TABLE OF CONTENTS
Refrigerator Temperature Semen Diluents
Refrigerator temperature semen diluents are special solutions used to preserve semen at low temperatures, typically around 4 to 6°C. These diluents help maintain sperm viability and motility during short- to medium-term storage.
Here the semen is preserved under refrigeration temperature. Until the invention of frozen semen technology this was the most common method used to preserve the semen.
Various semen diluents have been evolved by different workers. The semen is preserved in these diluents at 4-6°C which can be used for insemination up to 72 hours. Some of the diluents are described.
Refrigerator temperature semen diluents are as follows:
- Egg Yolk Phosphate Extender (EYP)
- Egg Yolk Citrate Extender (EYC)
- Caprogen
- Kampschmidt or Egg Yolk Glucose Bicarbonate Extender (EYGB)
- D2 Dilutor
- D5 Dilutor
- Glycine Dilutor
- Milk Dilutor
- Citric Acid Whey
- Tris-yolk Extender
1. Egg Yolk Phosphate Extender (EYP)
Philips and Lardy (1940) developed this dilutor.
Composition
| Components | Quantity |
|---|---|
| Disodium hydrogen phosphate | 2.0 gm |
| Potassium dihydrogen phosphate | 0.2 gm |
| Distilled water | 100 ml |
- Equal parts of this buffer solution and egg yolk are mixed together to constitute the dilutor.
- This dilutor preserves bull semen for 72 to 96 hours and buffalo semen for an average of 48-72 hours.
- In this dilutor, visibility of sperm motility was not very clear when examined under microscope.
- In field tests this extender provided good fertility.
2. Egg Yolk Citrate Extender (EYC)
Salisbury et al. (1941) evolved egg yolk citrate dilutor which has become popular for diluting and preservation of bull and buffalo bull semen.
The citrate available in this diluent is chelating agent and it disperses the fat globules of the egg yolk and improves visibility when examined under microscope.
| Components | Quantity |
|---|---|
| Sodium citrate dihydrate | 2.9 gm |
| Distilled water | 100 ml |
- Equal parts of this buffer and egg yolk are mixed.
- Swanson (1949) observed good results by mixing 3 parts of this buffer and one part of egg yolk.
- Subsequent works identified that 20 percent egg yolk is satisfactory.
- EYC dilutor present very clear film of semen under microscope.
- This dilutor is widely used for the preservation of bull and buffalo bull semen up to 72 hours.
- This is the basic extender from which various new extenders developed.
3. Caprogen
The citrate-buffered yolk diluent was improved by gassing with nitrogen and adding caproic acid. This led to an extender called “caprogen”.
The composition is as follows:
Caprogen was used routinely in New Zealand for extending sperm stored at 5o. Later this was modified for ambient temperature use by reducing the egg yolk content.
4. Kampschmidt or Egg Yolk Glucose Bicarbonate Extender (EYGB)
Kampschmidt or Egg Yolk Glucose Bicarbonate Extender (EYGB) was developed by Kampschmidt et al. (1951). These workers identified that the reduction of electrolyte contents in the medium and its replacement with metabolisable sugar will increase the viability of spermatozoa at 5oC.
The workers evolved a dilutor with the following composition:
This medium has been reported superior to egg yolk citrate for buffalo semen.
5. D2 Dilutor
Singh and Tomar (1959) modified the above dilutor and named it as D2 dilutor.
The composition is as follows:
| Components | Solution | Quantity |
|---|---|---|
| Sodium bicarbonate | 1.3% solution | 10 parts |
| Glucose anhydrous | 5% solution | 40 parts |
| Fructose | 5% solution | 25 parts |
| Egg yolk | 25 parts |
Tomar and Desai (1961) claimed it to be efficient medium for buffalo semen. Tomar et al. (1968) used the above D2 medium in routine practice for buffalo and Zebu semen.
Buffalo semen was maintained fit for use for 4 days while Zebu semen was preserved for 5-6 days.
Fertility ranged around 45% in Zebu and 40% in buffaloes.
6. D5 Dilutor
Another bicarbonate dilutor named as D5 was reported by Tomar and Desai (1961) for buffalo semen.
The medium was observed to be better than D5. It consists of the following ingredients:
| Components | Quantity |
|---|---|
| Potassium bicarbonate | 0.25 gm |
| Sodium citrate dihydrate | 11.222 gm |
| Glucose anhydrous | 18.750 gm |
| Fructose | 5.125 gm |
Distilled water added to make up to one litre. 80 ml of the buffer solution is mixed with 20 ml of egg yolk.
7. Glycine Dilutor
Knoop and Krano (1944) evolved a dilutor for bull semen with the following composition:
| Components | Quantity |
|---|---|
| Glycine | 1.09% |
| Sodium phosphate | 0.20% |
| Potassium phosphate | 0.08% |
Equal volumes of buffer and egg yolk are mixed. Roy and Bishop (1954) used equal parts of egg yolk and 4% glycine solution. This dilutor is inferior for buffalo semen preservation.
This dilutor is not used even for the bull semen for the reason that it affects the morphology of spermatozoa particularly the acrosome is damaged and the fertility is poor (Strom, 1956; Mukherjee and Dott, 1960).
8. Milk Dilutor
Thacker and Almquist (1951) have shown that boiled homogenized milk and boiled pasteurized skim milk are excellent dilutors for bull semen and presented the first of a series of papers on their successful use of heated milk as semen diluent.
Boiled, filtered milk gave highly satisfactory results at extension ratios up to 1:25.Thacker and Almquist (1953) showed that spermicidal action of unheated milk is associated with albumin containing fraction of milk.
Philips et al. (1954) indicated that “Lactenin” an antistreptococcal agent is normally present in milk in its albumin fraction which is toxic to sperm.
This can be inactivated by heating to 92:C for 10 minutes only. Use of whole or skim milk in semen extenders also protects sperm against cold shock.
The milk protein, casein, has been established as the agent responsible for prevention of cold shock.
Salisbury (1957) has reviewed the use of liquid whole milk, skim milk and powdered milk as bull semen extenders. Milk combined with sugar, glycine, glycerol and egg yolk were reviewed.
Milk dilutor is prepared as follows:
Obtain fresh, homogenized, pasteurized cow’s milk or fresh pasteurized skim milk from the reliable dairy.
Using an accurate thermometer, heat the milk to 92 to 95:C and hold at this temperature exactly for 10 minutes in the top portion of a covered glass double boiler or in a covered vessel placed directly on a hot plate.
Cool the milk to room temperature and remove the fat layer; or milk may be drawn with pipette from its deeper layer by piercing through fat layer.
Add the following to the above milk:
- Suphanilamide: 0.3 gm/ml
- Penicillin G sodium: 1000 units/ml
- Dihydrostreptomycin sulphate: 1000 µg/ml
The protective fraction of the milk is composed of phospholipids. The toxic fraction of milk (lactenin) is removed with the removal of albumin from milk.
Michajilov apparently considered that heating process released glucose from the disaccharide, lactose, in the milk, thus providing available carbohydrate substrate for the cells.
The release of sulpha-hydryl (SH) groups from milk by heating process produces an intensely reduced condition, which protect the SH containing enzymes of the spermatozoa and by limiting the available oxygen in storage tubes, forces the cells into anaerobic metabolic activity, which is one important form metabolic control.
Egg yolk which contains sulpha-hydryl group containing, compounds improves the quality of heated milk as a semen extender.
A level of 10 to 20% of egg yolk greatly improves the keeping quality of semen. Joshi et al (1967) used heated cow milk for preservation of buffalo semen up to 4 days.
The use of skim milk dilutors for preserving buffalo semen was reported by Tomar and Desai (1961) and the significant improvement in the efficiency of skim milk medium was observed when egg yolk and glycine or fructose were incorporated in it.
Kale (1963) also reported successful preservation of buffalo semen by incorporating egg yolk and glucose in boiled milk.
In skim milk dilutor visibility of sperm was better in comparison to whole milk.
Jesichen (1965) reported the use of powdered milk extender (Laiciphos) with 10% egg yolk and observed good fertility. “Spermasol” is another commercial preparation which has been used successfully.
9. Citric Acid Whey
Ganguli et al. (1973) and Bhosrekar and Ganguli (1974) developed a skim milk based diluents known as citric acid whey [CAW] at NDRI, Karnal and claimed to be superior to some conventional extenders.
However, reports from elsewhere have not confirmed such claim of superiority for the CAW extender (Fluckiger et al., 1976, Singh 1977, Sall et al., 1980 and Sidhu and Guraya 1979).
10. Tris-yolk Extender
Davis et al. (1963) and Good et al. (1966) studied certain organic compounds and found excellent buffering capacity when used as semen extenders.
Tris (hydroxymethyl amino methane (C4H11NO3) is an organic compound which in combination with glucose or fructose proved an ideal semen dilutor.
Yassen (1970) reported better preservation of buffalo semen with tris extender. Recently, Tris dilutor is being extensively used for bull and buffalo bull semen preservation by almost all the advanced countries.
Tris dilutor may be prepared as follows:
- 20% egg yolk is added and mixed properly.
- This dilutor was found to be superior for both Zebu and buffalo bull semen.
- The preservability and the fertility were found to be better those other dilutors.
