straws (IMV Technologies, L’Aigle, France), which were placed on a floating rack and equilibrated for 15 min on ice. After equilibration, the straws were frozen 3 cm above liquid nitrogen (in the vapor of liquid nitrogen) for 5 min in a Styrofoam box with an isolating Neopor block (Minitübe GmbH, Tiefenbach, Germany) and then placed in liquid nitrogen. The straws were then thawed by immersion in a water bath at 40°C for 10 s.

The cryopreservation of semen was performed in 2017–2018 resulting in the creation of a bank of cryopreserved semen from one line of sex-reversed female brook trout and nine lines of sex-reversed rainbow trout in cooperation with the Fish Hatchery “Dąbie”. A total of 19730 straws of cryopreserved semen were collected from 153 sex-reversed females ( Table 4 ). This quantity ensured unlimited availability of semen for hatchery operations and enabled the execution of breeding crosses. Post-thaw sperm motility ranged from 4 to 77%, and it is noteworthy that different lines exhibited varying qualities of sperm motility and concentration in fresh semen, thus differing significantly in their suitability for cryopreservation. The lines designated for freezing were selected by the hatchery staff, and the cryopreserved semen is stored at the “Dąbie” Fish Hatchery.

The experience gained during the development of cryopreservation procedures for different fish species led to the realization of the project titled “Protection of Genetic Resources of Farmed and Environmentally Valuable Freshwater and Dual-Environment Fish” in cooperation with the National Inland Fisheries Research Institute in Olsztyn. Within this project, a sperm cryobank is being established for rainbow trout (both spring and autumn lines), Atlantic salmon, whitefish, sea trout, grayling, and Atlantic sturgeon farmed in the Institute’s Stations ( Table 5 ). The project commenced in 2022, and the cryopreservation of semen for each species will be conducted over the next few years. To date, approximately 1380 straws of cryopreserved semen from Salmonid fish and sturgeon are stored in this bank.

Each cryopreservation trial started with the evaluation of fresh semen characteristics, such as sperm concentration and sperm motility parameters. Additionally, a fin clip was collected from each male to assess genetic variability, which will be utilized for more effective breeder selection in the future. The primary cryopreservation protocol involves freezing semen using a glucose-methanol extender and 0.5 ml plastic straws (as indicated above). However, it should be noted that species-specific differences in cryopreservation protocols were identified. For example, the optimal glucose concentration for almost all Salmonid fish species (except sex-reversed females brook trout) was found to be 0.15 M, whereas for Atlantic sturgeon, it appears to be 0.05 M. Moreover, also differences in the optimal values of final spermatozoa concentration were determined, ranging from 1 to 2×10⁹ spermatozoa ml⁻¹ (see Table 5 ). These differences can be attributed to variable values of sperm concentration in fresh semen for each species. Under optimal conditions, post-thaw sperm motility values ranged from approximately 30% for whitefish semen to 80%, which were recorded for sea trout.