Diluent for cryogenic storage of bovine spermatozoa

Abstract

A diluent for the cryogenic storage of bovine spermatozoa includes a phospholipid, a liposoluble vitamin accompanied by an emulsifier, an antioxidant and a polyol.

Description

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            qs

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The examples which follow illustrate the invention in a nonlimiting manner.

EXAMPLE 1

Following the procedure described above, a diluent according to the invention having the composition below was formulated:

Tris                        1.8490 g
Trisodium citrate dihydrate 7.3965 g
Potassium chloride          0.2955 g
Fructose                    0.8875 g
Lactose monohydrate         0.2220 g
Glycine                     2.7735 g
Anhydrous glucose           0.3695 g
Taurine                     0.0037 g
Gentamycin sulfate          0.2400 g
Tylosin tartrate            0.0330 g
Lincospectin 100            0.3830 g
Glycerol                    40.2240 g
Calcium lactate hydrate     0.0455 g
Lecithin 100                2.6000 g
Lecithin 130                1.0400 g
Citric acid monohydrate     1.0400 g
Ultra-pure water            0.0650 l

The above formulation produced 100 ml of concentrated diluent which was diluted to 500 ml for use.

EXAMPLE 2

Comparative in vitro tests: anomalies and test of osmotic strength

In this example, the diluent of Example 1 of the invention was compared with the diluent of the prior art described in EP-A-685 556 as regards anomalies and osmotic strength in vitro.

Following the procedure described in EP-A-685 556, a diluent according to the prior art having the composition below was formulated:

Trimethylolmethylamine      3.4 g to 4.2 g
Trisodium citrate dihydrate 13.7 g to 16.75 g
Potassium chloride          0.55 g to 0.67 g
Fructose                    1.65 g to 2.0 g
Glucose                     0.68 g to 0.84 g
Lactose                     0.41 g to 0.50 g
Calcium lactate             0.09 g to 0.11 g
Glycine                     5.15 g to 6.25 g
Glycerol                    64 ml to 78 ml
Soya lecithin               6.75 g to 8.25 g

This vehicle was diluted with 750 ml to 900 ml of water prior to use.

Table I gives comparative results for the diluent of the prior art (DILUENT A) and the diluent of the invention (DILUENT I) for various bulls: NIZAGO, NOMEL, NORRIS, NOVAK, OCARINA and OKAVANGO.

TABLE I
	% major
IDENTIFICATION	anomalies	% abnormal	HOST
NIZAGO DILUENT A	12.0%	23.5%	51.24%
NIZAGO DILUENT I			65.05%
NOMEL DILUENT A	13.5%	30.0%	34.31%
NOMEL DILUENT I			54.50%
NORRIS DILUENT A	6.0%	17.5%	50.50%
NORRIS DILUENT I			53.00%
NOVAK DILUENT A	20.0%	26.0%	34.80%
NOVAK DILUENT I			47.71%
OCARINA DILUENT A	7.5%	21.0%	50.00%
OCARINA DILUENT I			47.76%
OKAVANGO DILUENT A	8.5%	18.5%	67.68%
OKAVANGO DILUENT I			67.65%
Average results for	11.0%	23.0%	48.09%
DILUENT A at
7 × 10 6 spz/dose
Average results for			55.95%
DILUENT I at
7 × 10 6 spz/dose
HOST = osmotic strength test
spz = spermatozoa

Table I clearly indicates the superiority of the diluent of the invention over the diluent of the prior art since, for 7×10 6 spermatozoa per dose, a very much higher percentage of osmotic strength in vitro is obtained for the diluent of the invention.

EXAMPLE 3

Comparative in vitro tests, results after thawing and temperature resistance test

In this example, the diluent of Example 1 of the invention was compared with the diluent of the prior art described in EP-A-685 556 used in Example 2, as regards results after thawing and in vitro temperature resistance.

Table II gives comparative results for the diluent of the prior art (DILUENT A) and the diluent of the invention (DILUENT I) with various bulls: NIZAGO, KOMEL, NORRIS, NOVAK, OCARINA and OKAVANGO.

Table II clearly indicates the superiority of the diluent of the invention over the diluent of the prior art since, for 7×10 6 spermatozoa per dose, a higher percentage of mobiles and higher motility are obtained, except for the percentage of mobiles during the thawing after 1 h 30, which is very much inferior.

EXAMPLE 4

Comparative in vivo tests: results of in vivo artificial insemination for a nonreturn rate at 25 days

In this example, the diluent of Example 1 of the invention was compared with the diluent of the prior art described in EP-A-685 556 used in Example 2, as regards the results of in vivo primary artificial insemination (PAI), fertilization by primary artificial insemination (PAI fert) and percentage of fertilization by primary artificial insemination (% PAI fert), for a nonreturn rate (NRR) at 25 days.

Table III gives comparative results for the diluent of the prior art (DILUENT A) and the diluent of the invention (DILUENT I) with various bulls: NIZAGO, NOMEL, NORRIS, NOVAK, OCARINA and OKAVANGO.

	TABLE II
					TRT 1 h to
	Thawing	TRT + 30 min	TRT + 1 h 30	Thawing	1 h 45
	%	Moti-	%	Moti-	%	Moti-	%	Moti-	%	Moti-
IDENTIFICATION	mobiles	lilty	mobiles	lilty	mobiles	lilty	mobiles	lilty	mobiles	lilty
NIZAGO DILUENT A	55%	3	50%	2	30%	1.5	55%	3	0%	0
NIZAGO DILUENT I	55%	3.5	55%	3	45%	2	55%	3	45%	2
NOMEL DILUENT A	55%	3.5	55%	3	10%	1	45%	3	35%	2
NOMEL DILUENT I	45%	3	45%	2	10%	0.5	45%	3	40%	3
NORRIS DILUENT A	40%	2	40%	1	10%	0.5	50%	2.5	0%
NORRIS DILUENT I	45%	3	45%	2	45%	3	30%	3	5%
NOVAK DILUENT A	30%	2	30%	1.5	10%	0.5	45%	2.5	0%
NOVAK DILUENT I	35%	3	35%	2.5	35%	3	40%	3.5	0%
OCARINA DILUENT A	55%	3.5	50%	3	50%	3	45%	3	5%
OCARINA DILUENT I	60%	3.5	55%	3	50%	3.5	55%	4	10%
OKAVANGO DILUENT A	55%	3.5	45%	2.5	30%	2	55%	3	45%	2.5
OKAVANGO DILUENT I	55%	3.5	60%	3.5	50%	3	55%	3	45%	3
Average results for	48%	2.92	45%	2.17	23%	1.42	49%	2.83	14%	1.50
DILUENT A at
7 × 10 4 spz/dose
Average results for	49%	3.25	49%	2.67	39%	2.50	47%	3.25	24%	2.67
DILUENT I at
7 × 10 6 spz/dose
TRT = temperature resistance test
spz = spermatozoa

	TABLE III
	NRR at 25 days
		PAI	% PAI		AI2	% IA2		AI3 +	% AI3 +	Total	Total	Total %
IDENTIFICATION	PAI	fert	fert	AI2	fert	fert	IA3+	fert	fert	AI	AI fert	AI fert
NIZAGO DILUENT A	66	44	66.67%	48	35	72.92%	74	55	74.32%	188	134	71.28%
NIZAGO DILUENT I	68	55	80.88%	33	27	81.82%	89	71	79.78%	190	153	80.53%
NOMEL DILUENT A	76	60	78.95%	45	37	82.22%	71	59	83.10%	192	156	81.25%
NOMEL DILUENT I	72	59	81.94%	48	40	83.33%	72	60	83.33%	192	159	82.81%
NORRIS DILUENT A	74	61	82.43%	42	31	73.81%	75	56	74.67%	191	148	77.49%
NORRIS DILUENT I	60	51	85.00%	41	33	80.49%	73	54	73.97%	174	138	79.31%
NOVAK DILUENT A	80	63	78.75%	34	31	91.18%	63	50	79.37%	177	144	81.36%
NOVAK DILUENT I	68	52	76.47%	50	45	90.00%	77	67	87.01%	195	164	84.10%
OCARINA DILUENT A	73	60	82.19%	41	38	92.68%	73	60	82.19%	187	158	84.49%
OCARINA DILUENT I	72	65	90.28%	47	37	78.72%	74	59	79.73%	193	161	83.42%
OKAVANGO DILUENT A	74	57	77.03%	34	29	85.29%	60	49	81.67%	168	135	80.36%
OKAVANGO DILUENT I	71	52	73.24%	47	39	82.98%	75	63	84.00%	193	154	79.79%
Average results for	443	345	77.88%	244	201	82.38%	416	329	79.09%	1103	875	79.33%
Diluent A at
7 × 10 6 spz/dose
Average results for	411	334	81.27%	266	221	83.08%	460	374	81.30%	1137	929	81.71%
Diluent I at
7 × 10 6 spz/dose
Average results for	2028	1630	80.37%
the test bulls over
the same period at
20 × 10 6 spz/dose
DILUENT A
NRR = nonreturn rate
spz = spermatozoa

Table III clearly indicates the superiority of the diluent of the invention over the diluent of the prior art, since a very much higher percentage PAI is obtained in all cases but one (for the bull OKAVANGO). The average results for 7×10 6 spermatozoa per dose reveal a very much higher PAI percentage for the diluent of the invention.

Surprisingly, the diluent of the prior art shows very inferior average results, even for 20×10 6 spermatozoa per dose, than the diluent of the invention for only 7×10 6 spermatozoa per dose.

EXAMPLE 5

Comparative in vivo tests: results of in vivo artificial insemination for a nonreturn rate at 60 days

In this Example, the diluent of Example 1 of the invention was compared with the diluent of the prior art described in EP-A-685 556 used in Example 2, as regards the results of in vivo primary artificial insemination (PAI), fertilization by primary artificial insemination (PAI fert) and percentage of fertilization by primary artificial insemination (% PAI fert), for a nonreturn rate (NRR) at 60 days.

Table IV gives comparative results for the diluent of the prior art (DILUENT A) and the diluent of the invention (DILUENT I) with various bulls: NIZAGO, NOMEL, NORRIS, NOVAK, OCARINA and OKAVANGO.

Surprisingly, the diluent of the invention even has, for only 7×10 6 spermatozoa per dose, average results that are better than those for the diluent of the prior art for 20×10 6 spermatozoa per dose.

	TABLE IV
	NRR at 60 days
		PAI	% PAI		AI2	% IA2		AI3 +	% AI3 +	Total	Total	Total %
IDENTIFICATION	PAI	fert	fert	AI2	fert	fert	IA3+	fert	fert	AI	AI fert	AI fert
NIZAGO DILUENT A	55	24	43.64%	40	15	37.50%	68	39	57.35%	163	78	47.85%
NIZAGO DILUENT I	68	40	58.82%	33	22	66.67%	87	60	68.97%	188	122	64.89%
NOMEL DILUENT A	72	41	56.94%	43	27	62.79%	64	41	64.06%	179	109	60.89%
NOMEL DILUENT I	62	38	61.29%	38	24	63.16%	63	36	57.14%	163	98	60.12%
NORRIS DILUENT A	69	39	56.52%	41	24	58.54%	72	40	55.56%	182	103	56.59%
NORRIS DILUENT I	55	35	63.64%	34	19	55.88%	65	38	58.46%	154	92	59.74%
NOVAK DILUENT A	61	36	59.02%	47	37	78.72%	72	47	65.28%	180	120	66.67%
NOVAK DILUENT I	74	44	59.46%	30	21	70.00%	57	37	64.91%	161	102	63.35%
OCARINA DILUENT A	44	27	61.36%	31	24	77.42%	53	35	66.04%	128	86	67.19%
OCARINA DILUENT I	68	50	73.53%	44	28	63.64%	72	46	63.89%	184	124	67.39%
OKAVANGO DILUENT A	39	26	66.67%	23	15	65.22%	42	27	64.29%	104	68	65.38%
OKAVANGO DILUENT I	70	36	51.43%	46	29	63.04%	75	52	69.33%	191	117	61.26%
Average results for	340	193	56.76%	225	142	63.11%	371	229	61.73%	936	564	60.26%
Diluent A at
7 × 10 6 spz/dose
Average results for	397	243	61.23%	225	143	63.56%	419	269	64.20%	1041	655	62.92%
Diluent I at
7 × 10 6 spz/dose
Average results for	1771	1097	61.94%
the test bulls over
the same period at
20 × 10 6 spz/dose
DILUENT A
NRR = nonreturn rate
spz = spermatozoa

EXAMPLE 6

Comparative in vivo tests: results of in vivo artificial insemination for a nonreturn rate at 90 days

In this example, the diluent of Example 1 of the invention was compared with the diluent of the prior art described in EP-A-685 556 used in Example 2, as regards the results of in vivo primary artificial insemination (PAI), fertilization by primary artificial insemination (PAI fert) and percentage of fertilization by primary artificial insemination (% PAI fert), for a nonreturn rate (NRR) at 90 days.

Table V gives comparative results for the diluent of the prior art (DILUENT A) and the diluent of the invention (DILUENT I) with different bulls: NIZAGO, NOMEL, NORRIS, NOVAK, OCARINA and OKAVANGO.

Table V clearly indicates the superiority of the diluent of the invention over the diluent of the prior art, since a higher percentage of PAI is obtained in all cases but one (for the OKAVANGO bull). The average results for 7×10 6 spermatozoa per dose reveal a markedly higher percentage of PAI for the diluent of the invention.

Surprisingly, the diluent of the invention even shows, for only 7×10 6 spermatozoa per dose, average results that are higher than those for the diluent of the prior art for 20×10 6 spermatozoa per dose.

Although the invention has been described for specific embodiments, it will be understood that it encompasses all the implementation variants in the scope of the claims.

	TABLE V
	NRR at 90 days
		PAI	% PAI		AI2	% IA2		AI3 +	% AI3 +	Total	Total	Total %
IDENTIFICATION	PAI	fert	fert	AI2	fert	fert	IA3+	fert	fert	AI	AI fert	AI fert
NIZAGO DILUENT A	65	24	36.92%	47	19	40.43%	74	35	47.30%	186	78	41.94%
NIZAGO DILUENT I	68	37	54.41%	33	19	57.58%	89	55	61.80%	190	111	58.42%
NOMEL DILUENT A	76	36	47.37%	45	24	53.33%	71	42	59.15%	192	102	53.13%
NOMEL DILUENT I	72	37	51.39%	48	22	45.83%	72	38	52.78%	192	97	50.52%
NORRIS DILUENT A	74	37	50.00%	42	23	54.76%	75	37	49.33%	191	97	50.79%
NORRIS DILUENT I	60	31	51.67%	41	21	51.22%	73	42	57.53%	174	94	54.02%
NOVAK DILUENT A	68	33	48.53%	50	35	70.00%	77	43	55.84%	195	111	56.92%
NOVAK DILUENT I	78	41	52.56%	32	23	71.88%	62	38	61.29%	172	102	59.30%
OCARINA DILUENT A	72	42	58.33%	41	25	60.98%	73	49	67.12%	186	116	62.37%
OCARINA DILUENT I	72	46	63.89%	47	27	57.45%	74	42	56.76%	193	115	59.59%
OKAVANGO DILUENT A	73	37	50.68%	34	20	58.82%	60	34	56.67%	167	91	54.49%
OKAVANGO DILUENT I	71	31	43.66%	47	29	61.70%	75	47	62.67%	193	107	55.44%
Average results for	428	209	48.83%	259	146	56.37%	430	240	55.81%	1117	595	53.27%
Diluent A at
7 × 10 6 spz/dose
Average results for	421	223	52.97%	248	141	56.85%	445	262	58.99%	1114	626	56.19%
Diluent I at
7 × 10 6 spz/dose
Average results for	1833	1009	55.05%
the test bulls over
the same period at
20 × 10 6 spz/dose
DILUENT A
NRR = nonreturn rate
spz = spermatozoa

Claims

1. A diluent for cryogenic storage of bovine spermatozoa, comprising a phospholipid, a liposoluble vitamin accompanied by an antioxidant and a polyol, wherein said vitamin is vitamin A, said diluent further comprising a sterol and cyclodextrins to render said sterol soluble.

2. The diluent claimed in claim 1 in which said antioxidant is an amino acid with antioxidant properties.

3. The diluent claimed in claim 2 in which said amino acid with antioxidant properties is taurine.

4. The diluent claimed in claim 1 in which said antioxidant is an antioxidant peptide.

5. The diluent claimed in claim 4 in which said antioxidant peptide is reduced glutathione.

6. The diluent claimed in claim 1 in which said sterol is cholesterol.

7. The diluent claimed in claim 1 wherein said polyol is glycerol.

8. The diluent claimed in claim 1 further including salts and carbohydrates.

9. A process for preparing a diluent for cryogenic storage of bovine spermatozoa, comprising a phospholipid, a liposoluble vitamin accompanied by an antioxidant peptide or amino acid with antioxidant properties, and a polyol, including the following steps:a) preparing a dispersion of a phospholipid in the form of particles in a polyol; b) stirring the dispersion from step a) to micronize said 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, yielding a preparation referred to as the preparation from step c), d) preparing an aqueous phase including an amino acid with antioxidant properties or an antioxidant peptide, and a liposoluble vitamin, yielding a phase referred to as the aqueous phase from step d), e) combining said preparation from step c) and said aqueous phase from step d), yielding a preparation referred to as the preparation from step e), and f) sterilizing said preparation from step e).

10. The process claimed in claim 9 wherein said sterilization in step f) is carried out by ionizing radiation in an irradiation dose range from about 5 Kgy to about 20 Kgy.

11. The process claimed in claim 10 wherein said irradiation dose range is from about 15 Kgy to about 20 Kgy.

12. The process claimed in claim 10 for preparing the diluent claimed in claim 9 wherein a sterol is added to said preparation from step d).

13. The process claimed in claim 10 for preparing the diluent claimed in claim 11 wherein carbohydrates are added to said preparation from step d).

14. The process of claim 9 wherein said antioxidant is an amino acid with antioxidant properties.

15. The process of claim 14 wherein said amino acid with antioxidant properties is taurine.

16. The process of claim 9 wherein said antioxidant is an antioxidant peptide.

17. The process of claim 16 wherein said antioxidant peptide is glutathione.

18. The process of claim 9 wherein said vitamin is vitamin A.

19. The process of claim 9 wherein said diluent further comprises a sterol.

20. The process of claim 19 wherein said diluent further comprises cyclodextrins to render said sterol soluble.

21. The process of claim 9 wherein said wherein said polyol is glycerol.

22. The process of claim 9 wherein said diluent further comprises salts and carbohydrates.