Reproductive Techniques in Aquatic Animals

### **Chapter 5**

## Cryopreservation and Its Application in Aquaculture

*Judith Betsy C, Siva C and Stephen Sampath Kumar J*

### **Abstract**

Aquaculture is the major aquatic animal production segment. Problems like inbreeding depression, genetic drift, introgressive hybridization, etc. have been influencing the production of quality seeds negatively. Cryopreservation serves as a way-out for these problems and a possible answer to produce quality seeds and genetically improved varieties. It has been considered as a major strategy for conservation of fish genetic resources. Cryopreservation of fish gametes has been in vogue since 1953 and the technology is well studied and validated for many species. So far the milt of 200 fish species has been cryopreserved successfully. In this chapter, the importance of aquaculture in overcoming malnutrition, genetic issues affecting quality seed production, cryopreservation protocol employed for various fish species, problems faced in cryopreserving fish eggs and embryos and future of cryopreservation in aquaculture have been discussed.

**Keywords:** Cryopreservation, aquaculture, fish, spermatozoa, egg, inbreeding

### **1. Introduction**

In the World Summit on Food Security held at Rome during 16–18 November 2009, it was committed to eradicate hunger from earth by increasing investment in agriculture involving public and private enterprises. Food and Agriculture Organization (FAO) defined food insecurity as a situation that exists when people lack secure access to sufficient amounts of safe and nutritious food for normal growth and development and an active and healthy life.

The projections of FAO for the next 15 years indicate that, if agricultural innovation continues at a reasonable rate, food production can increase by 2 percent per year in the developing countries. Another report by World Bank mentioned that the world needs to produce at least 50% more food to feed 9 billion people by 2050. If the natural resources offer good potential for agricultural development, supporting agriculture research can bring big benefits in reducing food insecurity and malnutrition [1]. In solving the above issues, along with grains and vegetables, fish can also play a major role.

Fish and other aquatic products provide at least 20% of protein intake for one third of the world's population and the dependence on fish is high in developing countries [1]. Small-scale fisheries are considered to be more important for food security

because they supply more than half of the protein and minerals for over 400 million people in the food deficit countries of Africa and South Asia [2].

Fish is not an energy food, but it is an essential food for the human being. It is an extremely important source of protein, minerals and oils in many under developed countries. Fish protein constitutes around 30% of the Micronesian diet and 15% of the Polynesian diet [2]. Fish is more nutritious than other staple foods, providing quality animal protein, essential fatty acids and micronutrients. Interventions related to fish intake and aquaculture production include, utilizing fish as complementary food to improve nutritional status of children, encouraging children and women to eat nutrient-dense fish through nutrition education at community level [3] and increasing production of more demanded fish species through effective dissemination of the technology [4].

Fish being an important food for mankind, its production through all possible means has been explored. Besides exploitation of natural resources like sea and natural water impoundments, culture production through structured methods deserve due attention now. This shift in the population in the natural waters has impacted the availability of fish through capture and driving the people to develop ways to produce the fish through aquaculture. Nevertheless, the impacts of climate change on aquaculture also could not be pushed off.

Studies in Asia suggest that low-income households consume lesser quantities of fish than rich households [5], but they still depend on fish as a major source of animal protein [6]. This invariably suggests that fish supply should be sufficient to wade off the malnutrition from the low-income group of people in order to keep the life free from nutritional disorders.

It is at this point anthropologists in the world insist that apart from concentrating on improving agriculture production, agencies like FAO and World Bank must establish projects in aquaculture of species that are positively impacted by climate change and at the same time contains high nutrients to eradicate malnutrition. Some of the fishes rich in nutrition include carps, catfish, murrels, tilapia and prawn [7].

Globally, fish provides 20% of average per capita intake of animal proteins to more than 3.3 billion people. In some countries like Bangladesh, Cambodia, the Gambia, Ghana and Indonesia, fish contribute to 50% or more of animal protein. The global fish consumption per capita food grew from 9.0 kg (live weight equivalent) in 1961 to 20.5 kg in 2018 which is about 1.5% increase per year [8].

The total world fisheries and aquaculture production has reached 179 million tonnes in 2018 which was recorded as highest of all times and estimated at USD 401 billion [8]. The aquaculture sector was the main driver that led to the increase in production of aquatic animals, and the total aquaculture production was estimated to be 82.1 million tonnes valued at USD 250 billion with average growth of 5.3% per year. The contribution of world aquaculture to global fish production has increased from 25.7% in 2000 to 46% in 2018.

### **2. Genetic issues affecting seed quality**

Aquaculture in many countries depends on the adequate supply of quality seeds. However, it is not always possible since many hatcheries have reported stock deterioration due to poor brood stock management, inbreeding depression, genetic drift, introgressive hybridization, unconscious selection, lack of effective population size (Ne) and genetic erosion of domesticated stock.

### **2.1 Prevention of detrimental effect of inbreeding and genetic drift via cryopreservation**

Inbreeding and genetic drift cause undesired changes in the genome and result in lower viability and growth and increase developmental instability in fishes [9]. Unplanned and uncontrolled breeding often lead to inbreeding depression which lead to decreased growth rate, low fecundity and poor survival which are usually accompanied by loss of alleles via genetic drift [10]. Uncontrolled inbreeding and genetic drift occur together in closed hatchery populations and these factors are determined by the population's Ne. Hence maintaining the desired Ne will prevent adverse effects on productivity and profits [10].

Due to high fecundity in fishes, inbreeding is more prevalent in aquaculture than other domesticated animals. This applies especially to highly fecund species like Indian Major Carps (IMC) (catla, *Catla catla,* rohu, *Labeo rohita,* mrigal, *Cirrhinus mrigala*) and Chinese carps (silver carp, *Hypophthalmicthys molitrix*, grass carp, *Ctenopharyngodon idella*, common carp, *Cyprinus carpio*) where few broodstock are necessary to meet demands for fry and broodstock replacement. The detrimental effects of inbreeding are well documented and can result in 30% or more decrease in growth, survival and reproduction [11].

The problem of inbreeding and genetic drift can be reduced by spawning more fish than needed. Since the fecundity is high in some species, the required number of fingerlings can be produced by breeding one or two females and males. But the ability to spawn relatively few fish must be moderated if inbreeding and genetic drift are to be controlled. Another way to increase Ne and reduce the rate of inbreeding and genetic drift is to spawn a more equal sex ratio. Most farmers and hatchery managers use skewed sex ratios when they spawn their fish. This is done because one male can usually be used to fertilize eggs from several females. This enables farmers to use and maintain fewer males, which lowers production costs. When a skewed sex ratio is used, the rarer sex has a disproportionate influence on the size of Ne. Cryopreservation can help in maintaining Ne by breeding more number of fish which leads to maintenance of fewer males in the hatchery [10].

### **2.2 Introgressive hybridization with cryopreservation**

It is the gradual infiltration of the germplasm of one species into that of another as a consequence of hybridization and repeated backcrossing [12]. Sarder et al. [13] reported that pure mrigal are severely being threatened by introgressive hybridization in Bangladesh. Moreover, unplanned hybridization, inbreeding depression and genetic drifts have been the causes of deteriorating quality of this species. They opined that cryopreservation is the simplest and most inexpensive method to preserve genomes that can be used to maintain future conservation options. Introgression of autochthonous populations with introduced ones is a common phenomenon in salmonids and it can result in outbreeding depression and replacement of possibly locally adapted populations by allochthonous ones [14].

Horvath et al. [15] applied cryopreservation as a conservation effort of two salmonid species such as the marble trout (*Salmo marmoratus*) and the Adriatic lineage of the grayling (*Thymallus thymallus*) autochthonous to the drainage of the Soča river in Slovenia. Populations of these species were greatly affected by hybridization and introgression with allochthonous species such as the brown trout (*Salmo trutta m. fario*) and the Danubian lineage of the grayling that were introduced to the

Soča drainage during the 20th century. Cryopreservation of sperm from the Adriatic grayling and the marble trout has constituted an integral part of the conservation activities. In case of the grayling, no pure population was available and hence the proportion of Adriatic genotype in the broodstock was increased. Genetic analyses of the populations were also conducted. Sperm and fin clips were collected from wild males on the spawning grounds. Sperm was cryopreserved and stored until the genetic analysis was completed on each sample. Cryopreserved sperm of individuals containing higher than a pre-defined proportion of Adriatic genotype was thawed and used for fertilization of eggs from Adriatic females. The resulting progeny was developed as broodstock and 70–80% of the local grayling broodstock originated from cryopreserved sperm. In case of the marble trout, cryopreservation was used to create "sanctuary" streams. Sperm is collected from wild males of a given pure population prior to the spawning season (early November) and cryopreserved. Sperm is stored in liquid nitrogen (LN2) until the spawning season (December–January) and then eggs of females from the identical population are fertilized with the cryopreserved sperm. Eyed eggs are then stocked into artificially created nests in the prepared "sanctuary" stream. Thus, a high number of males of the given pure population participate in the creation of the new population.

From a management perspective, the desirability of introgressive hybridization in response to environmental change depends on the circumstances. It is desirable when the resulting adaptation has the potential to rescue a native species from extinction, such as adaptation to the sudden climate shifts that might become more frequent or extreme with climate change. In such cases, management actions to protect hybridization, such as the protection of hybrid zones, might enhance the potential for species to respond to environmental change [16].

### **3. Aquaculture and cryopreservation**

Fish breeding depends on many factors and failure or partial success in the breeding is a reality for many successful hatchery operators. In order to get the required quantity of seeds, induced breeding is considered as a viable tool that makes the fishes maturing and spawning despite to low or poor rainfall and worst climatic conditions. Nevertheless, the health of brooders is severely affected by repeated breeding attempts within its confined life time. Exchange of brooders is not a simple task due to difficulties and physiological factors associated with the transportation of the brooders. Therefore shipping of gametes is considered as a possible alternative that may have its own advantages as witnessed in the animal husbandry.

It is necessary to introduce biotechnological tools in fish breeding programme to ensure continuous seed production. Cryopreservation may be a possible answer to produce quality seeds and genetically improved varieties. FAO has endorsed cryopreservation as a major strategy for conservation of fish resources [17]. Cryopreservation increases the longevity of gametes for several years without any drastic change in the fertilizing capacity of the gametes by lowering the temperature usually to −196°C [18] which arrests all biological activities, including biochemical reactions that lead to cell death and DNA degradation [19].

In fishes, Blaxter [20] is believed to be the first successful scientist who did the cryopreservation of herring spermatozoa and proceeded up to artificial fertilization with the cryopreserved spermatozoa. It has been reported that so far milt from over 200 species of freshwater and marine fish have been cryopreserved [21, 22].

### **4. Principle of cryopreservation**

The basic principle of cryopreservation is exposure of living cells to sub-zero temperature as low as −200° C through a perfect process thereby arresting its activities without damaging the life of it. A series of complex and dynamic processes of heat and water transport between cells and their surrounding medium is involved during the freeze–thaw process of biological material. The effect of the process depends on the speed at which the cells are frozen or thawed. When cells are frozen in an aqueous solution, both cells and the solution get super cooled leading to freezing that will be followed by heterogeneous nucleation, usually in the extracellular solution. The same condition can be seen in the cell solutions also. If such condition occurs intracellularly, the resultant nuclei will be isolated by plasma membranes from the unfrozen cell components and leads to separation of ice crystals inside the cell. As water gets frozen, the extracellular solution becomes progressively more concentrated leading to slow dewatering conditions in the cells. This results only when the cooling is slow and there is sufficient time for the cells to lose enough water so as to remain in osmotic equilibrium with the concentrating extracellular solution leading to water loss inside the cells. If that occurs, that will lead to cell death otherwise called as freeze killing or chill killing. While this may take time in large and multi cellular organisms, in small micro-organisms and single cells much of water can be withdrawn during freezing leading to desiccation and the death of the cell instantly [23].

In contrary to the above situation, if the rate of cooling is faster and rapid, there will be less time for the intracellular water to diffuse out of the cells. A balancing situation will emerge under such fast or rapid cooling. This leads to survival of the cells by minimizing the time or exposure duration to concentrated solution. The cooling rate also ensures there is no formation of intracellular ice. This process is called vitrification and it is the process that is happening inside the cell in the cryopreservation process [23].

During thawing, the same cellular physiological processes occur in reverse order. The thawing rate should also be rapid and fast enough that of the corresponding cooling rate. Nevertheless, recrystallization invariably occurs during thawing, forming lethal intracellular ice. A high warming rate is usually employed to minimize the degree of recrystallization when thawing is rapid to provide insufficient time or least possibility for the dehydrated cells to absorb the amount of water lost during freezing [23].

### **5. Cryopreservation of fish spermatozoa**

### **5.1 Milt collection**

Cryopreservation success depends on the milt quality and hence, quality of milt must be evaluated based on the condition of spermatozoa prior to cryopreservation. Milt should be always collected from oozing ripe brooders by stripping method in ice cold, sterilized cryovials [19]. Milt must be collected in clean, dry and sterile vials and immediately stored on ice [19]. Collected milt should be in quiescent form and should be free from contaminants, such as water, mucus, blood, and gut exudates. Prior to stripping, the urinary bladder can be emptied by gentle squeezing in order to avoid milt contamination with urine.

Employing stripping method for collecting fish milt might result in contamination with urine which may seriously influence milt characteristics and quality [24]. The contaminated milt can deteriorate the spermatozoa quality and have detrimental effects on post thaw viability as the contaminants such as urine, blood, mucus, etc. can change the seminal fluid composition and induce sperm motility [25]. Urine contamination can lead to lower percentage of fertilized eggs [26].

Using a catheter for milt collection can avoid urine and fecal contamination [27–29]. Researchers have suggested that anesthetizing the donors prior to milt collection was advantageous [30, 31]. Anesthetizing agents like Tricaine methane sulphonate (MS-222) can be used before milt collection [32]. Fish can be anesthetized by immersing in 2-phenoxyethanol for 2 min at a dose of 0.5 ml/l of water [33].

For instance, *O. mykiss* was anesthetized with MS 222 in a 1:10,000 dilution water bath during milt collection and the milt was collected by gently massaging the abdomens of the fish [34]. *C. carpio* brooders were anesthetized with a 1:1000 aqueous solution of 2-phenoxyethanol before handling [35]. *C. carpio* males were anesthetized with 2-phenoxyethanol at a dose of 0.5 ml before milt collection [36].

### **5.2 Spermatological properties**

Sperm quality evaluation is very important as it provides necessary information for optimal handling and storage protocols for sperm used in artificial fertilization [25, 37]. The fish milt composition and its physical characteristics vary with species and are important from the aspect of milt quality [38]. The quality of the milt is species specific [39] and can be affected by the feeding regime, feed quality, rearing temperature and spawning season of males [40, 41]. Spermatozoa motility, milt volume and the spermatozoa concentration are considered to be good indicators for milt quality [42, 43]. Sperm quality can be evaluated based on the sperm volume, spermatozoa density, motility of spermatozoa [25]. The appearance, color and nature of milt are also used to assess the quality of milt. The milt volume of fishes is found to vary with species [44–46].

### *5.2.1 Sperm motility and motility duration*

Motility is one of the most important parameters which is most frequently used to assess milt quality after cryopreservation and generally presents a positive correlation with fertilizing capacity [47]. Sperm motility is considered as the best biomarker of milt quality [48]. Motility depends on various aspects of the cell, such as the physiological state of the mitochondria, ATP production, plasma membrane channel integrity and flagellum structure [49]. Relationship between percentage motility and fertilization capacity of spermatozoa was reported in many fishes [50–55]. The motility, velocity and fertilizing ability of sperm was found to vary according to seasonal variations in osmolality of seminal plasma [56–58]. Sperm motility was also found to vary in vigor and duration among individual male depending on ripeness [45].

The spermatozoa are in immobile phase before ejaculation and it was reported that the osmolarity and ion content of the aquatic medium are central factors in activating motility [59, 60]. It was observed that in some of the fish species, the changes in the osmotic pressure (0–300 mosmol/l) could initiate spermatozoa motility [61]. In carp testes and seminal plasma, inhibition of sperm motility was observed due to high osmolality (approximately 300 mosmol/kg) surrounding spermatozoa [50]. Various researchers observed that the spermatozoa usually remain motile for

less than 2 min and sometimes they are only highly active for less than 30 s in most of the freshwater fishes [62–64].

### *5.2.2 Sperm pH*

Milt pH can affect spermatozoa motility and maturation [25]. Hence determination of variation in sperm pH provides information on fertilization capacity of spermatozoa. The milt of most of the freshwater fish species exhibit slightly alkaline pH [65]. When intracellular pH is below 7.5, sperm cells remain immotile with low respiration rate, but in response to an internal alkalinisation, they become motile, concomitantly with an increase in oxygen consumption [66, 67]. The initiation and duration of sperm motility is influenced by the extracellular and intracellular pH [68]. The external pH affects intracellular proton concentration which modifies the membrane potential and motility behavior [69].

### *5.2.3 Sperm density*

Traditionally the density of sperm has been used for the assessment of milt quality. It is an important parameter which has an impact on fertilization success and is a characteristic feature of fish species [70]. Spermatozoa density is usually reflected by sperm volume [71]. Various methods like using Sysmex Microcell counter CC-120 [72], spectrophotometric method [73], haemocytometric method [74] were employed to estimate sperm density in fishes.

### **5.3 Extender**

For successful cryopreservation, it is essential to prevent activation of spermatozoa during preservation. Undiluted milt is unsuitable for storage at cryogenic temperatures, so it should be diluted with an appropriate medium [51]. Because motility of fish spermatozoa is mostly a one-time event, this medium should not induce motility and at the same time must not interfere with the ability of the spermatozoa to be activated subsequently during utilization. Media that satisfies these conditions is called "Extender" [75].

Extender is a salt solution which helps to maintain the viability of cell during cryopreservation, which supplies sources of energy to sperm cells, protect the cells from temperature related damage, and maintain a suitable environment for the sperm to survive during the period of cryopreservation [76, 77]. Based on the inorganic composition of seminal plasma, extender is prepared as a buffered physiological saline solution [78] and hence extender composition differs between species. Extenders maintains the inactivity of spermatozoa when milt is diluted before freezing due to stabilization of physicochemical properties [79]. Sperm typically need to be maintained in an extender with proper osmolality (usually nearly isotonic to the plasma osmolality) to inhibit undesired sperm activation during refrigerated storage or cryopreservation [80].

A large number of extenders such as Ringer's solution, Cortland's solution, Alsever's solution, etc. have been tried for the cryopreservation of spermatozoa of fish which were proven successful for milt cryopreservation in mammals [29]. Several simple extenders which are isotonic in nature, with inorganic salts like NaCl, KCl, CaCl2, NaHCO3, NaHPO4, MgSO4, MgCl2 and others with organic compounds such as fructose, mannitol, lecithin, glycine have been used with varying levels of success [29]. Extenders have been developed using saline and sugar-based diluents [81].

Tris-egg yolk gave higher post-thaw motility percentage (50%) during cryopreservation of milt of *C. carpio* and *L. rohita* [82]. Use of glucose-based extender containing 10% dimethyl sulfoxide (DMSO) could be successfully used for *Oncorhynchus mykiss* milt cryopreservation and fertilization rate similar to that of fresh spermatozoa can be achieved [45]. Sperm diluted with 0.3427 g NaCl, 3.4314 g sucrose, 100 ml DW, 21 μl NaOH solution, 0.5 ml antibiotic (10,000 unit/ml penicillin and 10,000 μg/ml streptomycin) and DMSO gave the best post-thaw motility (94.5 ± 3.3%) in *C. carpio* [83]. The feasibility of three extenders namely, Freshwater Fish Saline, Modified Fish Ringer and Physiological Saline was compared in cryopreserving *C. carpio* milt and the motility duration obtained was 57.28 ± 9.21 s, 64.78 ± 8.84 s and 67.39 ± 4.79 s for Physiological saline, Freshwater Fish saline and Modified Fish Ringer respectively [84].

### **5.4 Cryoprotectant**

Cryoprotectants are low molecular weight compounds that penetrate cells and lower the freezing points of solutions. Cryoprotectants in combination with an effective dilution ratio can also improve the cryo-resistance of spermatozoa [85]. Cryoprotectants need time to penetrate to the cells (equilibration), however, prolonged exposure before cryopreservation can be toxic for sperm [85]. At higher concentrations, cryoprotectants can suppress most of cryoinjuries but at the same time, it can become toxic to the cells [86]. Therefore, suitable cryoprotectant concentration is needed for the development of cryopreservation protocol. The protective effect of cryoprotectants varies in different fish species [87].

Cryoprotectants are very essential for the survival of spermatozoa during cryopreservation. There are two different types of cryoprotectants; permeating and non–permeating [88]. Permeating cryoprotectants such as DMSO, glycerol, methanol, propanediol etc., are believed to lower the freezing point of the solution, which minimize osmotic shock by replacing the water inside the cell, and reduce formation of destructive intracellular ice [89]. Non-permeating cryoprotectants include protein like milk, egg yolk, bovine serum albumin (BSA); sugars such as glucose, sucrose; synthetic polymers like polyethylene glycol and polyvinylpyrrolidone and are believed to stabilize the membrane during cryopreservation [90]. Use of insufficient cryoprotectant before cooling reduces effectiveness, whereas excessive cryoprotectant causes osmotic swelling and rupture during thawing and dilution [91]. Cryoprotectants were found to prevent the formation of ice crystals during freezing [48].

Due to ice crystal formations at low temperatures very few spermatozoa survive without cryoprotectant and same levels of those cryoprotectants can be lethal to unfrozen cell [92]. Cryoprotectants were most effective when they could rapidly penetrate the cell during freezing, which resulted in delay in intracellular freezing and led to minimization of the solution effect [93]. Common cryoprotectants used for fish sperm include DMSO, methanol and propylene glycol (PG) [94].

Regarding these cryoprotectants, PG used for sperm cryopreservation in yellowtail flounder (*Pleuronectes ferrugineus)* resulted to be an effective cryoprotectant [95] but showed moderately good post-thaw motility in *Clarias gariepinus* [96]. Methanol at 10% was found suitable for cryopreservation of bitterling milt [97], bagrid catfish [98] and *C. gariepinus* [99] and 5% methanol was reported to be suitable for tilapia (*Oreochromis niloticus*) milt cryopreservation [94]. DMSO was established to be very successful for cryopreservation of sperm in various freshwater species [100, 101] and has been considered as a universal cryoprotectant [102, 103].

The milt of *C. mrigala* when cryopreserved with glucose as co-cryoprotectant at 0.5% concentration egg yolk at 10% concentration gave the highest post-thaw motility duration [104, 105]. BSA at 2% gave the highest post-thaw motility duration in *C. carpio* [106]*.*

### **5.5 Dilution ratio**

The process of milt dilution is carried out as a means to increase the number of eggs that can be fertilized with a small volume of milt [107]. In fish spermatozoa cryopreservation, dilution of the sperm fluid is one of the most important steps which has been reported to improve fertilization rate as compared with results obtained with undiluted milt [108]. Milt dilution ratio is very important for fish sperm to survive after cryopreservation [109]. The dilution process is very important to increase the volume of milt, so that it can be used for multiple inseminations. Milt is generally diluted 3–20 folds for Salmonid, carp and tilapia [24].

In Cyprinids, full sperm motility is activated at osmolalities <50 mosmol/kg [50]. Using cryopreserved milt, full activation of sperm motility was obtained at ratios of milt to fertilization media of 1:10 for all types of media since at this ratio, the osmolality of the extender–water mixture was high enough to stabilize sperm viability [110]. Too low dilution ratio do not activate full sperm motility and too high ratios results in insufficient low sperm concentrations in the fertilization solution [110]. However, reports also suggest 1:25 [111, 112] and 1:20 [113] as the optimal ratio of milt to fertilization medium.

In this regard, when *C. carpio* milt was diluted with Kurokura medium at 1:5 ratio, it gave best results [114]. Dilution ratio of milt to extender of 1:7 resulted in highest hatching rates while at lower (1,3) and higher dilutions (1,10) fertility was significantly decreased in bleak (*Chalcalburnus chalcalburnus)* [110]. When dilutions of 1:25, 1:50 and 1:100 were evaluated on European perch (*Perca fluviatilis)*, best result was obtained at 1:50 dilution [115]. The highest mean post-thaw motility duration, motility score, percentage of fertilized eggs, and hatching rate was obtained with 1:40 dilution ratio in *C. carpio* [116].

### **5.6 Equilibration period**

Equilibration period is the optimum time that must be allowed to facilitate the penetration of permeating cryoprotectants into the cells while minimizing the toxicity for effective protection during freezing [117].During cryopreservation of milt, an equilibration time of 45–60 min for IMC [118], 10 min for *C. carpio* [119], 60 min for *Tor putitora* [120], 5 min for bleak (*C. chalcoides*) [100], 10 min for *L. rohita* [121], 10 min for *O. mykiss* [122] has been employed with successful results.

*Salmo gairdneri* milt stored for 20 min after dilution gave significantly higher percentage of fertilization than that stored for 65 min or longer and therefore, cryopreservation of milt should be done as soon as possible after the collection [123]. Poor post-thaw motility was recorded in *C. carpio* at 20 min of equilibration time than that frozen immediately [124]. When the diluted milt was equilibrated for 15 min, there was no adverse effect on the post-thaw fertility of Salmonid milt [48].

### **5.7 Freezing**

Too high freezing rate result in the formation of small ice crystals within the cell due to limited time for the free water to separate from the cytoplasm which

puncturescell membraneand the membranes of the cell organelles. Too low freezing rate exposes the cell to the concentrated cytoplasm for a long time resulting in pickling effect and the biomolecules in the cell get denatured due to the high salt concentration and subsequent changes in the pH [18]**.**

The optimum freezing rate is a moderate rate between the two extremes of the freezing rate [125] which depends on cell type and size, cryoprotectant type and concentration, equilibration time, final temperature prior to plunging in LN2, fish species and associated interactions [99, 126]. Optimal cooling rate should be rapid enough to minimize the duration of exposure to prevent the occurrence of concentrated solute and slow enough to allow water osmosis to prevent intracellular ice crystal formation [127].

The freezing rate is a critical factor and it was reported that instant immersion in LN2 may significantly decrease the post-thaw motility duration of fish spermatozoa [128]. Freezing can be performed by programmable temperature changes or simple immersion in LN2 vapor above the surface of LN2 [83]. Freezing can also be done using methanol-dry ice bath [129] or by freezing the extended milt with cryoprotectant over crushed dry ice [130]. The pelletization technique in which specific volumes of diluted milt is placed over dry ice (solid CO2) also served to freeze the milt and it was used by many workers [32, 131, 132].

In a protocol, straws were frozen for 4 min on a stainless steel tray (−80°C) suspended over LN2 and was immersed into LN2 [133]. When *C. carpio* milt was frozen 3 cm above the surface of LN2 for 3 min before plunging in LN2 it resulted in high post-thaw motility as well as fertilization and hatching rate [134]. During cryopreservation of *C. carpio* milt, the 0.5 ml straws were placed horizontally onto a 3 cm high styroframe raft (−130°C) for 20 min, which was floating on the surface of LN2, before immersing the straws into LN2 and it did not negatively affect the fertility of frozen–thawed sperm [35].

Programmable freezers was also used by many researchers for freezing the diluted milt samples of several fish species [135–137]. Programmable freezing allows the pre-setting of different freezing programs, the monitoring of precise temperature during the cooling sections and the continuous biological examination of cells during the freezing stages [138]. Different programmes and different final temperatures can be attained in programmable freezer [119, 139–141]. The use of programmable freezer allows the evaluation of spermatozoa motility at different rates of cooling during freezing [141]. Incorporation of fast freezing rates using the controlled-rate programmable freezer was successfully used in earlier studies for cryopreservation of carp sperm [111, 113, 142].

For cryopreservation of milt of *C. carpio,* the most efficient freezing rate was 5°C/ min from 2°C to −7°C and 25° C/ min from −7° C to −70°C [135]. A slower cooling rate at 4°C/min from 00 C to −4°C and 11°C/min from −4°C to −80°C can also be used for cryopreservation of *C. carpio* milt successfully [113]. A cooling program of 4°C to −9°C at a rate of 4°C/min and then from −9° C to −80° C at a rate of 11°C/min, which was held for 6 min at −80°C, and transferred into LN2 was followed for *C. carpio* and high motility (69 ± 14%) and moderate fertilization rate (56 ± 10%) was reported [111]. *C. gariepinus* spermatozoa can be frozen at the rate of −5°C/min initially from +5°C to −35°C and then from −35°C to −50°C or − 70°C [99].

Three different cooling methods were employed during the cryopreservation of *C. carpio* [142]. Two of them used 3 steps, initially from 2°C to −7°C then −7°C to −30°C and finally −30°C to −80°C with two different cooling rates (3 and 6°C/min) after which the sample was transferred to LN2. In the third method, a one-step method (2°C to −50°C) with faster average cooling rate (10°C/min) was applied and was reported that faster cooling rates (6 and 10°C/min) were more efficient for cryopreservation and the highest fertilization recorded with 10°C/ min was 99%.

### **5.8 Thawing**

The rate of thawing is an important step which is said to be a decisive factor for the success of cryopreservation procedure. It is the reverse of freezing but rapid thawing after the cooling procedure is preferred however, too high and too low rates of thawing are detrimental for the cryopreserved spermatozoa [18]. Thawing rates should be high enough to avoid recrystallization as its rate is very critical for preservation of spermatozoa viability [143]. It appeared that the ideal thawing procedure almost avoided or reduced either recrystallization and ice crystal formation during thawing. The temperature change should allow movement of water and cryoprotectants while preventing intracellular ice recrystallization [144].

In Cyprinid fishes, the highest mean fertilization percentage of 57% was obtained in *C. idella* when thawed at 20°C quickly in a water bath [145]. The cryopreserved milt of freshwater carps (*L. rohita*, *C. carpio*, *Puntius gonionotus, C. idella*, *Aristichthys nobilis* and *Pangasius sutchi*) was thawed by swirling the frozen ampoules in tap water at 29°C [146]. Similarly, the frozen milt of IMC and *H. molitrix* was thawed by swirling the straws in tap water at 30°C [147]. High post-thaw motility percentage of 92–98% and high hatching percentage of 25.7% was obtained after thawing the cryopreserved milt of *T. khudree* at 37 ± 1°C for 5–10 s [148]. The highest mean motility (83.4 ± 2.1) and fertilization rate (85.6 ± 2.8) was obtained in *C. Idella* when the milt was thawed at 35° C for 30 s [149]. The highest post-thaw motility of 52.6 ± 1.4 s was recorded in *C. carpio* when thawed at 30°C for 30 s [150].

In Salmonid fishes (*O. mykiss, Salmo trutta lacustris, S. trutta fario* and *Salvelinus fontinalis*), the highest fertilization rates obtained was when milt was thawed at 25°C in water bath for 30 s and change of the thawing period for only 5 s or the thawing temperatures for 5°C led to reduce of post-thaw fertilization ability of milt [45]. Cryopreserved milt of *T. khudree* was thawed at 37°C for 40 s in a water bath [120]. Cryopreserved milt of *O. mykiss* was thawed at 25°C in water bath for 30 s for 0.5 ml and 1.8 ml straws and at 60°C for 30s/ 80°C for 20 s for 5 ml straws and was reported that thawing at 25°C in water bath for 30 s was best for thawing of *O. mykiss* milt [42]. The cryopreserved milt of *O. mykiss* was thawed at 10°C for 30s in water bath [130]. The cryopreserved milt of Salmonid fishes was thawed at 25°C for 30 s for 0.5 ml straws and at 30°C for 30 s for 1.2 ml and 5.0 ml straws in a water bath [151].

### **6. Cryopreservation of fish eggs and embryos**

Cryopreservation of fish eggs and embryos are still in its infant stage. Unlike cryopreservation of spermatozoa, very few studies are available on cryopreservation of eggs. Attempts have been made to cryopreserve the eggs of rainbow trout [152, 153] and embryos of Japanese medaka fish, *Oryzias latipes* [154], rainbow trout, *O. mykiss* [155], zebra fish, *Brachydanio rerio* [156], common carp, *C. carpio* [157] and rohu, *L. rohita* [158].

Many of the attempts to cryopreserve fish eggs were failure due to dehydration, relatively large size of eggs, presence of large amount of yolk and different water permeability rate of membranes [159–161]. The major hindrances recorded in the

cryopreservation of egg and embryos of teleost fishes [162] are the large size of fish egg and embryos which results in low surface/volume ratio and lower membrane permeability to water and cryoprotectant solutions that makes the embryos difficult to cool and warm uniformly without damage and ice formation, low permeability of the membrane due to the presence of chorionic layer, sensitivity of fish egg and embryosto low temperatures and the presence of multi-layered membrane structure which hinders the osmotic properties for each compartment of the egg/embryos which finally affects the transport of the cryoprotectant solutions.

Studies have been carried out by different researchers to overcome these issues and some of the efforts made are microinjection of cryoprotectants directly into the cytoplasm [163], use of negative pressure on the egg/embryos to increase permeability of the cryoprotectants [164], microinjection of anti-freeze protein [165] and application of hydrostatic pressure on the egg/embryos [166]. Precise knowledge of embryo permeability is essential for successful cryopreservation of egg/embryos [167].

Herring embryos did not survive after cooling below −10°C when DMSO was used [168]. Methanol was a better cryoprotectant for zebrafish embryo when compared with DMSO or ethanediol since it penetrates theentire embryo within 15 min while other cryoprotectants could not penetrate into yolk even after 2.5 h [169, 170]. Similarly, PG also could not protect the zebrafish embryos upon immersion of it into LN2 as it resulted in mitochondrial damage, disorganization of ribosomes and plasma membrane of the yolk syncytial layer [171].

### **7. Application of cryopreservation in aquaculture**


### **8. Demerits of frozen milt in aquaculture**


### **9. Conclusion**

Cryopreservation technology has been developed for many fish species. However, standard species specific cryopreservation protocols must be developed and the success rate of using cryopreserved sperm in artificial fertilization program of every fish species has to be determined for commercializing the technology. Even though standard protocols of cryopreservation are followed, cryoinjuries are unavoidable. Ways to overcome the cryoinjuries by establishing proper freeze–thaw cycle is essential. The oxidative stress in the cryopreserved sperm must be clearly addressed and methods to reduce the production of reactive oxygen species (ROS) must be evolved. The possible effects of cryopreservation on the energy production, ROS production, mitochondrial DNA of the spermatozoa and the structure of spermatozoa must be documented. Unlike in animals, very few fish sperm banks have been established for fishes. More research is needed to make the sperm banks for fishes a reality in the developing countries. Addressing the research needs mentioned above will help to establish successful fish sperm banks for many commercially important fish species.

### **Conflict of interest**

The authors declare that they have no conflict of interest.

*Animal Reproduction*

### **Author details**

Judith Betsy C1 \*, Siva C2 and Stephen Sampath Kumar J3

1 TNJFU - Fisheries College and Research Institute, Tuticorin, Tamil Nadu, India

2 Molecular Genetics Laboratory, ICAR-Directorate of Coldwater Fisheries Research, Nainital, Uttarakhand, India

3 TNJFU - Directorate of Sustainable Aquaculture, Thanjavur, Tamil Nadu, India

\*Address all correspondence to: betsy@tnfu.ac.in

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Cryopreservation and Its Application in Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.99629*

### **References**

[1] Béné C, Macfadyen G, Allison EH. Increasing the contribution of smallscale fisheries to poverty alleviation and food security. Food & Agriculture Org.; 2007.

[2] Beare D. Fisheries and aquaculture: The importance of fish for food and nutritional security. Beare, D., 2012. Fisheries and aquaculture: The importance of fish for food and nutritional security. In: Thornton, P. and Cramer, L. (Eds.), Impacts of climate change on the agricultural and aquatic systems and natural resources within the CGIAR's mandate. CCAFS Working Paper 23. CGIAR Research Program on Climate Change, Agriculture and Food Security (CCAFS). Copenhagen; 2012. pp. 55-67.

[3] Roos N, Wahab MA, Chamnan C, Thilsted SH. The role of fish in foodbased strategies to combat vitamin A and mineral deficiencies in developing countries. The journal of Nutrition. 2007;137(4):1106-9.

[4] Thilsted SH, Roos N, Hassan N. The role of small indigenous fish species in food and nutrition security in Bangladesh. Naga, the ICLARM quarterly. 1997;20(3-4):82-4.

[5] Hossain M, Naher F, Shahabuddin Q. Food security and nutrition in Bangladesh: progress and determinants. eJADE: electronic Journal of Agricultural and Development Economics. 2005;2(853-2016-56126):103-32. DOI: 10.22004/ag.econ.110131

[6] Islam FU. Self-recruiting species (SRS) in aquaculture: their role in rural livelihoods in two areas of Bangladesh [thesis]. University of Stirling; 2007.

[7] Kawarazuka N. The contribution of fish intake, aquaculture, and small-scale fisheries to improving nutrition security: a literature review. 2010: p. 51

[8] FAO. The State of World Fisheries and Aquaculture. Food and Agriculture Organization of the United Nations, Rome. 2020: p. 206.

[9] FAO. Aquaculture development. 3. Genetic resource management. FAO Technical Guidelines for Responsible Fisheries. No. 5, Suppl. 3. Rome, FAO. 2008: p. 125.

[10] Tave D. Inbreeding and brood stock management. *Fisheries Technical Paper*. No. 392. Rome, Food & Agriculture Org.; 1999: p. 122.

[11] Dunham RA, Majumdar K, Hallerman E, Bartley D, Mair G, Hulata G, Liu Z, Pongthana N, Bakos J, Penman D, Gupta M. Review of the status of aquaculture genetics. In Aquaculture in the Third Millennium. Technical Proceedings of the Conference on Aquaculture in the Third Millennium, Bangkok, Thailand 2000: pp. 137-166.

[12] Anderson E. Introgressive hybridization. Biological Reviews. 1949:28(3),pp.280-307. doi:10.1111/ j.1469-185x.1953.tb01379.x

[13] Sarder MR, Rafiquzzaman SM, Sultana R, Islam MF. Cryopreservation of spermatozoa of Mrigal, *Cirrhinus cirrhosus* with a view to minimize inbreeding and hybridization. Journal of the Bangladesh Agricultural University. 2009;7(1):211-218. DOI: 10.3329/jbau. v7i1.4986

[14] Horváth Á, Jesenšek D, Csorbai B, Bokor Z, Raboczki É, Kaczkó D,

Bernáth G, Hoitsy G, Urbányi B, Bajec SS, Snoj A. Application of sperm cryopreservation to hatchery practice and species conservation: A case of the Adriatic grayling (*Thymallus thymallus*). Aquaculture. 2012 Aug 15;358:213-215*.* doi:10.1016/j.aquaculture.2012.07.012

[15] Horvath A, Bokor Z, Bernath G, Urbanyi B, Snoj A, Susnik Bajec S, Jesensek D. Application Of Sperm Cryopreservation To The Culture And Conservation Of Salmonid Species: A Slovenian-Hungarian Collaboration. Serbia, 2015.

[16] Baskett ML, Gomulkiewicz R. Introgressive hybridization as a mechanism for species rescue. Theoretical Ecology. 2011;4(2):223-239. DOI 10.1007/s12080-011-0118-0

[17] Khoshoo TN. Conservation of India's endangered mega animals: Tiger and lion. Current Science. 1997;73(10): 830-42.

[18] Diwan AD, Ayyappan S, Lal KK, Lakra WS. Cryopreservation of fish gametes and embryos. The Indian Journal of Animal Sciences. 2010;80(4):109-24.

[19] Agarwal NK. Cryopreservation of fish semen. Himalayan Aquatic Biodiversity Conservation & New Tools in Biotechnology. Transmedia Publication, Srinagar (Garhawal) Uttarakhand, India. 2011:104-27.

[20] Blaxter JH. Sperm storage and cross-fertilization of spring and autumn spawning herring. Nature. 1953;172 (4391):1189-90.

[21] Blesbois E, Labbé C. Main improvements in semen and embryo cryopreservation for fish and fowl. In Atelier sur la Cryoconservation des Ressources Génétiques Animales en

Europe. Bureau des Ressources Génétiques. Paris. 2003. ISBN 2-908447- 25-8: 55-65.

[22] Hiemstra SJ, van der Lende T, Woelders H. The potential of cryopreservation and reproductive technologies for animal genetic resources conservation strategies. InThe role of biotechnology in exploring and protecting agricultural genetic resources, FAO, 2006. pp.45-60.

[23] Kumar JSS and Betsy CJ. Cryopreservation of fish gametes and its role in enhancing aquaculture production. In: Santhanam P, Thirunavukkarasar AR, Perumal P, editors. Advances in Marine and Brackish water Aquaculture. India: Springer Publishers; 2015. p. 241-246.

[24] Rana KJ. Cryopreservation of fish spermatozoa. In Cryopreservation and Freeze-Drying Protocols. Humana Press, Totowa, NJ. 1995. pp. 151-165.

[25] Billard R, Cosson J, Perchec G, Linhart O. Biology of sperm and artificial reproduction in carp. Aquaculture. 1995;129(1-4):95-112.

[26] Bokor Z, Müller T, Bercsényi M, Horváth L, Urbányi B, Horváth Á. Cryopreservation of sperm of two European percid species, the pikeperch (*Sander lucioperca*) and the Volga pikeperch (*S. volgensis*). Acta Biologica Hungarica. 2007;58(2):199-207.

[27] Alderson R, Macneil AJ. Preliminary investigations of cryopreservation of milt of Atlantic salmon (*Salmo salar*) and its application to commercial farming. Aquaculture. 1984;43(1-3):351-4.

[28] Cabrita E, Alvarez R, Anel L, Rana KJ, Herraez MP. Sublethal damage during cryopreservation of rainbow trout sperm. Cryobiology. 1998;37(3):245-53.

*Cryopreservation and Its Application in Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.99629*

[29] Rao B, Soufir JC, Martin M, David G. Lipid peroxidation in human spermatozoa as relatd to midpiece abnormalities and motility. Gamete research. 1989;24(2):127-34.

[30] Kurokura H. Cryopreservation of rainbow trout sperm. Nippon Suisan Gakkishi. *Bulletin* of the *Japanese Society* of *Scientific Fisheries.* 1980;46:1493-5.

[31] Lakra WS. Cryogenic preservation of fish spermatozoa and its application to aquaculture. Indian Journal of Cryogenics. 1993; 18, pp. 171-176.

[32] Piironen J. Cryopreservation of sperm from brown trout (*Salmo trutta* m. lacustris L.) and Arctic charr (*Salvelinus alpinus* L.). Aquaculture. 1993;116(2-3): 275-85.

[33] Perchec G, Jeulin C, Cosson J, Andre F, Billard R. Relationship between sperm ATP content and motility of carp spermatozoa. Journal of Cell Science. 1995;108(2):747-53.

[34] Bozkurt Y, Akcay E, Tekin N, Secer S. Effect of freezing techniques, extenders and cryoprotectants on the fertilization rate of frozen rainbow trout (*Oncorhynchus mykiss*) sperm. *Israeli J. Aqua.– Bamidgeh*. 2005;57:125-130*.*

[35] Boryshpolets S, Dzyuba B, Rodina M, Li P, Hulak M, Gela D, Linhart O. Freezethawing as the factor of spontaneous activation of spermatozoa motility in common carp (*Cyprinus carpio* L.). Cryobiology. 2009;59(3):291-6.

[36] Cejko BI, Krejszeff S, Sarosiek B, Żarski D, Judycka S, Kowalski RK. Biochemical factors of common carp *Cyprinus carpio* L. 1758, seminal plasma and its relationship with sperm motility parameters. Journal of Applied Ichthyology. 2015;31:10-7.

[37] Linhart O, Gela D, Rodina M, Kocour M. Optimization of artificial propagation in European catfish, *Silurus glanis* L. Aquaculture. 2004;235(1-4):619-32.

[38] Kruger JD, Smit GL, Van Vuren JH, Ferreira JT. Some chemical and physical characteristics of the semen of *Cyprinus carpio* L. and *Oreochromis mossambicus* (Peters). Journal of Fish Biology. 1984;24(3):263-72.

[39] Piironen J. Variation in the properties of milt from the Finnish landlocked salmon (*Salmo salar* m. sebago Girard) during a spawning season. Aquaculture. 1985;48(3-4):337-50.

[40] Bromage N, Roberts RJ. Broodstock management and seed quality-general considerations. Brood-stock management and egg and larval quality. 1995:1-24.

[41] Rurangwa E, Kime DE, Ollevier F, Nash JP. The measurement of sperm motility and factors affecting sperm quality in cultured fish. Aquaculture. 2004;234(1-4):1-28.

[42] Cabrita E, Robles V, Alvarez R, Herráez MP. Cryopreservation of rainbow trout sperm in large volume straws: application to large scale fertilization. Aquaculture. 2001;201(3-4):301-14.

[43] Tekin N, Secer S, Akcay E, Bozkurt Y. Cryopreservation of rainbow trout (*Oncorhynchus mykiss*) semen. 2003; 55(3), pp. 208-212.

[44] Baynes SM, Scott AP. Seasonal variations in parameters of milt production and in plasma concentration of sex steroids of male rainbow trout (*Salmo gairdneri*). General and comparative endocrinology. 1985;57(1):150-60.

[45] Lahnsteiner F, Patzner RA, Weismann T. Semen cryopreservation of salmonid fishes: influence of handling parameters on the postthaw fertilization rate. Aquaculture Research. 1996;27(9):659-71.

[46] Lin F, Ciereszko A, Dabrowski K. Sperm production and cryopreservation in muskellunge after carp pituitary extract and human chorionic gonadotropin injection. The Progressive Fish-Culturist. 1996;58(1):32-7.

[47] Kime DE, Van Look KJ, McAllister BG, Huyskens G, Rurangwa E, Ollevier F. Computer-assisted sperm analysis (CASA) as a tool for monitoring sperm quality in fish. Comparative Biochemistry and Physiology Part C Comparative Pharmacology and Toxicology, 2001;130, pp.425-433.

[48] Lahnsteiner F, Mansour N, Berger B. Seminal plasma proteins prolong the viability of rainbow trout (*Oncorynchus mykiss*) spermatozoa. Theriogenology. 2004;62(5):801-8.

[49] Cosson J, Groison AL, Suquet M, Fauvel C, Dreanno C, Billard R. Studying sperm motility in marine fish: an overview on the state of the art. Journal of Applied Ichthyology. 2008;24(4): 460-86.

[50] Morisawa M, Suzuki K, Shimizu H, Morisawa S, Yasuda K. Effects of osmolality and potassium on motility of spermatozoa from freshwater cyprinid fishes. Journal of Experimental Biology. 1983;107(1):95-103.

[51] Stoss J. 6 Fish Gamete Preservation and Spermatozoan Physiology. Fish physiology. 1983;9:305-50.

[52] Harvey B, Kelley RN, Ashwood-Smith MJ. Cryopreservation of zebra fish spermatozoa using methanol. Canadian Journal of Zoology. 1982;60(8):1867-70.

[53] Cosson MP, Billard R, Gatti JL, Christen R. Rapid and quantitative assessment of trout spermatozoa motility using stroboscopy. Aquaculture. 1985;46(1):71-5.

[54] Billard R, Cosson MP. Some problems related to the assessment of sperm motility in freshwater fish. Journal of Experimental Zoology. 1992;261(2): 122-31.

[55] Ohta H, Shimma H, Hirose K. Relationship between fertility and motility of cryopreserved spermatozoa of the amago salmon *Oncorhynchus masou ishikawae*. Fisheries science. 1995;61(5):886-7.

[56] Benau D, Terner C. Initiation, prolongation, and reactivation of the motility of salmonid spermatozoa. Gamete Research. 1980;3(3):247-57.

[57] Akcay E, Bukan N, Bozkurt Y, Tekin N, Secer S. Correlation between biochemical and spermatological parameters in rainbow trout (*Oncorhynchus mykiss*) semen. Israeli Journal of Aquaculture-Bamidgeh. 2004;56:20390.

[58] Babiak I, Ottesen O, Rudolfsen G, Johnsen S. Quantitative characteristics of Atlantic halibut, *Hippoglossus hippoglossus* L., semen throughout the reproductive season. Theriogenology. 2006;65(8): 1587-604.

[59] Mochida K, Kondo T, Matsubara T, Adachi S, Yamauchi K. A high molecular weight glycoprotein in seminal plasma is a sperm immobilizing factor in the teleost Nile tilapia, *Oreochromis niloticus*. Development, growth & differentiation. 1999;41(5):619-27.

*Cryopreservation and Its Application in Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.99629*

[60] Cherr GN, Morisawa M, Vines CA, Yoshida Y, Smith EH, Matsubara T, Pillai M, Griffin FJ, Yanagimachi R. Two egg-derived molecules in sperm motility initiation and fertilization in the Pacific herring (*Clupea pallasi*). University of the Basque Country Press, 52, pp.743-752.

[61] Morisawa M, Suzuki K. Osmolality and potassium ion: their roles in initiation of sperm motility in teleosts. Science. 1980;210(4474):1145-7.

[62] Billard R, Weil C, Bieniarz K, Mikolajczyk T, Breton B, Epler P, Bougoussa M. Testicular and some hormonal changes during the first four years of life in the mirror carp, *Cyprinus carpio* L. Journal of fish biology. 1992;41(3):473-87.

[63] Perchec G, Jeulin C, Cosson J, Andre F, Billard R. Relationship between sperm ATP content and motility of carp spermatozoa. Journal of Cell Science. 1995;108(2):747-53.

[64] Cosson J. Fish sperm physiology: structure, factors regulating motility, and motility evaluation. Biological Research in Aquatic Science. 2019;1: 1-26.

[65] Verma DK, Routray P, Dash C, Dasgupta S, Jk JE. Physical and biochemical characteristics of semen and ultrastructure of spermatozoa in six carp species. Turkish Journal of Fisheries and Aquatic Sciences. 2009;9(1):pp.67-76

[66] Christen R, Schackmann RW, Shapiro BM. Metabolism of sea urchin sperm. Interrelationships between intracellular pH, ATPase activity, and mitochondrial respiration. Journal of Biological Chemistry. 1983;258(9): 5392-5399.

[67] Shapiro BM, Schackmann RW. Coupled ionic and enzymatic regulation of sperm behavior. Modulation by

Molecular Interactions: Current Topics in Cellular Regulation, Vol. 26. 2013;26:97-113.

[68] Márián TE, Krasznai ZO, Balkay L, Balazs M, Emri M, Bene L, Tron L. Hypo-osmotic shock induces an osmolality-dependent permeabilization and structural changes in the membrane of carp sperm. Journal of Histochemistry & Cytochemistry. 1993;41(2):291-297.

[69] Boitano S, Omoto CK. Trout sperm swimming patterns and role of intracellular Ca++. Cell motility and the cytoskeleton. 1992;21(1):74-82.

[70] Agarwal NK. Developing cryopreservation protocols for the spermatozoa of snow trout and other endangered coldwater species. [Final report]. 2005.

[71] Bozkurt Y, Öğretmen F. Sperm quality, egg size, fecundity and their relationships with fertilization rate of grass carp (*Ctenopharyngodon idella*). Iranian Journal of Fisheries Sciences. 2012;11(4):755-64.

[72] Kruger JD, Smit GL, Van Vuren JH, Ferreira JT. Some chemical and physical characteristics of the semen of *Cyprinus carpio* L. and *Oreochromis mossambicus* (Peters). Journal of Fish Biology. 1984;24(3):263-272.

[73] Suquet M, Dreanno C, Fauvel C, Cosson J, Billard R. Cryopreservation of sperm in marine fish. Aquaculture Research: Original Articles. 2000;31(3):231-43.

[74] Bozkurt Y, Öğretmen F, Kökçü Ö, Ercin U. Relationships between seminal plasma composition and sperm quality parameters of the *Salmo trutta macrostigma* (Dumeril, 1858) semen: with emphasis on sperm motility. Czech J. Anim. Sci. 2011;56(8):355-64.

[75] Routray P, Verma DK. Utilization of milt cryopreservation technology in aquaculture-a strategy. In *National Workshop On "Portable FRP Carp Hatchery Technology"*, 2011:11, pp.28.

[76] Graybill JR, Horton HF. Limited fertilization of chinook and coho salmon eggs with cryopreserved sperm. J. Fish. Res. Board Can. 1969;26(5):1400-4.

[77] Kamaruding NA, Embong WK, Abdullan RB. Frozen-thawed sperm motility characteristics of African catfish (*Clarias gariepinus*) by using glycerol or DMSO based extender. International Journal of Environmental Science and Development. 2012;3(1):49.

[78] Borchard B, Schmidt GW. Versuche mit Regenbogenforellensperma. IV. Die Tiefkuhlkonservierung. Beobachtungen zum Einsatz bei praktischen und wissenschaftlichen Arbeiten, Fischwirt. 1979;29:49-51.

[79] Ohta H, Izawa T. Diluent for cool storage of the Japanese eel (*Anguilla japonica*) spermatozoa. Aquaculture. 1996;142(1-2):107-18.

[80] Bates MC, Wayman WR, Tiersch TR. Effect of osmotic pressure on the activation and storage of channel catfish sperm. Transactions of the American Fisheries Society. 1996;125(5):798-802.

[81] Urbanyi B, Horvath A, Varga Z, Horvath L, Magyary I, Radics F. Effect of extenders on sperm cryopreservation of African catfish, *Clarias gariepinus* (Burchell). Aquaculture Research. 1999;30(2):145-51.

[82] Lakra WS, Krishna G. Preliminary trials for cryopreservation of spermatozoa of selected carps and catfishes. Indian journal of animal sciences. 1997;67(1):90-2.

[83] Irawan H, Vuthiphandchai V, Nimrat S. The effect of extenders, cryoprotectants and cryopreservation methods on common carp (*Cyprinus carpio*) sperm. Animal Reproduction Science. 2010;122(3-4):236-43.

[84] Betsy CJ, Kumar JS. Effect of three different extenders on the spermatological parameters of *Cyprinus carpio* spermatozoa. Journal of Aquaculture in the Tropics. 2016;31(1/2):25.

[85] Cloud J, Patton S. Basic principles of fish spermatozoa cryopreservation. Methods in. 2008:237..

[86] Tekin N, Secer S, Akcay E, Bozkurt Y, Kayam S. Effects of glycerol additions on post-thaw fertility of frozen rainbow trout sperm, with an emphasis on interaction between extender and cryoprotectant. Journal of Applied Ichthyology. 2007;23(1):60-3.

[87] Yavaş İ, Bozkurt Y, Yıldız C. Cryopreservation of scaly carp (*Cyprinus carpio*) sperm: effect of different cryoprotectant concentrations on postthaw motility, fertilization and hatching success of embryos. Aquaculture international. 2014;22(1):141-8.

[88] Tiersch TR, Yang H, Jenkins JA, Dong Q. Sperm cryopreservation in fish and shellfish. Society of Reproduction and Fertility supplement. 2007;65:493.

[89] Leung LK. Principles of biological cryopreservation. Fish evolution and systematics: evidence from spermatozoa. 1991:231-44.

[90] Meryman HT. Cryoprotective agents. Cryobiology. 1971 Apr 1;8(2):173-83.

[91] Taylor R, Adams GD, Boardman CF, Wallis RG. Cryoprotection—Permeant vs *Cryopreservation and Its Application in Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.99629*

nonpermeant additives. Cryobiology. 1974;11(5):430-8.

[92] Chao NH. Fish sperm cryopreservation in Taiwan: Technology advancement and extension efforts. *Bulletin of the Institute of Zoology, Academia Sinica*, 1991;16:263-83.

[93] Simione F. American Type Culture Collection in cooperation with NALGE Nunc International Corp. Cryopreservation manual. 1998.

[94] Routray P, Dash SN, Dash C, Swain P, Sarkar SK, Sarangi N. Cryopreservation of silver barb *Puntius gonionotus* (Bleeker) spermatozoa: effect of extender composition, cryoprotective agents and freezing rate on their postthawing fertilization ability. Aquaculture Research. 2008;39(15):1597-605.

[95] Richardson GF, Wilson CE, Crim LW, Yao Z. Cryopreservation of yellowtail flounder (*Pleuronectes ferrugineus*) semen in large straws. Aquaculture. 1999;174(1-2):89-94.

[96] Horvath A, Urbanyi B. The effect of cryoprotectants on the motility and fertilizing capacity of cryopreserved African catfish *Clarias gariepinus* (Burchell 1822) sperm. Aquaculture Research. 2000;31(3):317-24.

[97] Ohta H, Kawamura K, Unuma T, Takegoshi Y. Cryopreservation of the sperm of the Japanese bitterling. Journal of Fish Biology. 2001;58(3):670-81.

[98] Muchlisin ZA, Muhammadar. Long-term cryopreservation of baung spermatozoa, Mystus nemurus: effect of various cryoprotectants on motility and fertility. Torani. 2002;12:204-10.

[99] Viveiros AT, So N, Komen J. Sperm cryopreservation of African catfish,

*Clarias gariepinus*: cryoprotectants, freezing rates and sperm: egg dilution ratio. Theriogenology. 2000;54(9):1395-408.

[100] Lahnsteiner F, Berger B, Horvath A, Urbányi B, Weismann T. Cryopreservation of spermatozoa in cyprinid fishes. Theriogenology. 2000;54(9):1477-98.

[101] Kwantong S, Bart AN. Cryopreservation of black ear catfish, *Pangasius larnaudii*, (Bocourt) sperm. Aquaculture Research. 2006;37(9):955-7.

[102] Chao NH, Liao IC. Cryopreservation of finfish and shellfish gametes and embryos. Reproductive Biotechnology in Finfish Aquaculture. 2001:161-89.

[103] Vuthiphandchai V, Chomphuthawach S, Nimrat S. Cryopreservation of red snapper (*Lutjanus argentimaculatus*) sperm: effect of cryoprotectants and cooling rates on sperm motility, sperm viability, and fertilization capacity. Theriogenology. 2009;72(1):129-38.

[104] Betsy CJ, Kumar JS. Effect of egg yolk on enhancing quality of mrigal (*Cirrhinus mrigala*) spermatozoa during cryopreservation. Indian Journal of Fisheries. 2015;62(4):140-3.

[105] Betsy CJ, Kumar JS, Rajagopalasamy CB. Role of glucose in enhancing life and potency of *Cirrhinus mrigala* spermatozoa during cryopreservation. Journal of Applied and Natural Science. 2015;7(1):159-64.

[106] Betsy CJ, Kumar JS. Influence of bovine serum albumin (BSA) on the spermatological parameters of *Cyprinus carpio* during cryopreservation. Journal of Aquaculture in the Tropics. 2016; 31(1/2):103.

[107] Billard R. Artificial insemination in salmonids. In: R.N. Iwamoto and S. Sower (Editors), Salmonid Reproduction. Washington Sea Grant Publication.Seattle, WA, 1985:pp. 116- 128

[108] Rieniets JP, Millard JL. Use of saline solutions to improve fertilization of northern pike eggs. The Progressive Fish-Culturist. 1987;49(2):117-9.

[109] McPartlin LA, Littell J, Mark E, Nelson JL, Travis AJ, Bedford-Guaus SJ. A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis. Theriogenology. 2008;69(5):639-50.

[110] Lahnsteiner F, Berger B, Weismann T. Effects of media, fertilization technique, extender, straw volume, and sperm to egg ratio on hatchability of cyprinid embryos, using cryopreserved semen. Theriogenology. 2003;60(5):829-41.

[111] Linhart O, Rodina M, Cosson J. Cryopreservation of sperm in common carp *Cyprinus carpio*: sperm motility and hatching success of embryos. Cryobiology. 2000;41(3):241-50.

[112] Babiak I, Brzuska E, Perkowski J. Fractional factorial design of screening experiments on cryopreservation of fish sperm. Aquaculture research. 2000;31(3):273-82.

[113] Magyary I, Urbanyi B, Horvath L. Cryopreservation of common carp (*Cyprinus carpio* L.) sperm II. Optimal conditions for fertilization. Journal of Applied Ichthyology. 1996;12(2):117-9.

[114] Linhart O, Rodina M. Cryopreservation of common carp *Cyprinus carpio* and tench *Tinca tinca* sperm for gene resources conservation. In International Symposium on the Reproductive Physiology of Fish 2000.

[115] Alavi SM, Rodina M, Policar T, Kozak P, Psenicka M, Linhart O. Semen of *Perca fluviatilis* L.: Sperm volume and density, seminal plasma indices and effects of dilution ratio, ions and osmolality on sperm motility. Theriogenology. 2007;68(2):276-83.

[116] Betsy CJ, Kumar JS, Jawahar KT. Effect of different extenders and dilution ratios on the spermatological and fertilization parameters of *Cyprinus carpio* during cryopreservation. Biopreservation and biobanking. 2019;17(2):157-62.

[117] Grout BW, Morris GJ. Effects of low temperatures on biological systems. Edward Arnold; 1986.

[118] Gupta SD, Rath SC. Cryogenic preservation of carp milt and its utilization in seed production. In Proc. 3rd Ind. Fish. Forum 1993:pp. 11-14.

[119] Ponniah AG, Lal KK, Gopalakrishnan A, Srivastava SK. Use of fertilisation protocols to enhance hatching percentage with cryopreserved milt of *Cyprinus carpio.* National Academy Science Letters. 1998;21(7-8): 256-60.

[120] Ponniah AG, Sahoo PK, Dayal R, Barat A. Cryopreservation of *Tor putitora* spermatozoa: Effect of extender composition, activating solution, cryoprotectant and equilibration time. *Proceedings-national academy of sciences India section B*. 1999;69(1):53-60.

[121] Rafiquzzaman SM, Sarder MR, Islam MS, Sultana R. Sperm cryopreservation of Indian major carp, *Labeo rohita*: cryodiluents, sperm: cryodiluent dilution ratio and

*Cryopreservation and Its Application in Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.99629*

cryoprotectant concentration. Bangladesh Journal of Fisheries Research. 2007;11(2):141-52.

[122] Pérez-Cerezales S, Martínez-Páramo S, Beirão J, Herráez MP. Evaluation of DNA damage as a quality marker for rainbow trout sperm cryopreservation and use of LDL as cryoprotectant. Theriogenology. 2010;74(2):282-9.

[123] Stoss J, Holtz W. Successful storage of chilled rainbow trout (*Salmo gairdneri*) spermatozoa for up to 34 days. Aquaculture. 1983;31(2-4):269-74.

[124] Linhart O, Billard R, Proteau JP. Cryopreservation of European catfish (*Silurus glanis* L.) spermatozoa. Aquaculture. 1993;115(3-4):347-59.

[125] Franks F. *Biophysics and biochemistry at low temperatures*. Cambridge University Press, Cambridge, 1985: pp. 37-61.

[126] Hu E, Yang H, Tiersch TR. Highthroughput cryopreservation of spermatozoa of blue catfish (*Ictalurus furcatus*): establishment of an approach for commercial-scale processing. Cryobiology. 2011;62(1):74-82.

[127] Watson PF. The causes of reduced fertility with cryopreserved semen. Animal reproduction science. 2000; 60:481-92.

[128] Young JA, Capra MF, Blackshaw AW. Cryopreservation of summer whiting (*Sillago ciliata*) spermatozoa. Aquaculture. 1992;102(1-2):155-60.

[129] Kurokura H, Hirano R, Tomita M, Iwahashi M. Cryopreservation of carp sperm. Aquaculture. 1984;37(3):267-73.

[130] Scheerer PD, Thorgaard GH. Improved fertilization by cryopreserved rainbow trout semen treated with

theophylline. The Progressive Fish-Culturist. 1989;51(3):179-82.

[131] Ciereszko A, Dabrowski K. Effect of a sucrose–DMSO extender supplemented with pentoxifylline or blood plasma on fertilizing ability of cryopreserved rainbow trout spermatozoa. The Progressive fish-culturist. 1996;58(2):143-5.

[132] Ritar AJ. Artificial insemination with cryopreserved semen from striped trumpeter (*Latris lineata*). Aquaculture. 1999;180(1-2):177-87.

[133] Tiersch TR, Goudie CA, Carmichael GJ. Cryopreservation of channel catfish sperm: storage in cryoprotectants, fertilization trials, and growth of channel catfish produced with cryopreserved sperm. Transactions of the American Fisheries Society. 1994;123(4):580-6.

[134] Horváth Á, Miskolczi E, Urbányi B. Cryopreservation of common carp sperm. Aquatic Living Resources. 2003;16(5):457-60.

[135] Cognie PF, Billard R, Chao NH. La cryoconservation de la laitance de la carpe, *Cyprinus carpio*. Journal of applied ichthyology,1989; *5*(4), pp.165-176.

[136] Sultana M, Nahiduzzaman M, Hassan MM, Khanam MU, Hossain MA. Fertility of cryopreserved common carp (*Cyprinus carpio*) spermatozoa. University Journal of Zoology, Rajshahi University. 2009;28:51-5.

[137] Vuthiphandchai V, Wilairattanadilok K, Chomphuthawach S, Sooksawat T, Nimrat S. Sperm cryopreservation of silver barb (*Barbodes gonionotus*): cryoprotectants, cooling rate and storage time on sperm quality. Aquaculture Research. 2015;46(10):2443-51.

[138] Gorin NC. Cryopreservation and storage of stem cell. Bone Marrow and Stem Cell Processing.1992;pp.292-308.

[139] Rana KJ, McAndrew BJ. The viability of cryopreserved tilapia spermatozoa. Aquaculture. 1989; 76(3-4):335-45.

[140] Linhart O, Billard R, Proteau JP. Cryopreservation of European catfish (*Silurus glanis* L.) spermatozoa. Aquaculture. 1993;115(3-4):347-59.

[141] Conget P, Fernández M, Herrera G, Minguell J. Cryopreservation of rainbow trout (*Oncorhynchus mykiss*) spermatozoa using programmable freezing. Aquaculture. 1996;143(3-4):319-29.

[142] Warnecke D, Pluta HJ. Motility and fertilizing capacity of frozen/thawed common carp (*Cyprinus carpio* L.) sperm using dimethyl-acetamide as the main cryoprotectant. Aquaculture. 2003;215(1-4):167-85.

[143] Lahnsteiner FR. Semen cryopreservation in the Salmonidae and in the Northern pike. Aquaculture Research. 2000;31(3):245-58.

[144] Richardson GF, McNiven MA, Mansour N. Effect of methanol concentration and thaw rate on the viability and fertility of cryopreserved Arctic char, *Salvelinus alpinus* (L.), spermatozoa. Aquaculture Research. 2011;42(8):1096-100.

[145] Durbin H, Durbin FJ, Stott B. A note on the cryopreservation of grass carp milt. Aquaculture Research. 1982;13(3):115-7.

[146] Withler FC. Cryopreservation of spermatozoa of some freshwater fishes cultured in South and Southeast Asia. Aquaculture. 1982;26(3-4):395-8.

[147] Kumar K. A comparative-study of various extenders for cryopreservation of carp spermatozoa. Indian Journal of Animal Sciences. 1988; 58(11): 1355-1360.

[148] Basavaraja N, Hegde SN. Cryopreservation of the endangered mahseer (*Tor khudree*) spermatozoa: I. Effect of extender composition, cryoprotectants, dilution ratio, and storage period on post-thaw viability. Cryobiology. 2004;49(2):149-56.

[149] Yavas I, Bozkurt Y. Effect of different thawing rates on motility and fertilizing capacity of cryopreserved grass carp (*Ctenopharyngodon idella*) sperm. Biotechnology & Biotechnological Equipment. 2011;25(1):2254-7.

[150] Bozkurt Y, Yavaş İ. Effect of extender compositions, glycerol levels, and thawing rates on motility and fertility of cryopreserved wild African Catfish (*Clarias gariepinus*) sperm. The Israeli Journal of Aquaculture. 2017;69:1357-1364.

[151] Lahnsteiner F, Weismann T, Patzner RA. Methanol as cryoprotectant and the suitability of 1.2 ml and 5 ml straws for cryopreservation of semen from salmonid fishes. Aquaculture Research. 1997;28(6):471-9.

[152] Zell SR. Cryopreservation of gametes and embryos of salmonids fishes. Ann. Biol. Anim. Biochem. Biophys. 1978;18: 1089-1099.

[153] Erdahl DA, Graham EF. Preservation of gametes of freshwater fish, in Proceedings of International Conference of Animal Reproduction and Artificial Insemination, Madrid. 1980:317-326.

*Cryopreservation and Its Application in Aquaculture DOI: http://dx.doi.org/10.5772/intechopen.99629*

[154] Arii N, Namai K, Gomi F, Nakazawa T. Cryoprotection of medaka embryos during development. Zoological science. 1987;4(5):813-8.

[155] Nilsson E, Cloud JG. Cryopreservation of rainbow trout (*Oncorhynchus mykiss*) blastomeres. Aquatic Living Resources. 1993;6(1): 77-80. doi: 10.1006/cryo.1998.2084

[156] Zhang T, Rawson DM, Morris GJ. Cryopreservation of pre-hatch embryos of zebrafish (*Brachydanio rerio*). Aquatic Living Resources. 1993;6(2):145-53.

[157] Ahammad MM, Bhattacharyya D, Jana BB. Stage-dependent hatching responses of rohu (*Labeo rohita*) embryos to different concentrations of cryoprotectants and temperatures. Cryobiology. 2003;46(1):2-16. doi: 10.1016/s0011-2240(02)00138-4

[158] Harvey B, Kelley RN, Ashwood-Smith MJ. Permeability of intact and dechorionated zebra fish embryos to glycerol and dimethyl sulfoxide. Cryobiology. 1983;20(4):432-9.

[159] Horton HF, Ott AG. Cryopreservation of fish spermatozoa and ova. Journal of the Fisheries Board of Canada. 1976;33(4):995-1000.

[160] Loeffler CA, Løvtrup S. Water balance in the salmon egg. Journal of experimental Biology. 2000;52(2):291-8.

[161] Diwan AD, Harke SN, Panche AN. Cryobanking of Fish and Shellfish Egg, Embryos and Larvae: An Overview. Frontiers in Marine Science. 2020;7:251. doi: 10.3389/fmars.2020.00251

[162] Rall WF. Advances in the cryopreservation of embryos and prospects for application to the conservation of salmonid fishes.

In Genetic conservation of salmonid fishes. Springer, Boston, MA. 1993:pp. 137-158

[163] Jamieson BG, Leung LP. Fish evolution and systematics: evidence from spermatozoa: with a survey of lophophorate, echinoderm and protochordate sperm and an account of gamete cryopreservation. Cambridge University Press; 1991.

[164] Routray P, Suzuki T, Strüssmann CA, Takai R. Factors affecting the uptake of DMSO by the eggs and embryos of medaka, *Oryzias latipes*. Theriogenology. 2002;58(8): 1483-96. doi: 10.1016/s0093-691x(02) 01076-2

[165] Robles V, Cabrita E, Anel L, Herráez MP. Microinjection of the antifreeze protein type III (AFPIII) in turbot (*Scophthalmus maximus*) embryos: toxicity and protein distribution. Aquaculture. 2006;261(4):1299-306.

[166] Valdez Jr DM, Miyamoto A, Hara T, Edashige K, Kasai M. Sensitivity to chilling of medaka (*Oryzias latipes)* embryos at various developmental stages. Theriogenology. 2005;64(1):112-22. doi: 10.1016/j.theriogenology.2004.11.006

[167] Streit Jr DP, Godoy LD, Ribeiro RP, Fornari DC, Digmayer M, Zhang T. Cryopreservation of embryos and oocytes of South American fish species. In Tech; 2014.

[168] Whittingham DG, DG W. Attempts to preserve herring embryos at subzero temperatures. Arc Fischeriwiss 1978; 29: 75-9.

[169] Zhang T, Rawson DM, Morris BJ. Cryopreservation of prehatch embryo of zebrafish. Aquatic Living Resources. 1993; 6: 145-53.

[170] Hagedorn M, Hsu E, Kleinhans FW, Wildt DE. New approaches for studying the permeability of fish embryos: toward successful cryopreservation. Cryobiology. 1997;34(4):335-47.

[171] Anchordoguy TJ, Rudolph AS, Carpenter JF, Crowe JH. Modes of interaction of cryoprotectants with membrane phospholipids during freezing. Cryobiology. 1987;24(4):324-31. Section 4
