Ultralow Temperature Semen Diluents

Ultralow Temperature Semen Diluents are specially formulated extender solutions used to preserve semen at extremely low temperatures (<0°C), typically during cryopreservation (−196°C in liquid nitrogen). These diluents are essential for maintaining the viability, motility, and fertility of sperm cells during freezing, storage, and thawing.

The advances in semen preservation techniques lead to the storing at ultra-low temperature.

The commonly used dilutor for the preservation of semen at subzero temperature are as follows:

1. Tris-Egg Yolk-Glycerol Diluent / Universal Diluent
2. Sodium Citrate Extender
3. Milk Extender
4. Lactose Egg Yolk Extender

1. Tris-egg Yolk-glycerol Diluent (Universal Diluent)

Inclusion of glycerol at room temperature was found as satisfactory as 5°C. Tris diluter allows filling and sealing of poly vinyl straws at room temperature while freezing the semen.

Importance of Egg Yolk as a Medium

• The value of the yolk of the hen’s egg as a diluting medium in semen preservation was found by Philips during 1939.
• It helped in rapid growth in recent years of artificial insemination throughout the world. Egg yolk protects lipoprotein sheath of sperm against cold shock.
• The active principle in yolk responsible for this is now thought to be lecithin or a similar phospholipid occurring either free or in combination with protein.
• It contains glucose, proteins, vitamins and required viscosity index which may be of advantage to the cells.
• The yolk contains the amino on oxidative deamination.
• The hydrogen peroxide formed, which is toxic to the sperm, may be destroyed by additions of the enzyme catalase to the yolk diluents, under anaerobic conditions the problem does not occur.

2. Sodium Citrate Extender

Egg yolk citrate is prepared as per the procedure given in refrigerated storage of semen (except sulphanilamide-because sulphanilamide crystallizes during freezing and hence it is not used in dilutors used for deep freezing).

• Sodium citrate dihydrate- 2.9 gm
• Penicillin G sodium- 1 lac unit
• Dihydrostreptomycin sulphate- 100 mg
• Distilled water-make up to 100 ml
• To the above solution 20% egg yolk and 10% glycerol.

3. Milk Extender

The preparation is explained as milk diluents for refrigerated storage. The sulphanilamide is not added but the glycerol is added at the level of 10%.

4. Lactose Egg Yolk Extender

The sugars like glucose, lactose, raffinose and sucrose have been used with semen for freezing bull spermatozoa. The results indicated that sperm survival rate increased as the molecular weight of the sugar is increased.

Extenders for Freezing of Semen

The thought that the vital processes might be suspended at low temperatures had inspired attempts to preserve living cells in the frozen state.

The damage during freezing is caused by:

1. Internal ice crystal formation that affects the structure of the spermatozoa
2. The increase in solute concentration as water is withdrawn from the suspension medium and by interaction of these two physical factors. Hence the successful freezing of sperm will depend upon the prevention of occurrence of the above factors. This could be aimed by suspending the sperm in an ideal extender that could afford cryoprotection during freezing.

The basic components of extenders for freezing of bovine spermatozoa are:

• Buffer
• Fructose
• Glycerol
• Egg yolk
• Antibiotic

Buffer

A variety of inorganic and organic buffers have been used for deep freezing of semen. An organic buffer known as Trishydroxymethyl amino methane has been successfully used for deep freezing bull, buffalo, ram, buck, dog, poultry and human spermatozoa and hence Tris could be named as ‘Universal diluent’. Tris prolongs the life of sperm at room temperature, penetrates cell and act as intracellular buffer, less toxic in critical temperature during freezing and provides better clarity under microscope.

Fructose

Addition of fructose provides glycolysable substrate for the sperm, prevents sperm agglutination, maintains required osmotic tension and electrolyte balance and gives added protection during deep freezing.

Glycerol

Glycerol is the most widely used cryoprotective agent for bull spermatozoa. The exact mechanism and the loci of activity is not clearly understood.

The possible modes of action proposed are:

• Modifies shape and size of ice crystals
• Binds water and decreases freezing point of solution and less ice is formed
• It acts through salt buffering mechanism
• Reduces the solute concentration
• It prevents the denaturation of proteins and rupture of plasma membrane.

Although a number of related compounds like ethylene glycol, polypropylene glycol and dimethyl sulfoxide (DMSO) have been tried, satisfactory results have not been achieved with these agents.

Egg Yolk

When mammalian sperms are cooled to 5ºC they are subjected to ‘cold shock’ which causes leakage of intracellular enzymes, potassium, lipoprotein, ATP and other material from the sperm. The most effective means of protecting the spermatozoa against the effects of cold shock is providing lecithin, proteins, lipoproteins and similar compounds found in egg yolk or milk. In addition to this, glucose, protein, vitamins present in yolk are utilized by the spermatozoa.