Cryopreservation of Male Gamete
Semen cryopreservation is useful to preserve the superior and threatened genetic resources, genetic exchanges between captive as well as wildlife conservation centers, and semen having low sperm concentration and motility.
Cryopreservation is one of the possible ways to establish germ-plasm bank. It is effective in those wild species where narrow genetic variations exist like cheetah. Semen of wild animals are to be preserved precisely as most (usually more than 60%) of the spermatozoa are pleomorphic and teratospermic in nature. The chemicals and processes are to be accurate during cryopreservation.24.2.1 Collection of Male Gamete
Artificial vaginas or vaginal condoms are generally used to collect the male gamete. Male gamete along with semen can be collected by electro-ejaculation method. Hazards may arise to restrain the animals by this process. Post-coital sperm recovery and epididymal spermatozoa from recently poached or dead animals are the alternate processes of semen collection from wildlife. C ryopreservation of testicular tissue is also used successfully in domestic bovines, porcines, cats, and mice and some wild species like monkeys, blackbuck (Antilope cervicapra), jungle cat (Felis chaus), lion (Panthera leo), leopard (Panthera pardus), Timor deer (Rusa timorensis), Tenasserim muntjac (Muntiacus feae), and Sumatran serow (Capricornis sumatraensis).
24.2.2 Cryopreservation of Epididymal Spermatozoa
The quality of epididymal spermatozoa remains almost unchanged if the collection occurs within 4 h after death. The rate of fertility is vigorously altered after 24 h. The rate of abnormality is reduced if the epididymis is preserved at 5 °C immediately after death and the cryopreservation process starts within 24 h. The epididymal spermatozoa are not fully matured and have certain abnormal features, like the presence of nonmotile spermatozoa of about 15%, cytoplasmic droplets of nearly 14%, dead and less intact plasma membrane of 13% in bull.
But, epididymal spermatozoa exhibited effective results following species-specific protocol of cryopreservation. Successful cryopreservation was done from the epididymal spermatozoa of a gaur (Bos gaurus) bull and pregnancy was confirmed after 41 days by ultrasonography inseminating to the Holstein cow twice with 80 ? 106 spermatozoa. Cryopreservation of epididymal spermatozoa followed by successful pregnancy was demonstrated in certain semi-domesticated and wild animals like alpaca (4 ? 106 sperm per insemination). Pregnancy has been achieved by inseminating 4 ? 106 sperm per dose in wild cheetahs, wolves, and other animals where higher litter size (three) was obtained with a higher dose at 6-16 ? 10 6 sperm. Cryopreserved post-thaw spermatozoa having 40-50% motility and 50-60% intact acrosomes are biologically viable. Isolation of effective motile spermatozoa: Certain species-specific substances sometimes are used to clear the unwanted substance from the semen samples before cryopreservation to get better post-thawed motile spermatozoa. Papain, a protease used in alpaca to dissolve the protein mucin 5B of the semen cause to reduce the viscosity without disturbing the motility, acrosome integrity, viability, and DNA integrity of the spermatozoa. The epididymal sperm can be preserved after separating the contaminated cells and cellular debris by single-layer centrifugation using speciesspecific colloid formulations. Androcoll-C, a glycidoxypropyltrimethoxysilane-coated silica optimized for dog spermatozoa (where “C” stands for canine,Androcoll-E is a similar substance used for equine species), may be used at 30% to separate the carcasses of the epididy- mal spermatozoa of canine wildlife like wolf, etc.
24.2.3 Cryopreservatives and Cryoprotectants
Cryopreservatives and cryoprotectants are used in the semen during cryopreservation to protect the spermatozoa from thermal shock, formation of ice crystals within spermatozoa, and reduce viscosity. The Cryopreservatives comprised a combination of various substances that are used as diluents or extenders during cryopreservation.
The effective extender has the characteristics of cryoprotectiveness, energy sources. Egg yolk with varying concentrations from 5% to 20% is used in different species. Egg yolk is used along with glycerol, dimethylsulfoxide (DMSO), and ethylene glycol are the common cryoprotectants, where glycerol is mostly effective but has some toxic effects on certain species. These substances act as intracellular cryoprotectants. Egg yolk and glycerol at 4-6% are mostly used in cryopreservation of domestic mammalian male gamete. This combination is also used to preserve the semen of some wild species like Asian elephants (Elephas maximus), rhinos (Ceratotherium simum, C. simum cottoni), leopards (Neofelis nebulosa), bears (Ursus arctos), rhesus monkeys, felids, and marine mammals. DMSO and dimethylacetamide are effective in saltwater crocodiles (Crocodylus porosus) and kangaroos (Macropus giganteus). Glycerol is to be added at a particular temperature and specific step of cryopreservation, otherwise it may drag the water causing to shrinkage of the sperm. In cheetah sperm, glycerol is generally added slowly for a period of 60 min at ambient temperature just before cryopreservation. Some monosaccharide, disaccharide, and polysaccharide sugars like lactose, maltose, glucose, fructose, and citrate- or tris-based (SHOTOR), skimmed milk is usually used to provide the energy and act as diluter as well as extracellular cryoprotectant. Sugars trap the salt in the unfrozen water caused to inhibit the eutectic freezing and to increase the viscosity resulting to prevent the fast cooling, pattern of crystallization, and protect the sperm membrane. Glucose and fructose are permeable to sperm membrane and provide energy. Hence, monosaccharides are comparatively less effective than lactose in respect of its cryoprotective role. Lactose is larger in size than glucose and fructose and cannot easily enter inside the sperm, rather protect from osmotic change during dilution resulting in better survivability of the spermatozoa. Thus, lactose is effective than other diluents. It is most suitable for epididymal spermatozoa cryopreservation and for those species which have less fructose in semen like alpaca. Lactose is also effectively used in buck and garut ram semen. Some efficient extenders are 5% egg yolk extender with glycerol diluted with lactose at 1:3. It is effective in alpaca. Glucose or fructose is routinely used as a diluter in many species like cattle, sheep, and pig. Successful results were also obtained by using raffinose (trisaccharide) in Etawah buck, trehalose (non-reducing sugar having di-glucose) in garut ram, trehalose and sucrose in bull, and maltose in garut ram semen. Better result has been obtained in lactose-based semen in camel. Skim milk is effective in Asian elephant (Elephas maximus indicus) and the Indian rhinoceros (Rhinoceros unicornis).24.2.4 Form of Freezing
Semen can be frozen in straw or in pellet form and can be preserved in liquid nitrogen at -196 °C. Preservation can also be possible on a dry ice block in the laboratory. Straws are more secure and easy to handle. It has a more surface-to- volume ratio, resulting in better post-thaw motility.
24.2.5 Rate of freezing
The rate of freezing should follow the surface-to-volume ratio of the spermatozoa and the permeability of the membrane. Thus, small-headed spermatozoa (like alpaca) require a faster freezing rate than large-headed (like bull). In general, slow freezing at -0.5 °C/min demonstrated optimum result in most of the mammalian species.
24.3