SEMEN PRESERVATION

Requirement of semen from sires of high genetic merit demands the need of semen

preservation. Transportation and prolonged use are the added advantages of semen preservation.
Basic principle of semen preservation is the reduction of metabolism of spermatozoa

either physically or chemically so that they maintain only minimum essential cellular activity.

Lowering of temperature is the generally accepted way of semen preservation in which the

process can be reversed.

There are mainly three methods of semen preservation:
1. Refrigeration temperature preservation
2. Room temperature preservation
3. Cryopreservation (freezing)

Extenders

Extender is the combination of substances which can be added to the semen for

preserving or extending the life span of the spermatozoa.

Features of an ideal extender:

1. It should be isoosmotic .with the seminal plasma of the respective species and be capable

of maintaining it during preservation

2. It should maintain a proper balance of mineral elements which is essential for viable

sperms.

3. It should provide nutrients for both aerobic and anaerobic metabolism of the

spermatozoa.

4. If the semen is preserved at low temperature, the extender should have a component to

protection the sperms from cold shock. Eg. lecithin or lipoproteins (egg yolk, whole milk,

skim milk etc.).

 5. It should provide chemical means for buffering the toxic end products of sperm

metabolism. Eg. Citrate, bicarbonate, Zwitter ionic buffers: Tris, TES, MES, HEPES,

PIPES, MOPS, BES etc. (mainly chlorides).

6. There should be a source of reducing substance to protect the sulphydryl containing

enzymes.

7. There should be some way of inhibition of bacterial multiplication.

8. If the mode of preservation is freezing/cryopreservation, cryoprotectant should be

included to prevent cryoinjuries of the spermatozoa.

Refrigeration temperature preservation

Here, the semen is preserved at refrigeration temperature or 5°C for 2-5 days. This was

the common method for semen preservation before the introduction of cryopreservation. Here,

the sperm metabolism is reduced by lowering the temperature. Several extenders have been used

for this mode of preservation.

Eg:-

1. Egg yolk phosphate extender: First extender (Philips and Lardy, 1940): Equal volume of egg

yolk and phosphate buffer (2g of Na 2HPO4 12H2O and 0.2 g of KH 2PO4 in 100 ml distilled

water)

2. Egg yolk citrate extender: This is most commonly used for bull semen preservation at

refrigeration temperature. Salisbury in 1941 found that citrate was more useful than PO4.

Chelating property of citrate depresses fat globules in egg yolk and improves the solubility of

protein fractions in egg yolk.

 2.96 % sodium citrate-80 ml (2.96 g of Trisodium citrate dihydrate in 100 ml of double distilled
water)

Egg yolk-20 ml

Benzyl penicillin- 500-1000 IU /ml

Streptomycin sulphate- 500-1000 µg/ ml

3. Milk based extenders: Milk based extender should be undergone heating to inactivate lactenin

(spermicidal factor) before use. Heating temperature varies with different milk preparations so

that lactenin should be inactivated whereas the proteins and sugar moieties should not be

damaged.

Eg.Goat milk diluent: This is used for buck semen preservation at refrigeration temperature.

Heat goat milk to 92-95°C by keeping it in water bath for 10 minutes, cool and separate the

cream layer-to destroy and remove lactenin. Take required quantity of goat milk and add Benzyl

penicillin @ 500-1000 IU /ml and Streptomycin sulphate@ 500-1000 µg/ ml.

4. Egg yolk Sodium bicarbonate goat milk diluent: This is mainly used for buffalo semen

preservation at refrigeration temperature.

1% NaHCO3 -1 ml

5% glucose - 24 ml

goat milk - 25 ml

Mix these together and replace 2.5 ml of the extender with 2.5 ml of egg yolk. Then add Benzyl

penicillin @ 500-1000 IU /ml and Streptomycin sulphate@ 500-1000 µg/ ml.

5. Tris Citric acid Fructose Egg Yolk extender:

Tris Hydroxy methyl amino methane: 2.42 g

Citric acid Mono hydrate: 1.68 g

Fructose/ glucose: 1.25 g

Egg yolk: 10 ml

Double distilled water upto 100 ml

Add Benzyl penicillin @ 500-1000 IU /ml and Streptomycin sulphate@ 500-1000 µg/ ml.

6. Cornell University extender (CUE)

In 1956, Foote et al described CUE as an improved semen extender. It contains 1 part egg

yolk, 4 parts of special buffer containing sodium bicarbonate, potassium chloride, glucose,

glycine, sodium citrate and antibiotics. This extender was found to be as good as egg yolk citrate

for storage of bull semen at refrigeration temperature for 2-3 days.

7. CAW (Citric acid whey diluent) : This extender was developed at NDRI, Karnal by Ganguly

et al (1973). The extender is available as packets. Contents of each packet are suspended in 100

ml of distilled water and stir well. Then, allow the curdled suspension to stand for 5-10 minutes

and filter through cotton plug and wait for 5 -10 minutes. Adjust the pH to 6.8 with 10%

NaOH/HCl. Add antibiotics.

The bull semen extended with 130 million sperms /ml gets a storage life of 4-5 days at 5°C.

Damage of spermatozoa during preservation at refrigeration temperature:

Cold shock: The irreversible damage occurring to the spermatozoa upon rapid reduction of

temperature is termed as cold shock. This is related to the lipid composition of the membrane

bilayer affecting the fluidity of the plasma membrane. As the temperature is lowered, restriction

of lateral movement of membrane phospholipids results in a transition from a fluid to a gel

phase. Due to the different transition temperatures for different membrane lipids, phase

separations may occur, whereby proteins become irreversibly clustered. The cholesterol:

phospholipid ratio is another factor which decides the cold shock resistance of spermatozoa. For

example, pig spermatozoa are highly susceptible to cold shock as they have very low cholesterol:

phospholipid ratio. Cold shock results in leakage of vital substances from the cells like enzymes,

lipoprotein, ATP, intracellular potassium, phosphorus etc. Respiration and glycolysis are

impaired/ abolished.

Cold shock can be prevented by the addition of lecithin or lipoprotein containing

components like egg yolk, whole milk, skim milk etc. By adopting optimum cooling rate of the

extended semen also, cold shock can be controlled. Eg. One degree per two minutes.

Production of reactive oxygen species also results in damage of spermatozoa during

preservation of semen at refrigeration temperature.

Room temperature preservation

Room temperature preservation of semen can be used if there is no refrigeration facility. The

nil chance of spermatozoan damage due to cold shock is another merit of ambient temperature

preservation of semen. The medium in which spermatozoa are suspended should inhibit those

pathways that are detrimental to their survival at higher temperatures. The optimum temperature

range for preservation is 18◦C to 24◦C.

Eg. for extenders:

 1. IVT : Van Demark and Sharma, 1957 proposed CO2 narcosis as an effective means to

maintain viability and fertilizing ability of bovine spermatozoa for 6 to 7 days at room

temperature. The first diluent designed on the basis of CO2 immobilization of spermatozoa was

the IVT diluent. Norman et al. (1958) suggested lowering the pH inhibits metabolic activity of

spermatozoa.

IVT-Composition for 1 L.

Sodium Citrate dihydrate: 20 g

Sodium Bicarbonate : 2.1 g

Pottasium Chloride : 0.4 g

Glucose : 3g

Sulphanilamide : 3g

Gassed with CO2 until pH falls to 6.35

Antibiotics added at the previously described levels

EY : 10%

Distilled water is added to make up the volume to 1L.

A decrease in pH down to 5.5 is well tolerated by spermatozoa, and the effect can be

reversed by alkaline conditions, but a pH below 5.5 is spermicidal and causes irreversible

enzyme denaturation.
 The main difference between the IVT diluent and the CUE is that CUE is self carbonating

and relies on the action of citric acid on bicarbonate to release CO2 with no major effect on the

pH of the medium

2. Coconut Milk Extender: Coconut milk maintains the viability of spermatozoa due to its rich

content of Potassium. Vitamin C in coconut water helps to reduce the oxidative stress during

storage. Coconut milk is also a rich source of nutrients like sugars, amino acids, minerals and

vitamins.

Coconut Milk Extender composition (100 ml)

Na Citrate dihydrate: 2.2 g

Sulphanilamide : 0.3g

Catalase : 15,000 CU (catalase Units)

Coconut water : 15 ml

EY : 7 ml

Polymixin B : 0.01g

Mycostatin : 1000 units

Pencillin G sodium : 0.06 g

Streptomycin Sulphate : 0.135 g

Distilled water:up to 100 ml

NaOH 10% or 0.1 N and HCl 0.1 N can be used to adjust pH to 7.4

3. Milovanov’s extender:

Potassium dihydrogen phosphate: 0.72g

Sodium citrate dihydrate: 20.276 g

Glucose: 5.7 g
 Sodium bicarbonate: 1.26 g

Sulphanilamide: 3 g

Egg yolk: 11 %

Distilled water: up to 1 L

Penicillin G Sodium: 10 lakh IU

Streptomycin: 1 g

4. CAPROGEN extender: Shannon (1965) proposed Nitrogen gassing to inhibit metabolic

activity of spermatozoa and developed the CAPROGEN diluent for bovine semen. This

extender replaced the self-carbonating concept with a method to reduce the dissolved O

levels in the medium with Nitrogen gas. This has no effect on the pH, but substantially

reduce the metabolic activity of spermatozoa.

CAPROGEN extender consists of Sodium citrate, glucose, glycine, glycerol, caproic acid

and antibiotics. It is gassed with N2 before mixing with semen. The quantity of egg yolk in

this extender has to be reduced to as little as 2 % v/v. Volatile fatty acids like caproic acid

maintains the membrane integrity of spermatozoa.

Damage of spermatozoa during preservation at room temperature:

Production of reactive oxygen species is the inevitable disadvantage of room temperature

preservation of semen. The most damaging are the superoxide anion (o2-), peroxide (H2O2) and

the hydroxyl free radical (OH) of which peroxide is the most pernicious. It is mainly produced

by the deamination of aromatic amino acids by the enzyme AAAO (Aromatic Amino Acid

Oxidase). This enzyme is mainly released from the dead spermatozoa and is specific for L-

aromatic amino acids- in particular, L-phenylalanine. Egg yolk is a rich source of L-

phenylalanine.
 The peroxide content in the preserved semen can be reduced by the addition of catalase to

the extender. Vitamin C in coconut water also helps to reduce the oxidative stress during storage.

The quantity of egg yolk to be included in the extender and also the rate of extension are

important to reduce the oxidative stress of spermatozoa under storage.