Evaluation of different sugar extenders and large volume cryotube for sperm cryopreservation of brown-marbled grouper (Epinephelus fuscoguttatus)
Abstract
Brown-marbled grouper (Epinephelus fuscoguttatus) is a large-bodied coral reef fish with high ecological and economic value in South-East Asia. Previous studies have developed cryopreservation protocols in 0.25- or 0.5-ml straws, but this small volume may limit the utilization of cryopreserved sperm for large-scale artificial fertilization, considering the high fecundity of brown-marbled grouper. To aid its commercial application, the main objective of the present study was to further elaborate cryopreservation protocol in this species, by evaluating the cryoprotective efficacy of different sugar extenders (glucose, sucrose or trehalose) and the feasibility of a 5-ml cryotube for cryopreservation. After frozen in a simple media containing 10% DMSO and 0.3-M sugar extender using a 0.5-ml straw, there was a deterioration in sperm motility, viability, mitochondrial membrane potential (MMP) and ATP content compared with fresh semen, but fertilization success of cryopreserved samples was not compromised at the ratio of 10,000 spermatozoa per egg. Besides, the type of sugar extender had no obvious effect on post-thaw sperm quality and fertility, thus it is suggested that glucose, sucrose or trehalose are fully interchangeable for the cryopreservation of brown-marbled grouper semen. By employing a faster freezing rate (freezing height: 2 cm vs. 3 cm) and longer thawing time (40°C, 90 s vs. 40°C, 7 s), samples cryopreserved in a 5-ml cryotube exhibited similar post-thaw motility parameters and fertility with those in 0.5-ml straw. Overall, our results demonstrate the effectiveness of new sugar extenders and the applicability of a 5-ml cryotube for sperm cryopreservation in brown-marbled grouper, which would allow for better flexibility for large-scale artificial fertilization in hatchery practice.
1 INTRODUCTION
Brown-marbled grouper (Epinephelus fuscoguttatus), also known as tiger grouper, is a large-bodied coral reef fish that is widely distributed within the Indo-Pacific (Rhodes et al., 2012). Due to its excellent flesh quality, rapid growth rate and hardiness in captivity, this species is not only of high market value in the live reef fish trade but also serves as one of the most promising mariculture grouper species, especially in South-East Asia (Chieng et al., 2020; Shapawi et al., 2019). As a result of overfishing and environmental degradation, the abundance of wild stocks has declined significantly over the past few decades, which is now classified as ‘Vulnerable’ using the IUCN Red List (Rhodes et al., 2018). The unprecedented increasing consumer demand has greatly encouraged grouper aquaculture in Asian countries; it is estimated by FAO that about 155,000 tonnes of groupers valued at USD 630 million were produced in 2015 (Rimmer & Glamuzina, 2019). It is presumed that aquaculture could lessen the pressure on wild resources of groupers and the coral reef ecosystem. Constrained by a limited number of broodstock, late sexual maturation, asynchronous spawning of gametes and other related problems, however, an adequate supply of high-quality seed is gradually becoming one of the main issues surrounding the sustainable development of grouper farming (Shapawi et al., 2019).
Numerous research regarding sperm cryopreservation of grouper species has been carried out; this biotechnology has proven to be with great application potential in the fields of assisted reproduction, selective breeding and biodiversity conservation (reviewed by Magnotti et al., 2018; Che-Zulkifli et al., 2020). A suitable extender is arguably an important chemical component required for a successful cryopreservation protocol. Sugar extenders are known to protect the cell from injury during the freezing–thawing process (Ahmad & Aksoy, 2012).
Recently, we have demonstrated that a simple cryopreservation media containing 0.3-M glucose secures high post-thaw sperm quality in 0.5-ml straw samples of brown-marbled grouper (Yang, Fan, et al., 2020). Sucrose and trehalose are also effective for fish sperm cryopreservation (Golshahi et al., 2018; Nynca et al., 2016; Sarosiek et al., 2016; Sarvi et al., 2006), but their efficiency has not been investigated in brown-marbled grouper. In addition, according to our knowledge, large volume sperm cryopreservation in brown-marbled grouper has not been conducted yet. This method has been successfully developed in several fish species, which provides the advantage of improving the packaging efficiency and lowers the costs for sample storage, as well as facilitates the handling of gametes for artificial insemination (Horváth et al., 2010; Maria et al., 2015; Molnár et al., 2020; Nomura et al., 2018).
Therefore, the aim of this study was to (1) compare the cryoprotective efficacy of different sugar extenders (glucose, sucrose and trehalose) and (2) test the feasibility of using 5-ml cryotube for sperm cryopreservation of brown-marbled grouper.
2 MATERIALS AND METHODS
2.1 Ethics statement
All animal handling and procedures were conducted in accordance with guidelines from the Institutional Animal Care and Use Committee of Sun Yat-sen University.
2.2 Broodstock and gametes sampling
Experimental fish used in this study were provided by the Hainan Chenhai Seawater Products (Dongfang, Hainan Province, China). During the experimental period (2019.5–2019.7; 2021.3–2021.6), sexually mature males and females (3–6-year old, mean weight 6.5 ± 3.1 kg, mean total length 52.8 ± 13.7 cm) were raised separately in indoor flow-through cement tanks (5 × 3 × 1.6 m) under a natural photoperiod; they were fed to satiation every 2 days with frozen fish. The water quality was maintained with a dissolved oxygen concentration of 4.8 ± 0.6 mg/L, pH 7.5 ± 0.3, salinity 30.1 ± 1.7‰ and temperature 28.0 ± 1.2°C.
For gametes collection, fish were anaesthetised by MS-222 at a dose of 150 mg/L. Next, the genital area was dried and gametes were sampled by gentle abdominal pressure. Special care was taken to avoid contamination with blood, excrement or mucus. Semen was collected into 10-ml tubes and then maintained at 4°C. The eggs were stripped into dry plastic beakers and stored at room temperature for no more than 1 h before artificial insemination, as longer post-stripping storage would compromise egg fertilizing capacity in brown-marbled grouper (Yang, Huang, et al., 2020).
2.3 Experiment 1. Effect of cryopreservation with different sugar extenders on sperm quality and fertilization ability
2.3.1 Experimental design
Before freezing, semen was diluted at a ratio of 1:3 by different sugar extenders (0.3-M glucose, 0.3-M sucrose or 0.3-M trehalose) with 10% DMSO (final concentration, v/v) as cryoprotectant. The suspension was loaded into 0.5-ml straws ((IMV Technologies, L'Aigle, France) and equilibrated at 4°C for 10 min. Afterwards, the straws were frozen in a covered Styrofoam box (inside dimension: 36 × 28 × 25 cm; liquid nitrogen depth: 5 cm) for 10 min at 3 cm above the surface of liquid nitrogen, as described previously (Yang, Fan, et al., 2020). After storage for 7 weeks, the straws were removed from liquid nitrogen and thawed in a water bath at 40°C for 7 s for further assessment.
2.3.2 Evaluation of sperm quality
2.3.2.1 Sperm motility and concentration
Motility parameters were examined with an Integrated Semen Analysis Systems (ISAS 2.0, Spain) as described earlier (Yang et al., 2021). Briefly, the semen sample was activated with filtered seawater (supplemented with 0.1% bovine serum albumin, w/v) in a 1.8-mL Eppendorf tube at a dilution of 1:200. After rapid mixing, 10 μl of the mixture was deposited in the 10 mm-depth Sperm Counting chamber (ISAS attached) for motility analysis under a ×10 negative-phase objective. At least 500 cells from 3 to 5 different fields were evaluated for each sample. Spermatozoa with a curvilinear velocity <10 μm/s were considered immotile. Several sperm kinetic parameters including total motility (TM, %), progressive motility (PM, %), curvilinear velocity (VCL, μm/s), straight-line velocity (VSL, μm/s) and average path velocity (VAP, μm/s) were chosen for further analysis. Fresh semen samples characterized by initial TM higher than 90% were selected for this and the subsequent experiment.
Sperm concentration was estimated by the haemocytometer method after diluted 1000 times with 1% NaCl.
2.3.2.2 Sperm viability and mitochondrial function
The percentage of viable sperm was assessed using the Live/Dead Sperm Viability Kit (L7011; Thermo Fisher Scientific, Waltham, USA) (SYBR-14/PI dye). For this purpose, about 2 × 105 spermatozoa that resuspended in 100-μl PBS were mixed with 1-μl SYBR-14 and 1-μl PI. After 10 min incubation at 4°C in the dark, another 400-μl PBS was added for flow cytometer analysis. Viability was determined as the proportion of SYBR-14 positive cells.
To evaluate mitochondrial activity, changes in mitochondrial membrane potential (MMP) were determined using JC-1 dye (JC-1 Assay Kit; Beyotime, Shanghai, China). Briefly, a 4-μl semen sample was washed by PBS and centrifuged at 800 g for 5 min. The sperm pellet was resuspended in 500-μl PBS and mixed with 500-μl JC-1 staining working solution (10 μg/ml), incubated at room temperature for 20 min. After which, the cell suspension was washed twice by the washing buffer and resuspended in 1-ml PBS and evaluated using a flow cytometer. Red-stained (JC-1 aggregates) cells were recorded as cell population with high MMP.
Samples were measured in a CytoFlex flow cytometer (Beckman Coulter, USA) equipped with a 488-nm argon laser. Green and red fluorescence were measured in the FITC channel (525/40 nm) and ECD channel (610/20 nm) respectively. At least 10,000 sperm-specific events from each sample were counted at a flow rate of approximately 400 cells/s. Acquisition of data was performed with CytExpert software (Beckman Coulter, USA).
2.3.2.3 ATP bioluminescence assay
ATP content in the sperm was assessed using the ATP Bioluminescence Assay Kit (Beyotime, Shanghai, China) according to the manufacturer's protocol. In brief, the spermatozoa pellet centrifuged from 50-μl semen sample was fully lysed by 200-μl lysis buffer; the media was subsequently centrifuged at 12,000 g for 5 min at 4°C. After mixing 20-μl supernatant with 100-μl ATP detection working solution, the fluorescence intensity was read using a multipurpose microplate reader (Thermo Fisher, USA). ATP content was calculated from a standard curve and expressed as nM/109 cells.
2.3.2.4 Fertilization
For artificial fertilization, eggs from three females were divided into batches of approximately 300 eggs in a plastic Petri dish. Next, an accurate volume of semen (1 × 104 spermatozoa/egg) was dripped over the eggs, following which 5-ml hatchery seawater was added to activate the gametes. After 10 min, eggs were rinsed twice and transferred to flow-through incubators at room temperature for further development. Five to seven hours post fertilization, 100–120 eggs per replicate were randomly sampled and observed to determine the fertilization rate. Hatched larvae were counted after 26 h of incubation and the hatching rate was calculated based on the initial number of eggs.
2.4 Experiment 2. The applicability of 5-ml cryotube for sperm cryopreservation
In this experiment, freezing device, freezing medium, dilution ratio and equilibration time were the same as described above. The freezing condition for diluted semen loaded into 5-ml cryotubes (Corning, USA) was 2 cm above the surface of liquid nitrogen for 15 min. One month later, the cryotubes were thawed at 40 °C for 90 s to test sperm motility and fertility as described in Section 2.2. A subsample (with glucose as extender) cryopreserved in 0.5-ml straws at 3 cm height from liquid nitrogen for 10 min was served as control.
2.5 Data analysis
Results were expressed as the mean ± SD. Percentage data were normalized by arcsine transformation prior to analysis. Differences in these values between treatments were analysed by one-way ANOVA, followed by Tukey's multiple comparison test. All statistical analyses were performed at a significance level of 0.05 using SPSS 20.0 software (SPSS, Chicago, IL, USA).
3 RESULTS
The sperm density of fresh semen in this study was ranging from 6.8 to 11.4 × 109 cells/ml; the sperm volume ranged from 3.4 to 7.2 ml individually.
3.1 Comparative cryoprotective efficacy of different sugar extenders
The effect of different sugar extenders on cryopreserved sperm quality in brown-marbled grouper is presented in Table 1. In comparison to fresh semen (TM: 95.1 ± 3.3%, PM: 32.1 ± 3.8%, VCL: 150.1 ± 4.6 μm/s, VSL: 69.7 ± 3.0 μm/s, VAP: 99.3 ± 4.7 μm/s), cryopreservation caused a significant decrease in motility parameters for all the tested extenders. With regard to viability, MMP and ATP content, a significantly lower value was also recorded in cryopreserved sperm as compared with fresh semen (Table 2). The type of sugar contained in the extender had no significant effect on sperm quality parameters in cryopreserved samples (Tables 1 and 2).
| Treatment | TM (%) | PM (%) | VCL (μm/s) | VSL (μm/s) | VAP (μm/s) |
|---|---|---|---|---|---|
| Control | 95.1 ± 3.3a | 32.1 ± 3.8a | 150.1 ± 4.6a | 69.7 ± 3.0a | 99.3 ± 4.7a |
| Glucose | 86.5 ± 2.6b | 24.8 ± 3.7b | 129.3 ± 11.3b | 49.5 ± 4.7b | 71.6 ± 6.9b |
| Trehalose | 85.7 ± 3.8b | 24.7 ± 1.5b | 121.8 ± 10.9b | 48.5 ± 8.0b | 70.0 ± 10.6b |
| Sucrose | 86.7 ± 2.5b | 26.9 ± 4.1b | 124.7 ± 11.8b | 49.1 ± 8.2b | 71.2 ± 9.8b |
- Note: Different superscripts within the same columns indicate significant differences (p < 0.05).
- Abbreviations: PM, progressive motility; TM, total motility; VAP, average path velocity; VCL, curvilinear velocity; VSL, straight-line velocity.
| Treatment | Viability (%) | High MMP (%) | ATP (nM/109 cells) | Fertilization (%) | Hatching (%) |
|---|---|---|---|---|---|
| Fresh | 92.3 ± 1.7a | 96.2 ± 4.3a | 158.2 ± 4.2a | 96.2 ± 1.1a | 93.3 ± 2.6a |
| Glucose | 83.4 ± 4.1b | 89.4 ± 6.3b | 107.0 ± 5.1b | 93.7 ± 1.2a | 87.4 ± 2.4a |
| Trehalose | 85.9 ± 2.6b | 88.3 ± 8.7b | 110.4 ± 7.1b | 93.1 ± 2.7a | 87.9 ± 3.2a |
| Sucrose | 84.3 ± 2.3b | 89.4 ± 5.7b | 103.8 ± 4.3bc | 93.5 ± 2.2a | 91.2 ± 2.1a |
- Note: The ratio for fertilization was 10,000 spermatozoa per egg. Different superscripts within the same columns indicate significant differences (p < 0.05).
Reproductive success of brown-marbled grouper showed a similar level (no statistical difference) in fresh and cryopreserved samples (Table 2) when 10,000 spermatozoa per egg were used for fertilization. The fertilization and hatching rate in these samples were ranged from 93.1% to 96.2% and 87.4% to 93.3% respectively.
3.2 The applicability of 5-ml cryotube for sperm cryopreservation
Compared with 0.5-ml straw, no significant difference was detected in either sperm kinematic parameters or fertilization capacity between different sugar extenders when the frozen volume was scaled up to 5 ml (Table 3), suggesting that 5-ml cryotube could be adopted for sperm cryopreservation of brown-marbled grouper.
| Treatment | TM (%) | PM (%) | VCL (μm/s) | VSL (μm/s) | VAP (μm/s) | Fertilization (%) | Hatching (%) |
|---|---|---|---|---|---|---|---|
| Control | 87.1 ± 4.1 | 25.7 ± 2.1 | 121.1 ± 6.6 | 54.1 ± 7.7 | 76.1 ± 10.8 | 95.7 ± 3.4 | 86.9 ± 4.3 |
| Glucose | 86.2 ± 4.0 | 26.3 ± 3.6 | 120.0 ± 5.8 | 48.7 ± 10.5 | 68.7 ± 13.1 | 93.9 ± 2.7 | 83.9 ± 3.6 |
| Trehalose | 86.9 ± 2.9 | 25.8 ± 6.3 | 118.7 ± 10.3 | 50.4 ± 4.8 | 70.3 ± 7.6 | 94.8 ± 3.0 | 83.5 ± 3.2 |
| Sucrose | 84.5 ± 3.8 | 27.3 ± 4.1 | 119.8 ± 9.2 | 50.1 ± 6.7 | 72.6 ± 8.9 | 94.7 ± 3.2 | 83.9 ± 2.8 |
- Note: The fertilization test was conducted at the ratio of 10, 000 spermatozoa per egg. No significant difference was detected between groups (P > 0.05).
- Abbreviations: PM, progressive motility; TM, total motility; VAP, average path velocity; VCL, curvilinear velocity; VSL, straight-line velocity.
4 DISCUSSION
Compared with fresh semen, the results of the presented study showed that cryopreservation had a notable effect on the sperm physiology of brown-marbled grouper, including motility, plasma membrane integrity, mitochondrial membrane potential and ATP content. Correspondingly, many authors also reported reduced sperm quality after thawing in grouper species (Ahn et al., 2018; Cabrita et al., 2009; Fan et al., 2014; He et al., 2011; Peatpisut & Bart, 2010). This may be caused by the cold shock, osmotic stress, ice crystallization and cryoprotectant toxicity during the process of freezing and thawing, thus leading to an inevitable impairment of cell function (Chao & Liao, 2001). Although with alterations in sperm physiology after thawing, it seems that the sugar extenders we tested combined with DMSO are effective for freezing brown-marbled grouper sperm, the fertilizing capability of frozen sperm is comparable to that of fresh semen when a sperm: egg ratio of 10,000 was used, implying this ratio is enough to ensure effective fertilization. Indeed, the beneficial effect that sugars have on sperm freezing has been reported in several grouper species. In agreement with this study, sperm cryopreservation with trehalose has achieved a good fertilization rate (>60%) in kelp grouper (E. moara) and orange-spotted grouper (E. coioides) (Miyaki et al., 2005; Peatpisut & Bart, 2010). Sugars like glucose and sucrose have also been reported to improve the post-thaw sperm motility of E. akaara (Ahn et al., 2018). According to Yusoff et al. (2018); brown-marbled grouper semen could also be successfully cryopreserved when using 15% PG and 85% FBS as cryomedium. This inconsistency is probably owing to the different combinations of cryoprotectant and extender, which may interact with each other as shown in sperm cryopreservation of Atlantic cod (Gadus morhua L.) (Butts et al., 2010). Compared with FBS, sugar extender is definite in composition and easier to store, making it more suitable for standardized and commercial-scale cryobanking.
As shown in Tables 1 and 2, our results confirmed that employing different types of sugar extender has no significant effect on the subsequent cryoprotective efficiency; thus, sucrose and trehalose can be interchangeable with glucose for sperm cryopreservation in brown-marbled grouper. But for commercial-scale cryopreservation, glucose and sucrose may be better choices as the prices of which are significantly lower than that of trehalose (http://www.sigmaaldrich.com, accessed on May 30, 2021). Simple sugar-based extenders are found to be as effective as or even better than saline solutions for sperm cryopreservation in several fish species, for example, African catfish, common carp and Arctic char (Horváth et al., 2003; Mansour et al., 2006; Urbanyi et al., 1999). Like saline solutions, sugar-based extender could maintain the osmotic pressure and immotile spermatozoa before freezing. In the meanwhile, the success of sugars in the cryomedium can be further explained by their role in stabilizing membrane bilayers and decreasing intracellular ice formation (Tsai et al., 2018).
Although the addition of sugars to the freezing medium has produced positive results in a wide range of aquatic species, it is worth noting that not all sugar is beneficial in certain cases. Similar to our results, previous research regarding salmonidae species did not find a distinct effect of various sugars (glucose, sucrose or trehalose) on cryopreserved semen from brown trout, brook trout and sex-reversed rainbow trout (Nynca et al., 2016). Conversely, Golshahi et al. (2018) examined the effect of different disaccharides (maltose, lactose, trehalose and lactulose) on sperm cryopreservation in Persian sturgeon and Beluga sturgeon, amongst which lactulose was less effective for the preservation of post-thaw motility in both species. This evidence showed that the cryoprotective effect of various types of sugars is variable and highly species specific. It is also observed that its suitability in semen extenders could differ according to the molecular weight, composition or structure of the sugar and the type of buffer used (An et al., 2000; Fernández-Santos et al., 2007). Moreover, the beneficial effect of sugar could be influenced in a dose-dependent manner; sugar concentration out of the range of optimal concentration may produce a dramatic decrease in post-thaw sperm quality, such as the case in rainbow trout and Siberian sturgeon (Judycka et al., 2015, 2020). It is, therefore, recommended that the type and concentration of sugar for cryopreservation need to be adjusted according to the species.
Straws with a volume of 0.25 or 0.5 ml have been successfully developed for sperm cryopreservation in brown-marbled grouper (Yang, Fan, et al., 2020; Yusoff et al., 2018). Considering that brown-marbled grouper is characterized by large body and broodstocks with good maturity have high fecundity, small straws may have limitations for large-scale artificial insemination in practice; it would be labour and time consuming for sperm packaging and thawing since numerous straws have to be handled at the same time. Although sperm freezing with large volume has many advantages in practice, its potential for use may be limited which can cause longer freezing point plateau and less uniform cooling rate in the container, resulting in compromised post-thaw sperm quality (Bwanga et al., 1991). As both types of container and frozen volume can influence the heat exchange efficiency, it is necessary to adjust the freezing method when scaling up the storage volume (Nomura et al., 2018). In this study, cryopreservation of 5 ml volume yielded similar results as those in 0.5-ml straw by adopting lower freezing height (2 cm vs. 3 cm), demonstrating that the protocol we adopted here is acceptable for large volume cryopreservation without compromising the post-thaw sperm quality. This is consistent with results from yamú (Brycon amazonicus), sperm cryopreserved in 1.8-, 2.5- and 4.0-ml straws had similar fertility to 0.5-ml straws when using a fast freezing rate (Velasco-Santamaría et al., 2006). It has also been reported in other fish species that the potential negative effect of semen cryopreservation in large volume could be reduced using higher cooling rate (Ding et al., 2011; Liu et al., 2006; Molnár et al., 2020). Besides, the success of large volume semen cryopreservation in brown-marbled grouper may also have a relationship with its high freezing tolerance; our previous research found that sperm frozen ranged from 1 to 5 cm above the liquid nitrogen surface produced high post-thaw motility (Yang, Fan, et al., 2020), which makes it has an advantage in large volume freezing over species with a narrow optimal sperm freezing rate.
In conclusion, our results indicate that semen from brown-marbled grouper can be cryopreserved efficiently by a simple media consisting of 10% DMSO and 0.3-M glucose, sucrose or trehalose; the type of sugar has no significant effect on the cryoprotective efficiency. Moreover, results obtained in this study demonstrated that 5-ml cryotube can be successfully used for brown-marbled grouper semen cryopreservation, fertility and post-thaw sperm motility which were similar to those in 0.5-ml straw, indicating its great potential in artificial fertilization. More work still needs to be done, however, to make these results more applicable in commercial fingerling production, such as the utility of larger container or lower dilution ratio, fertilization with large egg batch and assessment of the growth and development of larvae produced from cryopreserved sperm.
ACKNOWLEDGEMENTS
This study was supported by the National Natural Science Foundation of China (31872572); Agriculture Research System of China (ARS-47); Program of the China-ASEAN Maritime Cooperation Fund of the Chinese Government (42000-41170002); Program for scientific research start-up funds of Guangdong Ocean University.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
AUTHOR CONTRIBUTIONS
Sen Yang: Conceptualization, Methodology, Writing – original draft. Bin Fan: Formal analysis. Xinghan Chen: Data curation. Zining Meng: Writing – review & editing, Funding acquisition.
Open Research
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.
