Diluent for cryogenic storage of bovine spermatozoa

Details Diluent for cryogenic storage of bovine spermatozoa Diluent for cryogenic storage of bovine spermatozoa

2000-05-11

2002-04-09

Saucier, Sandra E. (Department: 1651)

Chemistry: molecular biology and microbiology

Maintaining blood or sperm in a physiologically active state...

C435S001100, C600S033000, C600S035000

Reexamination Certificate

active

06368786

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to a diluent for cryogenic storage of bovine spermatozoa. More specifically, the invention relates to an aqueous diluent medium which contains no substance of animal origin and which can be stored, ready for use, over prolonged periods.
2. Description of the Prior Art
Diluents for cryogenic storage of bovine spermatozoa are known in the art. FR-B-2 720 407 describes a diluent which contains nutrients, buffers and mineral salts, as well as a protective product consisting of soya lecithin.
The diluent described in the abovementioned patent is intended for diluting bovine semen which is then frozen pending subsequent use. This is an essentially “sanitary” approach. This diluent is relatively satisfactory.
It is easy to prepare and is very clear. The absence of products of animal origin ensures the absence of any infective agent.
However, the question of the biological efficacy of the product was not addressed. The efficacy in the concentration ranges used previously does not provide the flexibility required in an insemination center.
Specifically, it is currently required to optimize the production of semen, i.e. to produce a large number of doses stored in a “French straw” with a minimum concentration of spermatozoa.
In vivo tests with a very low concentration of spermatozoa in the straw indicate that the diluent of FR-B-2 720 407 does not perform well.
One object of the present invention is to provide a diluent for cryogenic storage of bovine spermatozoa which gives improved in vivo results.
Another object of the invention is to provide a diluent which assures satisfactory survival of the spermatozoa at low concentrations.
The present invention provides a diluent which satisfies the above objects.
SUMMARY OF THE INVENTION
The diluent for cryogenic storage of bovine spermatozoa of the present invention includes a phospholipid, a liposoluble vitamin accompanied by an emulsifier, an antioxidant and a polyol.
The vitamin is vitamin A, for example.
The vitamin is accompanied by an emulsifier, for example sorbitan monooleate, which is sold under the trade name Tween 80.
The antioxidant can be an amino acid with anti-oxidant properties, for example taurine or glycine.
The antioxidant can instead be an antioxidant peptide, for example reduced glutathione.
The diluent of the present invention can instead contain a sterol, in particular cholesterol.
When a sterol is present in the diluent, the diluent advantageously contains cyclodextrine which render it soluble in an aqueous medium.
The diluent of the present invention can additionally contain salts and carbohydrates.
The carbohydrates are glucose, fructose or lactose, for example.
The salts are buffers in particular, for example trimethylolmethylamine, which is usually referred to as Tris buffer or simply Tris.
The diluent of the invention contains an effective dose of a polyol capable of inhibiting the formation of ice crystals. The polyol is advantageously glycerol.
To prepare the diluent according to the invention, the following steps are carried out:
a) preparing a dispersion of a phospholipid in the form of particles in a polyol at a sufficient temperature,
b) stirring the dispersion from step a) to micronize the phospholipid particles,
c) leaving the dispersion obtained in step b) to stand for a period of at least about 12 hours to stabilize the emulsion obtained, i.e. to ensure no separation of phases, yielding a preparation referred to as the preparation from step c),
d) preparing an aqueous phase including an amino acid with antioxidant properties, an antioxidant peptide and a vitamin, yielding an aqueous phase referred to as the aqueous phase from step d),
e) combining the preparation from step c) and the aqueous phase from step d), yielding a preparation referred to as the preparation from step e), and
f) sterilizing the preparation from step e).
According to one preferred embodiment of the invention, the sterilization in step f) is carried out by ionizing radiation in an irradiation dose range from about 5 Kgy to about 20 Kgy, preferably from about 15 Kgy to about 20 Kgy.
Techniques for obtaining the dispersion from step a) include techniques using mechanical dispersants and emulsifiers which act by pressure.
In order to obtain a stable emulsion in step c), it is necessary to leave the dispersion to stand for at least 12 hours.
The diluent of the invention is preferably formulated with a small amount of water and is diluted for use with sterile water.
The diluent advantageously contains an antibiotic agent at the time of use, but if the antibiotic agent is added to the diluent during its preparation, its efficacy can be lost during the sterilization step.
According to one preferred embodiment of the present invention, the antibiotic agent is added extemporaneously at the time of use.
In this embodiment, the antibiotic-free diluent is advantageously contained in a receptacle closed by a stopper. For example, the antibiotic agent in powder form is present in the stopper and is released into the diluent by exerting pressure on the stopper.
The antibiotic agent is gentamycin in sulfate form, for example.
The diluent of the invention typically contains the following phospholipids in the weight proportions indicated below:
“Lecithin 100” having the following weight proportions of the following phospholipids:
phosphatidylcholine 20-24 wt. %
phosphatidylethanolamine 18-22 wt. %
phosphatidylinositol 12-15 wt. %
“Lecithin 130” having the following weight proportions of the following phospholipids:
phosphatidylethanolamine 14-20 wt. %
phosphatidylinositol 7-13 wt. %
phosphatidylcholine 30-35 wt. %
Lecithin 130 is a phosphatidylcholine-enriched lecithin.
It should be noted that the diluent of the invention is intended exclusively for cryogenic storage of bovine spermatozoa. It is not suitable for cryogenic storage of spermatozoa of other animal species or human spermatozoa.
In one preferred embodiment, a diluent according to the invention includes, per 100 ml of water:
Tris
1 to 2 g
Trisodium citrate dihydrate
 5 to 10 g
Potassium chloride
0.2 to 0.5 g
Fructose
0.6 to 1.0 g
Lactose monohydrate
0.18 to 0.30 g
Glycine
2.0 to 3.0 g
Anhydrous glucose
0.25 to 0.40 g
Taurine
0.0030 to 0.0040 g
Gentamycin sulfate
0.20 g to 0.30 g 
Tylosin tartrate
0.028 to 0.040 g
Lincospectin 100
0.30 to 0.35 g
Glycerol
30 to 45 g
Calcium lactate hydrate
0.03 to 0.05 g
Lecithin 100
2.0 to 3.0 g
Lecithin 130
0.80 to 1.10 g
Citric acid monohydrate
0.5 to 2 g  
Ultra-pure water
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DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The examples which follow illustrate the invention in a nonlimiting manner.


REFERENCES:
patent: 6130034 (2000-10-01), Aitken
patent: 6140121 (2000-10-01), Ellington et al.
patent: 0 685 556 (1995-05-01), None
patent: 2 720 407 (1994-05-01), None
patent: 1189448 (1985-11-01), None
Ollero et al., “Improvement of Ram Sperm Cryopreservation Protocols Assessed by Sperm Quality Parameters and Heterogeneity Analysis”, Cryobiology 37 : 1-12 (1998).*
Al-Hanak et al., “Effect of Vatamin A on some Bioloical Indexes of bull Spermatozoa on Cryoconservation”, Zhivotnov'd Nauki 26 (5) : 80-83 (1989).*
O'Brien et al., “Preservation of Motility in Modeled Bull Sperm Using a Glutathione-Based Antioxidant Medium”, J. Cell Biol. 25th Ann. Meeting ASCB 101 (5 part 2) 487A (1985).*
Slaweta et al., “Effect of Glutathione (GSH) on the Quality of Preserved Bull Semen”, Med. Weter. 42 (8) : 498-500 (1986).*
Gupta et al., “Comparative Study on Room Temperature Dilutors of Cross-Bred Bull Semen”, Indian J. Anim. Res. 18 (2): 81-85 (1984).*
Sanchez-Partida; Setchell; Maxwell; “Epididymal compounds and antioxidants in diluents for the frozen storaged of ram spermatozoa,” Reprod. Fertil. Dev., vol. 9, pp. 689-696, (1997).

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