12.1 Introduction
12.2 Quality of gametes
12.3 Methods of preservation of gametes
The literature on gametes of fishes, though voluminous, rarely deals with the quality of gametes in terms of motility and survival of spermatozoa or the. degree of ripeness of ova (see Kuo, Shehadeh and Milisen, 1973; Barnabé, 1976; Fujiya, 1979). Some information is, however, available on the salmonid fishes (Bagenal, 1969; Gall, 1974; Lindberg, 1979), where ova from females spawning for the first time are reported to be smaller and inferior as compared to those from the second and subsequent spawnings (Gall; 1974) even though their chemical composition is similar (Philipps and Dumas, 1959). Hulata, Moav and Wohlfarth (1974) have reported on gonad development and the size of ova from the European and Chinese races of common carp. According to Billard, Dupont and Barnabé. (1977) the duration and intensity of motility of spermatozoa of Dicentrarchus labrax decrease as the spawning season progresses.
The sperm, which is immotile in the testis, becomes motile on contact with the medium in which fertilization takes place. Osmotic pressure, pH, and ionic content of the medium, particularly calcium, may be important in the activation of spermatozoa (Sundararaj and Nayyar, 1969c; Nayyar and Sundararaj, 1970a; Hines and Yashouv, 1971). The duration of activity of spermatozoa varies with the species and is generally not longer than a couple of minutes (see Harvey and Hoar, 1979).
The most crucial aspects of gametes, which are of practical significance in aquaculture, pertain to survival and motility of spermatozoa. In the males of most species, dense semen having highly motile spermatozoa can often be obtained without injection (Hines and Yashouv, 1971; Billard, 1978b). Administration of pituitary extracts or gonadotropin brings about thinning of the seminal plasma which facilitates spermiation. The hormonal regulation of spermiation has been discussed in section 11.
The ripeness of eggs is of great significance in aquaculture. Overripeness of ova can be avoided by proper timing of stripping since ripe ova remaining unspawned for long periods after ovulation become overripe and do not develop normally (Nomura, Sakai and Takashima, 1974; Sakai et al., 1975; Takashima, Nomura and Sakai, 1975). Further, it is not uncommon to come across the phenomenon of "plugging" in gravid females subjected to overdoses of hormones during hypophysation. In such cases, maturation and ovulation takes place but natural spawning cannot be accomplished since a mass of ovarian eggs forms a plug at the urogenital opening preventing the free flow of eggs. The causes for "plugging" have not been investigated at all and merit study.
In a large number of cultivated species, the maturation of gonads in the two sexes is not synchronous. Often, the male shows testicular recrudescence earlier than the female (see section 2 on Reproductive cycles and environmental cues). This results in a situation where at the beginning of the spawning season the males are active while the females are not yet ready for spawning and vice versa towards the end of the breeding season. Under such circumstances, it would be prudent to preserve the gametes for use in artificial fertilization as and when required. Further, establishment of a reserve of genetic material having known and desired qualities is important for initiation of selective breeding programmes.
While considerable research has been conducted on preservation of spermatozoa in the salmonids, very little attention has been bestowed on the tropical species of cultivated fishes (see Wiltzius, 1973; Shehadeh, 1975; Horton and Ott, 1976; Erdahl and Graham, 1978; Mounib, 1978; Harvey and Hoar, 1979). Apart from rainbow trout (Billard and Jalabert, 1974; Billard et al., 1974; Horton and Ott, 1976; Zell, 1978), spermatozoa of carps, Cyprinus carpio (Moczarski, 1977; Stein and Bayrle, 1978; Pavlovici and Vlad, 1976), Aristichthys nobilis (Sin, 1974), Ctenopharyngodon idella (Harvey and Hoar, 1979), Labeo rohita (Harvey and Hoar, 1979), and Mugil cephalus (Pruginin and Cirlin, 1975; Chao, Chen and Liao, 1975) have been subjected to cryopreservation (cooling and storage at subzero temperatures of liquid nitrogen -196°C) by using either dimethyl sulfoxide, glycerine, ethylene glycol or other cryoprotectants and diluents; dimethyl sulfoxide gives superior results (Guest, Avault and Roussel, 1976). Details concerning the ratios of diluent and cryoprotectant used, freezing rate, storage and thawing techniques have been reviewed by Harvey and Hoar (1979). Since cryopreservation requires liquid nitrogen, fish spermatozoa may be frozen, like those of cattle and livestock at artificial insemination centres using the same facilities. The requirements for short-term preservation of spermatozoa are more easily met with than those for cryopreservation (see Harvey and Hoar, 1979). Semen from common carp has been successfully stored at 0° to 5°C for up to 5 h (Hulata and Rothbard, 1979), while that from rainbow trout has been stored at -2°C for up to three weeks (Stoss, Büyükhatipoglu and Holtz, 1978). Similarly, semen of Lates calcarifer has been stored under refrigeration (Manevonk et al., 1978). Milt from Labeo rohita maintains its fertilizing capacity for up to 4 h when stored in 1 percent glycerine in frog Ringer solution at 28°C (Bhowmick and Bagchi, 1971).
Attempts at cryopreservation of ova have not been as Successful as for sperm. Zell (1978) has reported the first successful cryopreservation of unfertilized ova and zygotes of salmonid fishes. Ova frozen in liquid nitrogen at -20°C for 5 min proved to be fertile, While zygotes frozen to -50°C survived the exposure. All other reports describe unsuccessful attempts to cryopreserve eggs of other species (see Harton and Ott, 1976). Davenport, Vahl and Lönning (1979) and Pullin and Bailey (1979) have described attempts to prolong the incubation of plaice ova at subzero temperature after prior embryonation to various stages. Prolongation of embryonation may spread the availability of ova over short spans of time.
