Extenders for use with frozen semen

Many of the extenders used for the cryopreservation of stallion semen are based upon those used for cool storage, as detailed earlier, but with the addition of cryoprotectants. Initially, heated whole cow's milk plus 10% glucose was used as an extender for freezing stallion semen. In fact this extender was used to store the semen that resulted in the first pregnancy, in a mare, as a result of AI with frozen semen (Barker and Gandier, 1957). Out of the seven mares inseminated, one pregnancy resulted. In this initial work semen was collected from the cauda epididymis and diluted in a single diluent prior to freezing.

Once the deleterious effects of seminal plasma had been noted (Pickett et al., 1975a; Varner et al., 1987; Webb et al., 1990) a double diluent procedure was proposed. It is common practice today that the preparation of semen for cryopreservation involves the use of two extenders: a primary extender for initial dilution, which is aspirated off after centrifugation, prior to the addition of a secondary extender and freezing. Numerous extenders have been used as primary or secondary extenders.

Primary extenders

The prime function of a primary extender is to maintain motility, but it also acts to protect spermatozoa during the process of centrifugation. There is no real requirement for a cryoprotectant in such extenders. Examples of primary extenders are given in Table 7.8. Investigations into the addition of other elements, including lecithin and phenylmethanesulphonyl fluoride, to skimmed milk primary extenders have been carried out, but with little advantage being reported (Heffe, 1993).

Secondary extenders

Many of the secondary extenders are also based upon those given for fresh and cooled semen storage and may be similar to the primary extenders used, but in order to protect spermatozoa from cold shock, the addition of a cryoprotectant is needed. These secondary extenders may be used in isolation without prior centrifugation, or used after centrifugation. After centrifuga-tion the secondary extender is normally (though not always) added after the primary extender has been aspirated off. Several cryoprotectants have been investigated for use with stallion semen.

Glycerol and egg yolk extenders were amongst the first to be used for freezing semen (Table 7.9) (Polge et al., 1949; Nishikawa, 1975; M. Boyle, Cambridge, 1996, personal communication). More recent work has demonstrated the cryoprotectant nature of many other components, including sugars and liposomes (Heitland et al., 1995). Many extenders used for freezing contain a mixture of many components in varying ratios. Examples of extenders for freezing include those based on skimmed milk with egg yolk, and egg yolk (Table 7.10), and are commonly used as secondary extenders. The addition of a detergent (or surfactant) to the extender was investigated, with some success, under the impression that it had a beneficial effect on the lipoproteins in egg yolk and within the plasma membrane of the spermatozoa, affording the spermatozoa additional cryoprotection (Jimenez, 1987; Pickett and Amann, 1993) (Table 7.11).

Other secondary extenders (Table 7.12) use sugars as a major component. Use of sugar-based secondary extenders has been reported both with and without initial dilution with a primary extender followed by centrifugation. If they are to be used without centrifugation then, as would be expected, the best results are obtained by fractionating semen and using the sperm-rich fraction (Tischner, 1979; Naumenkov and Romankova, 1981, 1983). The inclusion of mannitol and glucose in addition to lactose in the second extender given in Table 7.12 was reported to increase the percentage of motile spermatozoa post thaw. These last two extenders are popular for use in Eastern Europe. In comparative work carried out by Barsel (1994), an

Table 7.8. Examples of primary extenders for use at semen centrifugation prior to cryopreservation.

(a) BSA primary extender (Samper, 1995b)

Component

Quantity

Sucrose Glucose BSA

Distilled water

(b) Glucose-EDTA primary extender I (Cochran et al., 1984)

Component

Quantity

Glucose

Sodium citrate dihydrate Disodium EDTA Sodium bicarbonate Deionized water PH

Osmotic pressure (mOsm kg 1)

(c) HF-20 primary extender (Nishikawa, 1975)

Component

Quantity

Glucose

Lactose

Raffinose

Sodium citrate

Sodium phosphate

Potassium sodium tartrate

Egg yolk

Penicillin

Streptomycin

Deionized water (made up to)

5.0 g 3.0 g 3.0 g 0.15 g 0.05 g 0.05 g 0.5-2.0 g 25,000 IU 25,000 n g 100.0 ml

(d) EDTA Glucose extender II (Cochran et al., 1984)

Component

Quantity

Glucose

Sodium citrate dihydrate Disodium EDTA Sodium bicarbonate Polymixin B sulphate Deionized water pH

Osmotic pressure (mOsm kg 1)

59.985 g 3.7 g 3.699 g 1.2 g 10 x 106 IU 1000.0 ml 6.59 409.0

(e) Skimmed milk and sugar primary extender (Pickett and Amann, 1993)

Component

Quantity

Sterile skimmed milk Salt-and-sugar solution Gentamicin Penicillin

Salt-and-sugar solution: Glucose Lactose Raffinose

Sodium citrate dihydrate Potassium citrate Water (made up to)

Table 7.9. Glycine egg yolk extender (M. Boyle, Cambridge, 1996, personal communication).

Component

Quantity

Extender 1

Glucose monohydrate

12.Q g

Fructose

12.Q g

Distilled water

6QQ.Q ml

Extender 2

Sodium citrate

2Q.Q g

Glycine

9.4 g

Distilled water

1QQQ.Q ml

Mix extenders 1 and 2; supplement with 20% egg yolk and clarify.

Table 7.10. Skimmed milk egg yolk secondary extender (Samper, 1995b).

Component

Quantity

Skimmed milk

2.4 g

Egg yolk

8.Q ml

Sucrose

9.3 g

Glycerol

3.5%

Distilled water

1QQ.Q ml

Table 7.11. Secondary extenders, incorporating detergent (Equex STM, Nova Chemicals Sales, Scituate, Massachusetts), used for the cryopreservation of stallion semen (Cochran et al., 1984).

Component Quantity

Lactose solution (11% w/v) 50.0 ml

Glucose EDTA solution (EDTA Glucose extender II primary extender) 25.0 ml

Egg yolk 20.0 ml

Glycerol 5.0 ml

Equex STM 0.5 ml egg yolk, EDTA and lactose extender, similar to those given in Table 7.12, outperformed NFDSMG extender as far as motility and acrosome integrity were concerned.

A sugar-based extender with the addition of raffinose has been used with success in Japan (Table 7.13). This extender may be used without the 10% glycerol as a primary extender for centrifugation, or with the addition of the 10% glycerol as a freezing extender. Motility rates of between 49 and 73% were reported post thaw in 80% of the samples frozen, resulting in up to 100% conception rates in small trials (Nishikawa, 1975). Good success has been reported with the use of trehalose as a cryoprotectant within a skimmed milk-egg yolk extender. It is suggested that trehalose has a stabilizing effect on the spermatozoon plasma membrane (Steinmann, 1996). In France a

Table 7.12. Secondary extenders, with the addition of sugars, for use in the cryopreservation of equine semen.

(a) Lactose-based secondary extender (Tischner, 1979)

Table 7.12. Secondary extenders, with the addition of sugars, for use in the cryopreservation of equine semen.

(a) Lactose-based secondary extender (Tischner, 1979)

Component

Quantity

Lactose

11.0 g

Disodium EDTA

0.1 g

Sodium citrate dihydrate

0.089 g

Sodium bicarbonate

0.008 g

Deionized water

100.0 ml

Egg yolk

1.6 g

Glycerol

3.5 ml

Penicillin

50.0 IU

Streptomycin

50.0 mg

(b) Lactose-, mannitol- and glucose-based secondary extender (Naumenkov and

Romankova, 1981, 1983)

Component

Quantity

Lactose

6.6 g

Mannitol

2.1 g

Glucose

0.7 g

Disodium EDTA

0.15 g

Sodium citrate dihydrate

0.16 g

Sodium bicarbonate

0.015 g

Deionized water

100.0 ml

Egg yolk

2.5 g

Glycerol

3.5 ml

Table 7.13. HF-20 extender which may be used as a primary extender (without the 10%

glycerol) or as a secondary extender (with the 10% glycerol) (Nishikawa, 1975).

Component

Quantity

Glucose

5.0 g

Lactose

0.3 g

Raffinose

0.3 g

Sodium citrate

0.15 g

Sodium phosphate

0.05 g

Potassium sodium tartrate

0.05 g

Egg yolk

0.5-2.0 g

Penicillin

25,000 IU

Streptomycin

25,000 g

Deionized water (made up to)

100.0 ml

Glycerol

10%

skimmed milk, sugar and salt solution extender is popular for use as a secondary extender (Table 7.14). A very simple, sugar-based secondary extender (Table 7.15) has been used successfully, in a ratio of 1:1, after primary extension with a simple 11% sucrose solution followed by centrifuga-tion and aspiration (Piao and Wang, 1988).

As mentioned previously in the context of fresh and cooled semen storage, hydrogen ion extenders may also be used. Examples are given in Table 7.16. They may be used as primary extenders as they are, or as secondary extenders with the addition of 2-7% glycerol. Of these extenders, TCA 325 was reported to provide the best conception rates after storage in ampoules (Pace and Sullivan, 1975).

It is evident that many of these extenders use glycerol as the major cryoprotectant. Because the use of glycerol may itself be detrimental to spermatozoan viability (Pace and Sullivan, 1975; Demick et al., 1976; Fahy, 1986), a compromise has to be reached as regards the concentration of glycerol and the length of time that the glycerol is in contact with the spermatozoa prior to freezing, in order to maximize the beneficial effects of glycerol as a cryoprotectant but minimize its toxic effects. Many variations in contact time and concentration of glycerol in diluents have been investigated. In addition, the efficiency of glycerol may be affected by the diluent to which it is added, as well as the method of storage. Nagase (1967) reported that the

Table 7.14. A skimmed milk, sugar and salt solution secondary extender used in France.

Component

Quantity

Sterile skimmed milk

5Q.Q ml

Salt-and-sugar solution

5Q.Q ml

Egg yolk

2.Q ml

Glycerol

2.5%

Gentamicin

5.Q mg

Penicillin

5QQQ IU

Salt-and-sugar solution:

Glucose

2.5 g

Lactose

Q.15 g

Raffinose

Q.15 g

Sodium citrate dihydrate

Q.Q3 g

Potassium citrate

Q.Q4 g

Water (made up to)

5Q.Q ml

Table 7.15. A simple sugar-based secondary extender, used after primary extension with an

11% sucrose solution (Piao and Wang, 1988).

Component

Quantity

11% Sucrose solution

1QQ.Q ml

Skimmed milk

45.Q ml

Egg yolk

16.Q ml

Glycerol

6.Q ml

Table 7.16. Hydrogen ion primary extenders (without glycerol) and secondary extenders (plus 2-7% glycerol) (Pace and Sullivan, 1975).

(a) Basic components. For use as primary extenders all diluents contain the following four basic components, to which are added the specified extra ingredients, and the volume is made up to 100 ml with distilled water. For use as secondary extenders 2-7% glycerol is added to each one.

Component

Quantity

Fresh egg yolk Penicillin

Dihydrostreptomycin sulphate Polymixin B sulphate

+1 0

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