~ Dibakar Baruah and Sagarika Barman
The cryopreservation of mammalian sperm is a complex process that involves balancing many factors in order to obtain satisfactory results. To insure even minimal success, not only proper diluent, sperm dilution rate, cooling rate and thawing rate are required, but also an intricate knowledge of the sperm physiology for the species is essential to maximize post-thaw recovery of sperm and consequently the fertility.The cryopreservation of goat semen is a complex process that involves balancing of many factors to achieve optimum results. Goat sperm require special attention to maximize the post-thawing viability and fertility. For example,.the deleterious interaction between egg yolk and the bulbourethral gland secretions exists for goat semen that does not exist for other species, Due to these and other differences between the sperm of the domestic species, this review discusses are biochemical characteristics of semen, dilution rate, diluents, osmolality, buffering capacity and pH of diluents, sugars used in diluents, cryoprotectants, freezing and thawing of semen and traditional and recent additives used in goat semen cryopreservation.
Biochemical characteristics of semen
It has been reported that goat spermatozoa are susceptible to various biochemical and physiological hazards during freezing and thawing processes. Specific biochemical factors are present in seminal plasma, which prevent the damages caused by the cryogenic procedures. Thus, there is a need to develop biochemically defined species specific diluents and cryogenic procedures that may result in the improvement of viability and fertility of frozen thawed goat spermatozoa.
The purpose of a cryopreservation diluent is to supply the sperm cells with sources of energy, protect the
cells from temperature-related damage, and maintain a suitable environment for the spermatozoa to survive temporarily. Diluent for cryopreservation of goat sperm include a non-penetrating cryoprotectant (milk or egg yolk), a penetrating cryoprotectant (glycerol, propylene glycol or dimethyl sulfoxide), a buffer (Tris), one or more sugars (glucose, fructose, lactose or trehalose), salt (sodium citrate) organic acid (citric acid) and antibiotics (penicillin, streptomycin)..
Osmolality of diluent
While the osmolality of the medium may vary within limits, goat spermatozoa prefer a hyperosmotic medium for cryopreservation.Osmotic stress attributed to differences in relative permeability of cryoprotectants appears to be an important factor in cryopreservation. Goat spermatozoa survive cryopreservation and can remain fertile in diluent composed of a wide range of ingredients. As the permeability of most the cryoprotectants is much lower than that of water, there is a net influx of water with a resulting increase in cell volume. As the osmolality of the diluent may vary within limits, goat spermatozoa prefer hyperosmotic medium for cryopreservation.
Buffering capacity and pH of diluents
In order to sustain the viability and fertilizing ability of sperm it is essential to maintain proper environment by controlling pH fluctuations in cryopreservation media. Therefore, dilution of semen in a suitable buffer is a very crucial factor that affects sperm survivability during cryopreservation. Tris seems to be the buffer of choice for use with buck sperm, but this is not the case in other species where negative aspects of the buffer, such as increased capacitation and acrosome reaction rates, and swelling of the apical ridge of the sperm cell, have been documented .
Sugars play an important role in sperm respiration and provide osmotic balance and cryoprotection in
Diluent . The addition of sugars in diluent is logical as seminal plasma itself contains sugars, but of all the
sugars, fructose has the greatest molar concentration in neat goat semen.
The exact mechanism by which trehalose and other non-penetrating sugars act on the sperm plasma membrane is unknown, but it is supposed that these sugars penetrate into the plasma membrane of the spermatozoa and form hydrogen bonds with the polar head groups of the phospholipids.
It is essential that a semen sample be diluted properly so that there are sufficient numbers of sperm and sufficient diluent to accommodate the cells in an insemination straw, so that a high fertility rate can be achieved using the least number of inseminations and the lowest number of sperm per insemination. The site of semen deposition is the one of the important factors influencing the number of sperm required for acceptable fertility. Other factors which depends on acceptable fertility are the type of AI procedure, time of year (season/out of season), type of oestrus (natural or induced), category of animal (pluriparous or nulliparous) and physiological state (lactating or dry).
Freezing and Thawing of semen
During cryopreservation spermatic membrane suffers a series of changes in fluidity due to changes in temperature. Evidence demonstrated the detrimental effects of cryopreservation on sperm motility, the integrity of membrane, DNA function, and mitochondrial function. It is generally accepted that the cryopreservation process itself reduces more than 50% of the sperm viability. During cryopreservation, the spermatozoa are subjected to biochemical, osmotic, thermal and mechanical stresses, which are conspicuous at dilution, cooling, equilibration, freezing and thawing stages. Freezing of sperm in straws is more expensive and laborious than the pellet freezing technique, but each sample can be accurately labeled for inventory management.
Thawing of semen samples is based on the method used to freeze the semen sample. Sperm pellets should be thawed in a dry test tube at 37°Cwhile the straws can be thawed using various time and temperature combinations.
Thus, the survival of spermatozoa can be significantly influenced by cooling rate from temperature just above 0°C after freezing and thawing.
Future studies may direct towards evaluation of a simple, rapid and economical method for processingand freezing of buck semen in terms of determination of critical temperature, freezing and prevention of cryo-injury to acrosome during freezing-thawing process with acceptable fertility. In addition, use of sex-sorted sperm for AI should be promoted as a means of increasing the efficiency of reproduction in goats, especially in the dairy business where males have little commercial value. Sex-sorted semen has been used successfully in several species, including cattle, horses, pigs and sheep but not in goats in India.
The semen cryopreservation consists of semen samples collection, the assessment of the collected sample for concentration, motility, and normal morphology, the dilution of the semen sample. The diluted semen is loaded into plastic straws and sealed with polyvinyl acetate powder and the straws are dipped into liquid nitrogen for cooling and freezing purposes. There is need to identify the seminal characteristics which directly affects the freezing ability of spermatozoa. Critical studies to establish the fertility marker-based selection of the bucks for using in semen cryopreservation might be of great importance in years to come. Further research work will help to investigate the effect of factors such as reactive oxygen species
on the semen quality of freezing and thawing process.
Authors – Dibakar Baruah1 and Sagarika Barman2
- M.V.Sc Scholar , College of Veterinary Science, Khanapara
- M.V.Sc Scholar , College of Veterinary Science, Selesih, Mizoram