Development of Milt Cryopreservation Protocol for Economically Important Minor Carp Cirrhinus reba (Hamilton, 1822)

The carps constitute 95% of the total freshwater fish production in India (FAO, 2014) with the major carps being the choice of preference for decades. But recently the freshwater aquaculture system has started to diversify by including the medium and minor carps as well. The natural waters of the country inhabit several minor carp species that show high compatibility with the major carps and have the potential to enhance the biomass yield (Sahu et al., 2007). Studies have revealed that the incorporation of these minor carps in the conventional major carp culture has proved to be an effective strategy to enhance production with high economic return as they fetch 20-30% higher market price as compared to the IMCs (Das and Mishra, 2016). Among these minor carps, reba, has gained popularity among the fish farmers and proved to be a potential candidate species for culture and captive breeding (Ponniah and Sarkar, 2000; Ayyappan and Jena, 2001). The fish is popular for its nutritive value, as a rich source of calcium, protein and low fatty acid (Afroz and Begum, 2014) and relished for its flavour and due to low quantity of spines (Chondar, 1999). Researchers have revealed that the content of protein, carbohydrate and fat provided by C. reba is relatively higher than that of the IMCs (Khawaja, 1966; Sharma and Simlot, 1971). Induced breeding of reba was done by Chattopadhyay et al., (2013) using ovaprim @ 0.3 ml and 0.5 ml per kg body weight in males and females respectively. Thus, the introduction of this carp in the culture ponds along with major carps may turns out to be a successful step towards commercialization.
       
A technique that can be adopted to improve the gene pool of this highly- priced fish is cryopreservation which is the simplest method to preserve the genetic material at ultra- low temperature (-196°C) for a prolonged period. Cryopreservation of germplasm holds immense prospects in the aquaculture industry (Maria and Carneiro, 2012) as the hatcheries can supply seed round the year as per demand. To ensure this, milt cryobanking is considered to be one of the most established and commercialized tools. For success of cryobanking, species specific protocol is required (Diwan et al., 2010) and till date protocol for 200 fish species is reported (Diwan et al., 2020). In this background we evaluated three existing extenders, two cryoprotectants and two incubation methods for developing species specific fish milt cryopreservation protocol for aquaculture important reba carp.

Experimental animals
 
This study was approved by the Animal Ethics Committee of National Bureau of Fish Genetic Resources and was conducted in experimental farm of ICAR-NBFGR, Lucknow during July-Aug, 2020. Sexually mature brooders (100-350 g)  were selected for hormonal induction. Hormonal induction was performed via intraperitoneal injections (male: 0.2 ml/kg body weight) of commercial gonadotropin GnRH (Ovarim) to increase milt volume and decrease viscosity, as a routine method for Cyprinids. During administration of hormonal dose, fishes were anaesthetized using 2-Phenoxyethanol (0.4 ml/L) as per (Bernath et al., 2016) For hormonal induction of females, intraperitoneal injections were administered at 0.5 ml/Kg of body weight.
 
Milt collection and quality assessment
 
Live, running ripe milters (15 nos.) of reba were used for milt collection. After 4 hrs of hormonal induction, milt was collected in dry plastic boxes by applying abdominal pressure in anaesthetized condition. Sperm motility was assessed under a phase contrast microscope at 40 X.
 
Diluent preparation
 
Working extender 7 (NaCl 128 mM, KCl 0.0027 mM, CaCl₂.2H₂O 0.0014 mM and NaHCO₃ 0.0024) 7B (extender 7 with 2% egg yolk) and 9C (NaCl 111 mM, KCl 0.019 mM, CaCl₂.2H₂O 0.002 mM, MgSO₄.7H₂O 0.008 mM and Glucose 0.003 mM) were prepared and stored in chilled condition. Dimethyl sulphoxide and methanol were used as cryoprotectants. After quality assessment, non-activated sperm cells were pooled and diluted with diluent (extender with 10% cryoprotectant v/v) in dilution rate of 1:6. Cryoprotectants were added to the extender just prior to mixing with milt. French straws (Minitubes, Gmbh, Germany) of 0.5 ml capacity were filled with extended milt using manual filling system and equilibrated over ice for 10 min.
 
Freezing
 
After equilibration, they were exposed to liquid nitrogen (LN₂) vapour on a freezing stand at 3.0 cm above surface level of LN₂ in styrofoam box.  After 10 minutes of holding in liquid nitrogen vapour, the straws were plunged into liquid nitrogen (-196^oC) and stored in canisters in cryocan. 
 
Fertility trial
 
Eggs from running ripe females (4 nos.) were used for fertility trial purpose and it was collected approximately 6 hrs after hormonal induction. Eggs were stripped by applying abdominal pressure and collected individually for each female. Based on visual observation of eggs, all good quality eggs from different females were pooled and stored in covered condition. To thaw frozen milt, the straws were rapidly plunged into warm water (37°C) with vigorous shaking and time elapsed was recorded. Plastic tubs (10 L) were used for fertilization of measured volume of eggs (1 spoonful approx. 9 ml~16,320±560 eggs) and was calculated as 16,000 for all practical purposes. One spoonful egg containing 16,000 eggs were fertilized with 2 ml (4 numbers of straws of 0.5 ml capacity) cryopreserved milt (preserved for 2 weeks), mixed well with shaking and 5 ml of tap water was added to activate spermatozoa. In control, 0.3 ml fresh milt collected 4 hrs after hormonal induction was added, mixed well and activated using tap water for fertilization of eggs. Fertilized eggs were washed thrice with tap water to remove all milt debris and kept for incubation.
 
Incubation
 
For hatching, two types of incubation systems were used viz. bowl and flow through. For hatching estimation, 100-150 eggs were collected randomly and incubated in 0.5 L plastic bowl in replicates. Remaining eggs were incubated in flow-through system in replicates. Hatching estimation was done just prior to hatching. In bowl incubation system, all the eggs were counted. In flow-through incubation system, randomly 100-150 eggs were sampled three times from incubation tray and all the eggs were counted. Hatching took place after 14-16 hrs of fertilization. From the total number of eggs present in bowl/ trough at the start of the trial, numbers of hatchlings were counted to obtain the hatching percentage.  
Statistical analysis
 
Statistical tests were conducted with the IBM SPSS Statistics 26 software. The distribution of the residuals for each model was tested for normality and an arcsine transformation was performed when the data did not follow a normal distribution. The data were subjected to Levene’s test of homogeneity of variance. Levene’s test was rejected, Welch analysis of variance was applied and difference of means was compared by the Games-Howell test. Statistical significance was set at p<0.05.

Extenders and cryoprotectants play a vital role in cryopreservation and their suitability differs from one species to another (Muchlisin, 2005). The efficacy of the process of cryopreservation is highly increased if the milt suspension is diluted with a suitable diluent (extender and cryoprotectant). Extenders used for milt dilution are generally formulated to be compatible with the physico-chemical composition of the seminal fluid of the candidate species. An ideal extender reversibly inhibits sperm activation and provides suitable pH and isotonic conditions for their survival. Cryoprotectant protects the cells against the formation of intracellular ice crystals and prevents excessive dehydration (Cabrita et al., 2005).
       
The hatching percentage was used as a parameter for the comparative evaluation of different cryoprotectants and extenders. Between the two cryoprotectants used, 10% DMSO with extender 7 (44.6%) gave significantly better results compared to other combinations of cryoprotectant and extender at the post-thaw level which confirmed its suitability for sperm cryopreservation of the selected minor carp species (Table 1). The results are in accordance to previous observations reported for several freshwater species (Viveiros and Godinho, 2009). Dimethyl Sulfoxide at concentrations of 5-20% (v/v) has been used successfully for cryopreservation of several carps  (Routray et al., 2006).  While working on cryopreservation in silver carp and grass carp, Chen et al., (1992) reported efficiency of DMSO at 10-12%. Dimethyl sulfoxide (DMSO) has been found to show promising results in terms of fertility and motility rates and is considered the cryoprotectant of choice for most of the species (Suquet et al., 2000). According to Anchordoguy et al., (1987), the success achieved using DMSO is because of its low toxicity, fast rate of penetration and interaction with sperm membrane phospholipids. As compared to other potential cryoprotectants, DMSO has a higher efflux capacity and a lesser temperature-dependent process of cellular inflow making it one of the best and most preferred cryoprotectant (Huang et al., 2018). Methanol on the other hand, has been found to be more toxic with high permeation capacity (Noble, 2003). Lahnsteiner et al., (2004) and Glogowski et al., (2002) used methanol as a cryoprotectant for cryopreservation of Acipenser ruthenus and Acipenser baeri sperm respectively and observed lower motility and fertilization rate than DMSO. While working on cryopreservation of bata carp (Labeo bata), Noor et al., (2018) reported low post-thaw motility (<40%) with methanol as cryoprotectant in comparison to DMSO (70%). Our results are in accordance with this study. During fish milt cryopreservation protocol development of olive barb (Puntius sarana), Nahiduzzaman et al., (2011) reported 61% post-thaw motility with 10% DMSO as cryoprotectant. In the same study, 37% hatching was observed using cryomilt and 62 % hatching with fresh milt was observed. These results agree with our results. On the other hand, methanol was proved to be more efficient cryoprotectant than DMSO in common carp with 63 % post-thaw motility Horvath et al. (2003).


       
The comparison of results revealed the optimum extender required for successful milt cryopreservation in reba carp. Among Extenders 7, 7B and 9C, extender 7 resulted in significantly higher hatching i.e., 44.56% with DMSO as cryoprotectant (Table 1). Presence of egg-yolk in extender 7B did not exhibit any improved performance in the hatching rate.
       
Between the bowl and flow-through incubation systems, the hatching percentage was observed to be higher in the flow-through system for all combinations of extender and cryoprotectant (Table 1). Fertilized eggs of this species easily get settled at bottom and results in poor hatching, so flow-through system is recommended for proper incubation of eggs. This may be attributed to continuous circulation of water providing sufficient oxygen to the eggs along with proper removal of dead and decayed eggs.

The results from the present paper demonstrated successful sperm cryopreservation of Cirrhinus reba and concluded that extender 7 with DMSO (10% v/v) as cryoprotectant proves to be the best combination for freezing C. reba sperm with a hatching success of 44.6%. When results are expressed in comparison to control, it is nearly 75%. For commercialization of sperm cryopreservation protocol of this species, bigger cryovials should be used and bulk fertilization using cryomilt need to be tested. Cryomilt can be used for production of genetically diversified seed for commercial aquaculture of this species and it will help in species diversification.

This work was undertaken under Institute funded project “Up-scaling of fish milt cryopreservation tools for sustainability of aquaculture seed production”. Authors would like to thank Dr. Achal Singh, Principal Scientist, ICAR-NBFGR for his guidance in statistical analysis of data.

None.