Is catalase an effective additive to alleviate oxidative stress during cryopreservation of zebrafish sperm at the repository level?

Abstract

The goal of this study was to investigate whether supplementation of cryoprotective medium with catalase (CAT), an antioxidation enzyme, is efficient for zebrafish sperm cryopreservation from the viewpoint of high-throughput genetic repository operations. Three cryoprotectants (10%, v/v), dimethylacetamide (DMA), dimethylformamide (DMF), and methanol were used. The objectives were to evaluate the effects of CAT on sperm motility, plasma membrane integrity, and concentration: 1) fresh sperm at equilibration up to 60 min; 2) post-thaw sperm after cooling at 10, 20, and 40 °C/min), and 3) post-thaw fertilization and embryo survival rates. Catalase addition did not improve sperm motility, regardless of the cryoprotectants added. After 10-min exposure to DMA and methanol, membrane integrity was significantly decreased (70–75%) compared to controls. With catalase, sperm cells were maintained membrane integrity and after 50 min equilibration, cell concentrations were maintained with CAT compared to cryoprotectant-only test groups. However, after cryopreservation and thawing, CAT did not affect the outcome of motility, membrane integrity, cell concentration, fertilization, or embryo survival assays. Analysis of cooling rates also indicated that CAT did not affect 3-hpf fertilization or 24-hpf survival rates. Overall, addition of CAT could provide some protection of sperm from oxidative stress before freezing, but not after thawing. We propose that decisions concerning routine use of CAT for repositories, especially those handling tens of thousands of frozen samples per year, would depend on whether efficient high-throughput operation, or specific research questions are programmatic goals.

1. Introduction

Zebrafish (Danio rerio) is the most widely used aquatic organism in biomedical research [21]. Thousands of mutant lines have been created for gene function identification [9, 10, 26, 29], and additional lines are continuously created through efficient genome editing approaches such as CRISPR/cas9 [2]. Live maintenance of valuable lines carries the risk of accidental loss and is costly in terms of personnel effort, space, water, and power consumption. Therefore, germplasm cryopreservation coupled with repository development offer safe and cost-efficient maintenance of genetic resources [41]. To date, cryopreservation technology has been applied for research and repository purposes for zebrafish [68], and has become an efficient tool at the Zebrafish International Resource Center (ZIRC) [42] for management of genetic lines from large-scale mutagenesis studies and smaller-scale preservation of genetic lines from research laboratories [64]. To operate at high-throughput or large-scale levels, cost-benefit analyses are necessary to optimally support repository goals (e.g., thousands of samples per year with high quality management). It is necessary to establish reliable, reproducible, cost-effective, and easily trainable protocols and practices [59]. For zebrafish, the addition of catalase to sperm samples was found to alleviate oxidative stress [23]. Given that this is an additional reagent and step, we asked how a working repository could make an informed decision about the efficacy of incorporating research findings into an established cryopreservation pathway? A first step would be to evaluate function across the entire cryopreservation process from sperm collection to post-thaw fertilization. Such procedure-spanning evaluations are not typically performed in research studies but are essential for repository management decisions.

Reactive oxygen species (ROS) [31] play crucial roles for cells, ranging from beneficial to harmful, depending on their concentration, localization, and exposure duration [1, 22]. For example, the osmotic tolerance and flagellar activity of tilapia sperm (Oreochromis mossambicus) is thought to be regulated by an ROS-dependent mechanism [46]. Under normal physiological conditions, biological systems include protective antioxidants and enzymes to maintain a safe and biologically required ROS balance [4, 35, 55]. However, when exposed to stressors or pathogens, intracellular ROS can accumulate and damage nucleic acids, proteins, and fatty acids, creating a condition referred to as oxidative stress [15].

Conventional cryopreservation generates several conditions that potentially lead to oxidative stress conditions, such as dilution of sperm samples (and seminal fluid), exposure to toxic cryoprotectants, pH and osmotic changes induced by cooling to cryogenic temperatures, and heating for thawing. Significant increase of ROS production has been reported in sperm cells during cryopreservation of fish sperm [16, 30, 36, 37, 48] and molluscan bivalves [18, 19]. In these cases, the innate anti-oxidative agents of sperm cells were insufficient to counteract increased ROS levels, especially after suspension in cryopreservation extender solutions [5, 39]. In general, ROS production in sperm cells can occur at the plasma membrane by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system or in mitochondria by the NAD-dependent redox reaction [11]. The effects of increased ROS production in fish sperm cells during cryopreservation have been previously reviewed [54] and can be categorized as being involved in: lipid peroxidation [40], nucleic acid mutation and fragmentation [5] mitochondrial damage and dysfunction [16], protein oxidation [50] and sperm motility [8]. Because of these studies, exogenous antioxidants are frequently added to cryopreservation media to counter ROS generation during cryopreservation, including α-tocopherol and ascorbic acid [14, 39], amino acids and vitamins [5], or antioxidant enzymes. However, a protocol-spanning evaluation or validation does not exist between routine antioxidant supplementation of cryopreservation media and practical, i.e., cost-effective protection against ROS induced damage caused by the cryopreservation process [54].

Catalase (CAT, EC1.111.6) catalyzes the breakdown of H₂O₂ into water and oxygen, and plays a significant role controlling ROS levels in biological systems [31]. The use of CAT during sperm cryopreservation has shown improvement of post-thaw sperm quality in human [38], bull [17], boar [51], and ram [6] cells. However, meta-analysis of 23 studies indicated no significant impact of CAT (and other antioxidants) on total sperm motility during the freeze-thaw process [3].

The addition of CAT has been studied for sperm cryopreservation in six fish species (Table 1). For rainbow trout (Oncorhynchus mykiss) no effect or a moderate decrease in post-thaw sperm motility was observed [32, 34]. Similarly, decreased post-thaw motility and fertility, and increased DNA damage were detected for brook trout, Salvelinus fontinalis, sperm [34]. For sturgeon species, addition of CAT had no effect observed for post-thaw motility even though post-thaw membrane integrity improved [37].

Table 1.

Summary of the effects of catalase addition for sperm cryopreservation in various fish species (based on alphabetical ordering of the families). NE, no effect. n.a., not available.

Family/Species	Catalase (U/L)	Fresh sperm		Post-thaw sperm					Reference
		Motility	Membrane integrity	Motility	DNA damage	Membrane Integrity	Fertility	Hatching/ Survival
Acipenseridae
Acipenser baerii	100			NE	n.a.	Increased	n.a.	n.a.	[21]
	200			NE	n.a.	NE	n.a.	n.a.
	300			NE	n.a.	Increased	n.a.	n.a.
Acipenser dabryanus	25			NE	n.a.	Increased	NE	n.a.	[37]
Acipenser sinensis	25			NE	n.a.	NE	n.a.	n.a.	[37]
	50			NE	n.a.	NE	n.a.	n.a.
	100			NE	n.a.	NE	n.a.	n.a.
Cyprinidae
Danio rerio	100 (U/ml)	NE	NE	n.a	n.a.	NE	n.a.	n.a.	[23]
	200 (U/ml)	NE	NE	Improved	n.a.	NE	n.a.	n.a.
Salmonidae
Oncorhynchus mykiss	100			NE	n.a.	n.a.	NE	n.a.	[34]
	250			Decreased	n.a.	n.a.	n.a.	n.a.
Oncorhynchus mykiss	250			NE	n.a.	n.a.	NE	NE	[32]
Salvelinus fontinalis	100			NE	NE	NE	NE	n.a.	[34]
	250			Decreased	Increased	NE	Decreased	n.a.

Since the first published study [25], cryopreservation of zebrafish sperm protocols have undergone several modifications [68], such as changing the concentration of methanol as cryoprotectant [7, 42, 65, 67], or the use of dimethylformamide (DMF) [69], or dimethylacetamide (DMA) [47]. Production of ROS and DNA damage were observed after equilibration with cryoprotectants before freezing, and after thawing [53] and inclusion of skim milk, which was used as an additive in the first publication [25] about zebrafish sperm cryopreservation, resulted in moderate protection against oxidative stress [52]. Addition of CAT at 100 or 200 U/ml was found to alleviate experimentally induced oxidation by xanthine-xanthine oxidase [23]. Addition of CAT at 200 U/ml in fresh sperm showed no effects on membrane integrity and sperm motility during 10 min exposure to methanol compared to untreated controls, and CAT did not improve post-thaw motility or membrane integrity [23]. Despite these previous studies in zebrafish and other fish species, there is no evaluation that spans the entire process, from collecting, freezing, thawing and in vitro fertilization, because emphasis was placed specifically on improving cryopreservation and successful thawing at an experimental level, rather than making repository-level decisions for efficient large-scale applications.

The goal was to investigate whether supplementation of cryoprotective medium with catalase (CAT), an antioxidation enzyme, is efficient for zebrafish sperm cryopreservation from the viewpoint of high-throughput genetic repository operations. Three cryoprotectants (10%, v/v), dimethylacetamide (DMA), dimethylformamide (DMF), and methanol were used. The objectives were to evaluate the effects of CAT on sperm motility, plasma membrane integrity, and concentration: 1) fresh sperm (within 30 min after collection) at equilibration up to 60 min; 2) post-thaw sperm after cooling at 10, 20, and 40 °C/min), and 3) post-thaw fertilization and embryo survival rates. Procedure-spanning analyses such as these are essential at the repository level and could enhance the utility of research findings.

2. Materials and methods

2.1. Animals

Four-month-old zebrafish (AB wild types) were obtained from the Zebrafish International Resource Center (ZIRC) at the University of Oregon. Males were shipped to the Louisiana State University Agricultural Center, and maintained [62, 63] on a recirculating water system (Pentair Aquatic Eco-system) at 20 fish per 16-L tank at 26 °C. Fish were fed twice daily with commercial zebrafish diet (Zeigler®, Gardners, PA) and live Artemia nauplii hatched from cysts (INVE Group. Grantsville, UT, USA). The photoperiod was set at 14 h light and 10 h dark. System water quality was monitored twice a week for ammonia (< 0.01 mg/L), nitrite (< 0.01 mg/L), and nitrate levels (< 10 mg/L). Water hardness ranged between 75–200 mg/L [62, 63]. Animal handling procedures were approved and followed guidelines from the Institutional Animal Care and Use Committee of the Louisiana State University Agricultural Center and guidelines from the Institutional Animal Care and Use Committee of the University of Oregon.

2.2. Sperm collection

Live fish were selected from holding tanks and transferred to the laboratory for sperm collection within 30 min. Fish were anesthetized on ice for 30 sec, dried on a paper towel, and body length and weight were measured. Testes were removed while viewing with a dissecting microscope at 10× magnification and were weighed with tared 1.5-mL microcentrifuge tubes. Sperm cells were released by crushing testes with tweezers in Hanks’ balanced salt solution (HBSS) at an osmolality of 310 mOsmol/kg (HBSS310), and the remaining tissue was removed from the tube. Sperm suspension was used for experiments within 30 min after collection. Specific osmolality was obtained by adjusting the water volume based on the original recipe [24, 58]: 0.137 M NaCl, 5.4 mM KCl, 1.3 mM CaCl₂, 1.0 mM MgSO₄, 0.25mM Na₂HPO₄, 0.44 mM KH₂PO₄, 4.2 mM NaHCO₃, and 5.5 mM glucose, pH 7.2, and the final osmolality was confirmed with a vapor pressure osmometer (5520, Wescor, Inc., ELITech Group, Logan, UT). Samples were held on ice.

2.3. Concentration determination and adjustment

Sperm concentrations were determined by measurement of light absorption at 400 nm wavelength using a micro-spectrophotometer (NanoDrop 1000; www.thermofisher.com, Thermo Fisher) [56]. The concentration was calculated as: Y = (3×10⁸) X – 1×10⁷, where Y was the sperm concentration and X was the light absorption measurement. All samples were adjusted to 2×10⁸ cells/ml prior to experiments.

2.4. Motility estimation

Sperm samples were analyzed at 200× magnification using a dark-phase microscope (Optiphot 2, Nikon Inc., Garden City, NY, USA). To estimate motility, 1 μL of sperm suspension (1×10⁸ cells/ml after mixing with an equal volume of cryomedia for different treatments) was placed on a 20-μL reusable chamber slide (2X-CEL® disposable sperm analysis chamber, Hamilton Thorne, https://www.hamiltonthorne.com/), and 9 μL of distilled water was added to activate the sperm. A cover slide was applied before sperm motility estimation. Within 10 to 20 s after activation, 3 to 5 different fields were assessed visually. Sperm motility was expressed as the estimated percentage of cells which actively moved in a linear, forward direction, whereas sperm that vibrated in place were not included.

2.5. Flow cytometry

Plasma membrane integrity was analyzed as a characteristic of sperm quality. The LIVE/DEAD® SYBR-14/propidium iodide (PI) assay kit (Invitrogen, ThermoFisher Scientific, Eugene, OR, USA) was used following manufacturer instructions. Briefly, sperm samples were diluted to a concentration of 1 × 10⁶ cells/ml and filtered through a 20-μm nylon screen (folded and held by a pair of forceps). A 500-μl sample was stained with 100 nM SYBR-14 and 12 μM PI for 10 min in the dark and was analyzed using an Accuri C6 Flow Cytometer® (BD Biosciences, San Jose, CA, USA) equipped with 488-nm and 640-nm excitation lasers. Before sample analysis, flow cytometer performance was calibrated using fluorescent validation beads (Spherotech beads, BD Biosciences, San Jose, CA, USA) to ensure that the coefficient of variation (CV) values for the fluorescence detectors was < 3.0% (based on full peak height). Events from each sample were collected at a medium flow rate (35 μL per min) with a total event over 10,000. Sperm concentration was recorded simultaneously when collecting membrane integrity data [66].

Flow cytometry data was analyzed using the manufacturer-provided software (Cflow® version 1.0.202.1, BD Biosciences, San Jose, CA, USA). On plots of forward scatter (FSC) vs. side scatter (SSC), the sperm population was gated to exclude cell debris. Gated cells were analyzed on a scatter plot of FL1 (SYBR 14) vs. FL3 (PI) with fluorescence compensation (FL1 was compensated by FL3 with 0.1%, and FL3 was compensated by FL1 with 1.9%) to reduce spectral overlap. Sperm cells with intact plasma membranes were stained with SYBR-14, whereas cells stained with PI had damaged plasma membranes. Membrane integrity was expressed as the percentage of cells stained with SYBR-14 over the total cells stained with SYBR-14 and PI.

2.6. Experiment I: CAT protection of fresh sperm in cryoprotectants

To evaluate the effects of catalase (CAT), a final CAT concentration of 200 U/ml (Sigma, C3515) was tested (based on a previous study [23]) with 10% final concentrations of 3 cryoprotectants. Six treatment groups: 10% DMA, 10% DMF, and 10% methanol with (200 U/ml) and without CAT, and two controls: fresh sperm and fresh sperm with 200 U/ml CAT were tested. Briefly, sperm samples (100 μl) at 2×10⁸ cells/ml cells/ml were allocated equally into eight 1.5-ml microcentrifuge tubes, CAT at 400 U/ml was added into four of eight sample tubes, and finally two sperm samples with and without CAT were mixed with equal volumes (100 μl) of 20% of DMA, DMF, methanol or HBSS310 (control). At 0 (<1 min), 10, 20, 30, 40, 50, and 60 min after mixing, 5 μl of sperm sample (at 1×10⁸/ml cells/ml) from each group was sampled, diluted into 495-μl HBSS310, stained for membrane integrity estimation by flow cytometer, and for sperm motility estimation. All experimental tubes were blind coded for operators to eliminate bias. A total of 5 replicates was generated by pooling of sperm samples from 3–4 males to yield sufficient volume for each replicate (using a total of 16 fish) in this experiment.

2.7. Experiment II: CAT protection against cryoprotectants during cryopreservation

A three-factorial experiment was conducted: 1) cryoprotectants: 10% DMA, 10% DMF, 10% methanol, and no cryoprotectant (control); 2) without (control) and with CAT (at 200U/ml), and 3) cooling rates: 10°C/min, 20°C/min, and 40°C/min from 4°C to −80°C. Three replicates were produced for this experiment. To prepare each replicate, sperm from multiple males was pooled for each replicate (10–14 males for each replicate to yield sufficient volume). After collection and concentration adjustment, sperm samples were aliquoted into 24 tubes at 50 μl each. Each of these aliquots was mixed with an equal volume of pre-made double-strength cryoprotectant and CAT to yield the desired treatment levels in a total volume of 100 μl. Immediately after mixing, each of these samples was loaded into a 250-μl French straw (IMV International, Minneapolis, MN, USA). Samples in straws were cooled from 4 °C to −80 °C in a programmable freezer (Kryo 10 series II, Planer Products, Sunbury-on-Thames, UK) at the target cooling rates. Samples were removed from the freezer and were plunged into liquid nitrogen and placed into storage dewars. After storage for over one-week, post-thaw sperm motility and post-thaw membrane integrity was assessed as described above.

2.8. Experiment III: CAT effect on fertilization after freeze-thaw process

A set of frozen samples from Experiment II (24 treatments with 3 replicates) were coded and shipped to the ZIRC for thawing and fertilization trials. The performance of in vitro fertilization with thawed sperm at ZIRC followed the protocol described in previous publications [65, 67] with minor adjustment (see below). Briefly, eggs were collected from females after anesthesia in 0.01% MS-222 in fish water, blotted on a paper towel, and compressed gently along the belly with damp fingers in a sterile Petri dish. Yellowish translucent eggs were used for fertilization within 5 min after collection. Eggs from one or two females were pooled and divided evenly into two groups (> 75 eggs in each) using a fine brush. One group of eggs was used for fertilization with thawed sperm, and the other group for fertilization with fresh sperm as a control for testing of egg quality.

Cryopreserved samples were thawed at 40 °C for 5 s in a water bath and sperm from each straw were immediately released into a Petri dish with eggs. Fresh water from the fish culture system (350 μl, adjusted from 700 μl in previous publications) was added immediately to activate the gametes for fertilization. After 2 min, another 2 ml of system water was added, and the dishes were moved to a 28 °C incubator for culture. Fertilization rate was calculated as the percentage of developing embryos observed divided by the total number of viable eggs after 3 hpf (at late blastula stage). The survival rate was calculated as the percentage of visibly segmented embryos at 24–36 hpf divided by the total number of fertilized eggs.

2.9. Data collection and statistics

Microsoft Excel was used for data formatting and organization. Data analysis was performed by use of JMPpro 15 (SAS; Cary, NC). To meet the assumptions for data homogeneity, percentage data for sperm motility and membrane integrity were arcsine transformed, and sperm cell concentration data was log transformed before analysis and were tested using multi-way ANOVA. Repeated-measures ANOVA was used for analysis of the effects of duration time in Experiment I. The significance level was set at P = 0.050 for all experiments.

3. RESULTS

3.1. Experiment I: CAT protection against cryoprotectants for fresh sperm

After mixing, different cryoprotectants with or without CAT, exposure time (0 to 60 min, P < 0.001), and cryoprotectant type (control, DMA, DMF, and methanol, P < 0.001), but not the addition of CAT (P = 0.348) significantly affected fresh sperm motility. No interactions among these three factors (P ≥ 0.117) were found to affect fresh sperm motility. In general, sperm motility decreased over the 60-min period (Fig. 1). However, significant differences in motility within each of the 8 treatment groups were not observed at any time point (P ≥ 0.063).

Figure 1.

Motility of zebrafish fresh sperm after exposure to HBSS310 (control, white columns), 10% DMA (light gray columns), 10% DMF (dark gray columns), and 10% methanol (black columns) with catalase (+CAT, 200 U/ml) and without catalase. Addition of CAT did not affect sperm motility significantly (P = 0.348; three-way ANOVA).

For plasma membrane integrity, significant changes were found for cryoprotectant (P = 0.000), CAT addition (P = 0.000), and the interaction of cryoprotectant and CAT addition (P = 0.000), but not exposure time (P = 0.181) and its interactions with cryoprotectant and CAT (P ≥ 0.470) (Fig. 2A). Addition of CAT to fresh sperm without cryoprotectants did not change membrane integrity (P ≥ 0.990), and with cryoprotectants addition of CAT maintained the same membrane integrity as observed in controls (95%, P ≥ 0.940). After mixing with DMA and methanol and holding for 10 min, fresh sperm showed a significant decrease of membrane integrity (70% and 75%) compared to that of controls without cryoprotectants (95%, P ≤ 0.029). Addition of CAT did not suggest significant changes compared to controls (P ≥ 0.099) for sperm membrane integrity. At each time point, differences in membrane integrity among the test groups (control, DMA, DMF, and methanol with or without CAT addition) were analyzed and annotated (Fig. 2A).

Figure 2.

Plasma membrane integrity (A) and cell concentration (B) of zebrafish sperm over time after exposure to HBSS310 (control, white columns), 10% DMA (light gray columns), 10% DMF (dark gray columns), and 10% methanol (black columns) with catalase (+ CAT, 200 U/ml) and without catalase. Different letters above the bars indicate statistically significant differences at P < 0.050 (two-way ANOVA) among different groups at each exposure time.

Cryoprotectant, CAT addition, exposure duration, and their interactions (P ≤ 0.026, Fig. 2B) caused significant changes in sperm concentration. Even at 10-sec exposure i.e., almost immediately after mixing with DMA, DMF, or methanol (without CAT), sperm concentration decreased significantly compared to controls (P ≤ 0.0021). However, in the presence of 200 U/ml CAT, sperm concentrations did not change (P ≥ 0.4824). Over the equilibration duration from 10 to 50 min after mixing with DMA, DMF, or methanol, CAT addition yielded no differences in sperm concentration compared to controls (P ≥ 0.7030). However, in the absence of CAT, sperm concentration decreased significantly compared to controls and the test groups containing cryoprotectant and CAT (P ≤ 0.0001).

3.2. Experiment 2: No CAT protection against cryoprotectants during cryopreservation

A three-way ANOVA analysis indicated that post-thaw sperm motility was significantly affected by cryoprotectants (P = 0.000) and cooling rates (P = 0.023), but not CAT addition (P = 0.417) or interactions of the three factors (P ≥ 0.053) (Fig. 3). No motility was observed for control groups that did not include cryoprotectant, with or without CAT. DMF at 10 and 20 °C/min cooling rate showed the highest post-thaw motility, but was not significantly different from methanol or DMA at 10 °C/min. For each pairing of cryoprotectant treatment groups, addition of CAT had no significant effect (P ≥ 0.878). In general, the cooling rate of 40°C/min yielded the lowest post-thaw motility, which was significantly lower than 20°C/min (P = 0.042), but not lower than 10°C/min (P = 0.202). Overall, the post-thaw motility was low, and variance was high among replicates.

Figure 3.

Post-thaw motility of zebrafish sperm cryopreserved at 10, 20, and 40 °C/min cooling rates from 4 °C to −80 °C without cryoprotectant (control), and with 10% DMA, 10% DMF, or 10% methanol with or without catalase addition (+CAT) of 200 U/ml. Different letters above the bars indicate statistically significant differences at P < 0.050 (three-way ANOVA) among treatment groups.

The membrane integrity of post-thaw sperm was significantly affected by cryoprotectants (Fig. 4A, P = 0.000), cooling rates (P = 0.000), and interaction of cryoprotectant and cooling rate (P = 0.001), but not by CAT addition (P = 0.100) or interactions of these three factors (P ≥ 0.064). The 40°C/min cooling rate yielded the lowest post-thaw membrane integrity. However, the addition of CAT did not improve membrane integrity (P = 0.100). DMA and DMF at 10 °C/min or 20 °C/min, and methanol at 10 °C/min yielded significantly higher post-thaw membrane integrity than other groups (P ≤ 0.047).

Figure 4.

Plasma membrane integrity (A) and cell concentration (B) of post-thaw zebrafish sperm with HBSS310 only (control, white columns), 10% DMA (light gray columns), 10% DMF (dark gray columns), and 10% methanol (black columns) with (+CAT) and without catalase at cooling rates of 10, 20, and 40°C/min from 4°C to −80°C. Different letters above the bars indicate statistically significant differences at P < 0.050 (three-way ANOVA) among different groups.

As with the results for membrane integrity, cell concentration of thawed sperm showed significant differences between cryoprotectants (Fig. 4B, P = 0.000) and cooling rates (P = 0.014) and the interaction of cryoprotectant and cooling rate (P = 0.000), but not among CAT-containing and CAT-free test groups (P = 0.790) or other interactions of these three factors (P ≥ 0.246). Thawed sperm concentration of control groups without cryoprotectants was significantly lower than all other groups regardless of the presence of CAT or the cooling rate (P < 0.001).

3.3. Experiment 3: No CAT effect on fertilization after thawing

A three-way ANOVA revealed that fertilization rates at 3 h post fertilization (hpf, Fig. 5A) and survival rates at 24 hpf (Fig. 5B) were significantly affected by cooling rate (P = 0.0025, and P = 0.0094) and cryoprotectant type (P = 0.000, and P = 0.0004), but not CAT addition (P = 0.761, and P = 0.785) or the interactions of these three factors (P ≥ 0.522 and P ≥ 0.326). Cooling rates of 10 or 20 °C/min yielded significantly higher post-thaw fertilization and survival than did 40 °C/min. Without cryoprotectants (controls), post-thaw sperm yielded 0–1% fertilization and survival, while three cryoprotectants yielded similar post-thaw fertilization and survival (Fig. 5A and Fig. 5B). Cooling at 20 °C/min yielded the highest fertilization (55%) and survival (57%), and further analysis of cooling at 20 °C/min showed that CAT did not improve fertilization (P = 0.231) or survival rate (P = 0.519).

Figure 5.

In vitro fertilization of post-thaw sperm at 3 hpf (A) and survival at 24 hpf (B). Zebrafish sperm cells were cryopreserved with HBSS310 only (control, white columns), 10% DMA (light gray columns), 10% DMF (dark gray columns), and 10% methanol (black columns) with (+CAT) 200U/ml or without catalase at cooling rates of 10, 20, and 40°C/min from 4 °C to −80°C. CAT addition did not affect fertilization and survival rates (P > 0.760).

4. Discussion

This study explored the practical evaluation and subsequent decision making regarding the inclusion of supplemental reagents to existing protocols. Such decisions are essential for the functionality and efficiency of germplasm repositories and are often made using criteria different than those used to analyze research results. Specifically, we evaluated the effects of CAT addition to zebrafish sperm in combination with three commonly used cryoprotectants at processing stages of fresh sperm, after equilibration, and after thawing. A combination of sperm motility, plasma membrane integrity, sperm concentration, pre- and post-thaw, and post-thaw fertilization analyses were employed to evaluate CAT performance. This combination of parameters has not been used in other published studies and provides insights for aligning studies performed in basic research with practical repository operation.

Cryopreservation is a series of interconnected steps. Cell damage occurring at each step will accumulate and collectively can reduce post-thaw sperm viability and fertility. Therefore, it is necessary to evaluate the effects of additives such as CAT or other ROS antioxidants at steps spanning the entire procedure, as the evaluation of methanol concentration for zebrafish sperm cryopreservation [67].

Naturally occurring defense mechanisms against ROS have been grouped into four categories [28]: 1) neutralization of reactive molecules by oxidative primary and secondary enzymes such as superoxide dismutase (SOD), CAT, and glutathione peroxidase (Gpx); 2) scavenging of non-enzymatic antioxidants that capture active radicals and inhibit subsequent propagation of chain reactions (e.g., ascorbic acid, uric acid, glutathione, and lipophilic components such as α-tocopherol and ubiquinol); 3) cell repair mechanisms using polymerases, glycosylases, and nucleases to repair damage to DNA, proteins, and lipids, and 4) adaptation mechanisms mediated by cell and tissue signals.

Supplementation of antioxidants in cryoprotective solutions has been tested for sperm cryopreservation in terrestrial animals, for example, the addition of egg yolk and glutathione for bull sperm cryopreservation [60]. In fish, supplementation of antioxidants for sperm cryopreservation has been studied in 17 species since the first study in Russian sturgeon (Acipenser gueldenstaedtii) with ascorbic acid and lysine [45] and a comprehensive study in semen of species within the families Percidae, Salmonidae, Cyprinidae, and Lotidae [33]. Overall, use of these supplements produced varied effects on post-thaw sperm performance, and solid relationships were not identified between antioxidant defense and protection against the damage caused by cooling (see a comprehensive review [54]) or thawing.

Catalase is a high-molecular mass enzyme, typically located in peroxisomes and to a lesser degree in the cytosol of nearly all aerobic cells and organisms. The 200 – 340 kDa homotetramer [61] catalyzes the breakdown of hydrogen peroxide generated in many biological processes into water and oxygen without the generation of free radicals [20]. As such, the enzyme is not plasma membrane permeable, which limits its protective antioxidant function to the cell exterior when added to cryoprotective media, before cryogenic cooling. It was proposed that the inability of penetrating the cellular or mitochondrial plasma to gain access to sites of active radical formation might be one of the reasons for the universal lack of effectiveness [12]. In contrast, non-enzymatic antioxidants typically have smaller molecular mass, and are lipophilic and plasma membrane associated, or hydrophilic, cytosol associated metabolites, such as vitamins, minerals, fatty acids or amino acids [13].

Consistent with its presumed extracellular localization, addition of CAT to fresh sperm in this study showed some protection of cell plasma membranes against oxidative stress from DMA and methanol, even though this did not lead to statistically significant differences between test groups. For DMF, with or without CAT supplementation, sperm cell membrane integrity appeared similar to controls, suggesting DMF could be less damaging to cell membranes of fresh sperm. In the absence of CAT, significant decrease of fresh sperm concentration with all three cryoprotectants indicated that after exposure to these cryoprotectants sperm cells started lysing almost immediately, possibly by cell membrane damage or intracellular breakdown through other types of damage. However, addition of CAT maintained the concentration of fresh sperm after exposure to cryoprotectants, indicating that it provided protection from cellular breakdown. For sperm oxidation studies, sperm concentration has rarely been collected as a parameter [54]. Hence, comparisons are difficult, whether CAT showed similar ROS protection in other fish species for the same or other cryoprotectants.

Cooling to and heating from cryogenic storage temperature is a considerable stressor for sperm cells. Stresses includes physical damage due to ice crystal formation and heat transfer, chemical changes such as reduced pH, osmotic shock and exchange of solute across cell membranes, and biological changes, such as ROS generation. Based on the two-factor hypothesis [44], an optimal cooling rate can be empirically determined based on post-thaw survival or fertilization rates. Usually, optimal freezing rates vary depending on cell types, cryoprotectants, packaging containers and other factors. [43, 49]. Initially, an optimal cooling rate for zebrafish sperm cryopreservation was determined between 10–16 °C/min using 8% methanol as cryoprotectant [25, 65]. An optimal methanol concentration has been recently adjusted to 5% after systematic testing [42, 67]. In the present study, cooling of sperm samples was performed across a wide range of rates (10, 20, and 40 °C/min) with 10% of cryoprotectants to produce sub-optimal conditions that would encourage ROS induction within a practical scope such that effects of CAT (provided at a relatively high concentration, 200 U/ml) on post-thaw sperm samples could be clearly detected.

Addition of CAT did not produce significant differences in post-thaw performance (motility, membrane integrity, cell centration), indicating limited protective effects from CAT during the cooling-thawing process. This is consistent with the idea that its large molecular mass prevented CAT from entering sperm cells. Thus, it likely remained mainly in the extracellular environment, protecting only the outer face of the phospholipid membrane bilayer. This is further supported by the observation that anti-oxidative protection by CAT against cooling-thawing processes was not significant for fertilization rates, regardless of cryoprotectants. In a previous zebrafish study, CAT addition was found to alleviate the induced oxidant stress by xanthine-xanthine oxidase (X-XO, 270 kDa) [23], proposing CAT may serve as an effective antioxidant for sperm cryopreservation. However, the evaluation in the present study indicated that CAT only protected fresh sperm upon dilution. In combination, the results in this study suggest that the intracellular physical and chemical damage induced during cooling and thawing were sufficient to render the use of CAT (and maybe XO in [9]) ineffective when considered for the entire process.

Overall, this study highlights that the inclusion of new components in routine cryopreservation pathways should be evaluated for overall benefits. Even if the use of a substance, such as an antioxidant, offers encouraging results in an experimental setting for a particular step in a process, this does not automatically translate as beneficial for the outcome of the entire process. This is of particular importance for germplasm repositories. To effectively serve the scientific community, repositories have to operate efficiently to provide access to resources at reasonable cost. The current cryopreservation system at ZIRC yields approximately 70% post-thaw fertilization [42] virtually guaranteeing successful recovery of all preserved lines. ZIRC cryopreserved 8,256 samples in 2019 and 5,262 samples in 2020, and currently (as of June 2021) holds 698,866 samples (representing 45,944 alleles) in the repository. Without significant improvement of post-thaw outcomes, the addition of CAT would not be recommended, although it is relatively inexpensive. The addition of other antioxidants would need to be evaluated in a similar procedure-spanning manner, especially for the “bottom line” outcome – recovery of embryos from frozen sperm.

The results in this study suggest that antioxidant protection may be more complicated than simply responding to research results by adding a promising candidate to cryoprotective medium. Several intracellular and extracellular (and potentially lipophilic) antioxidants may merit evaluation alone or in combination for the ability to improve the overall post-thaw outcome. The addition of new components should also be evaluated in the context of existing components because modification of the entire process might be required to accommodate a new component. This can add significant effort and cost to Resource Center operation without guaranteed improvement of post-thaw outcomes. There are also practical considerations for an established method to accommodate new components, for example the preparation time and cost of a modified cryoprotective medium, training requirements, and unanticipated interactions (perhaps with specific genetic lines) that could increase failure rates. In addition, when post-thaw outcomes indicate a problem, troubleshooting becomes more complicated with every additional component that has been included in the procedure.

Finally, oxidative stress is known to induce DNA fragmentation and point mutations through the generation of unoccupied (abasic) sequence sites or thymine dimers which weaken and destroy DNA structure and generate mutations that can evade cellular repair mechanisms [15]. Such damage could affect the quality of preserved material significantly enough to warrant the inclusion of antioxidants. For example, a recent study indicated that skim milk powder reduced oxidative effects and DNA damage, but post-thaw motility and viability were not evaluated [52]. For zebrafish sperm, skim milk powder has been incorporated in the cryomedium from the first protocol [25] and has persisted through modifications [7, 10, 42] without a clearly defined function. However, milk powder was not included for cryopreservation of lines generated during recent large-scale mutagenic screens (with 10% DMA used as the cryoprotectant) [9, 29]. When these lines were acquired by ZIRC, considerable effort and time were expended to develop methods for their efficient recovery. It was observed that ([7]; Matthews and Varga, unpublished) reducing the overall volume and adding skim milk powder to the fertilization solution improved fertilization outcomes. Ultimately, this led to an improved thawing and in vitro fertilization protocol [7] but posed significant bottlenecks for operations to meet the subsequent demand for these lines while improving recovery. In scenarios like these, inclusion of antioxidants before freezing may be warranted for laboratories producing novel genetic mutations and may in the future contribute to standardizing the overall quality of samples. This could facilitate exchange among research laboratories and repositories, especially when samples are submitted by groups using sub-optimal conditions. However, the efficacy of any additives at the repository level needs to be tested and justified by significant improvements in post-thaw outcomes.

5. Conclusions

The incorporation of additional reagents or steps into an established cryopreservation procedure for large-scale repositories is a considerable decision in terms of cost, protocol stability, and program management. Research often presents new ingredients, devices, and applications that may be beneficial to stock center programs. However, repository-level evaluation must extend beyond testing hypotheses of mechanisms. A “beginning-to-end” experimental design is necessary to reach informed conclusions that provide efficiency at the level of the repository rather than at an individual step in a larger process. In this case, we made evaluations from sperm collection through the survival rate at 24 hpf. Because of the multiple steps involved, any additions to protocols or components can add significant costs. Therefore, in addition to the reliability of cryopreservation protocols, the design for repository-level validation should focus on simple and cost-effective solutions based on overall post-thaw sample quality and fertilization efficacy in relation to the scale of operations. For example, tens of thousands of zebrafish have been and are being processed at ZIRC for sperm cryopreservation by use of an established protocol [42]. This is in contrast to cryopreservation research, which typically is performed with experiments comprising a few dozen samples (and even fewer fish), an approach that can lack relevance to the scale of throughput at a stock center repository.

Based on the evaluation of CAT in this study, it may not be efficient for repositories to modify zebrafish sperm cryopreservation media under the studied conditions, even though we observed partial protection of fresh sperm against cryoprotectants, including methanol [42], and protection of cells from lysis. These features, if presented as typical research results, would likely be recommended as useful additions to cryopreservation protocols. Going forward it would be extremely useful to seek a more practical integration of research and repository level activities for aquatic species. In this case, we provide an example where additives to an established cryopreservation procedure require evaluation across the entire process for an effective cost-benefit analysis. This is essential for decision-making on the repository management level when dealing with large numbers of samples, especially for samples contributed by the research community. Other mechanisms are being addressed to standardize cryopreservation procedures and outcomes across user communities by custom development of open scientific hardware [27, 57]. Incorporation of new avenues such as these to repository development emphasizes the need to develop mechanisms to integrate research findings at the repository level through community-level approaches.