WO2006051200A1

WO2006051200A1 - Seed protection medium

Info

Publication number: WO2006051200A1
Application number: PCT/FR2005/002760
Authority: WO
Inventors: Olivier Gerard; Xavier Druart; Patrice Humblot
Original assignee: Union Nationale Des Cooperatives Agricoles D'elevage Et D'insemination Animale
Priority date: 2004-11-05
Filing date: 2005-11-04
Publication date: 2006-05-18
Also published as: FR2877575B1; FR2877575A1

Abstract

The invention relates to a seed protection medium comprising an antioxidant from the thiol group, which is preferably selected from the group comprising ß-mercaptoethanol, L-cysteine, dithiotreitol, reduced glutathione and mixtures thereof.

Description

Protective medium for semen

The invention relates to a protective medium for semen, a seed flake for artificial insemination comprising such a protective medium, a method for sorting sperm of such seed, and the use of such a protective medium or such a straw to protect a seed of a bovine, a pig, a bird, small ruminants, for example a sheep or a goat, and more generally, any mammal.

To provide breeders with high quality genes, the breeding and animal insemination cooperatives produce selected seeds. For this purpose, ejaculates of animals, or "semen", are collected and then undergo a set of elaborate controls in order to select those which have the best quality, in particular the least morphologically abnormal spermatozoa and the highest percentage of spermatozoa mobile. The seeds are then diluted in protective media still called

"Diluers". Protective media help maintain the viability and functional competence of spermatozoa that would otherwise decrease rapidly.

The protected seed is then sucked into flakes containing 10 to 30 million total spermatozoa. Flakes can be stored "cool" at a temperature of about 4 ° C or frozen according to an electronically controlled cooling and freezing program.

Fresh semen and thawed seed can be used for artificial insemination. The success of insemination depends on the fertilizing power of the seed. To improve this fertilizing capacity, the patent application WO 98/00125 describes the addition, in the protective medium, of an antioxidant to a sperm or a fluid surrounding the sperm. The antioxidant is preferably selected from vitamins, plant extracts and carotenoids.

There is, however, a continuing need for a protective medium further improving the fertilizing ability of the seed to be inseminated.

According to the invention, this goal is achieved by means of a seed protecting medium comprising an antioxidant of the thiol family. Preferably, the antioxidant is chosen from the group formed by β-mercaptoethanol, L-cysteine, dithiotreitol, reduced glutathione, and mixtures thereof, preferably from the group formed by β-mercaptoethanol, L-cysteine, and their mixtures.

Preferably, the protective medium according to the invention comprises a mixture of L-cysteine and β-mercaptoethanol. The combination of these two antioxidants indeed leads to a remarkable protective effect of the seed when subjected to oxidative stress. Such a protective medium is particularly advantageous in the case of frozen seeds.

As will be seen in more detail in the following description, the antioxidants of the thiol family limit the degradation over time of the quality of the seed, in particular the mobility and viability of the spermatozoa. They thus make it possible to maintain over time a fertilizing capacity of the satisfactory seed, whether it is fresh or frozen.

According to other preferred features of the protective medium according to the invention, the molecular concentration of said antioxidant in the protective medium is between 0.1 and 10 mM, preferably between 0.5 and 6 mM, preferably between 4 and 6 mM. or between 0.1 and 3 mM, more preferably about 5 mM when the protective medium is to be frozen or about 1 mM otherwise. These low doses advantageously make it possible to limit the manufacturing cost of the protective medium according to the invention. They also allow to move away from the concentration from which the antioxidant would have a toxic effect. The tolerance of the dosage is thus advantageously increased.

The invention also relates to a seed, fresh or frozen, comprising a protective medium according to the invention. Generally the dilution rate of the protective medium resulting from the addition of the protective medium in a seed is low. Thus, preferably the molecular concentration of the antioxidant in the seed is substantially the same as that in the protective medium and is between 0. , 1 and 10 mM, preferably between 0.5 and 6 mM, preferably between 4 and 6 mM or between 0.1 and 3 mM, more preferably about 5 mM when the seed is frozen or intended to be ^" frozen ^" to get to know ^" mM sinorr (seed

The invention also relates to a seed flake for artificial insemination comprising a protective medium according to the invention. The invention also relates to a method for sorting spermatozoa from a seed intended for fertilization in vitro or in vivo, in particular by artificial insemination, remarkable in that, after a possible thawing, said seed is diluted at least in a protective medium according to the invention. The invention also relates to a method of protecting a seed for the purpose of sorting spermatozoa of said semen, remarkable in that, after a possible thawing and before the sorting of said spermatozoa, said seed is diluted at least in a conformal protective medium to the invention.

The invention finally relates to the use of a protective medium according to the invention or, if appropriate, a straw according to the invention, for protecting a seed of a bird or a mammal, preferably of a bovine, an equine, a pig, a small ruminant such as an ovine or a goat, a rodent or a lagomorph, and said seed thus protected.

Other features and advantages of the invention will appear on reading the description which follows and on examining the appended figures.

A protective medium typically comprises more than 70% by volume of a saline buffer, for example TRIS. In the particular case of protective media for porcine seeds, the protective media comprise substantially only a saline buffer. Generally, a protective medium also comprises at least 15% by volume of one or more protective additives. Protective additives, protein and lipid, are intended to protect the membranes of spermatozoa. They may be of animal origin, for example consist of egg yolk, or not.

When a protective medium is intended to be used to freeze semen, it finally comprises an agent preventing the formation of crystals in the spermatozoa during freezing, conventionally glycerol, in an amount of at least 3% by volume.

FIG. 1 represents the evolution over time of the percentage of motile spermatozoa for different protective media, as well as the average of the percentages obtained for these different protective media. Figure 1 illustrates the effect of β-mercaptoethanol on the evolution of the percentage of motile spermatozoa over time. The molecules of the following nomenclature have been studied. The code code "code", used during the tests to proceed blindly, is also sometimes used in the following description.

Molecule Code

A ferulic acid

B Gallic acid

C Flavone

Curcumin

E Ethyl gallate

F Caffeic acid

G Rutine

H Quercetin

I Luteolin

J Catechin

K Epicatechin

L Resveratrol

M N-propyl gallate

N b-mercaptoethanol

O DTT

P Lauryl gallate

Q Hydroquinone

R Coenzyme Q10

S Epigallocatechine gallate

T 'octyl gallate

U Ethoxyquin

V 2,3 tert-butyl-4-hydroxyanisole (bha)

W Nordihydroguaiaretic acid

X Phloroglucinol

Y tannins

Z Vitaflavan

AA Glutathione reduced

AB oxidized Glutathione

AC L-cysteine

AD L-Glutamine

AE Carnitine

AF N-Acetyl Cysteine

In all trials, sperm motility, ie the percentage of motile spermatozoa, was measured objectively using an automated mobility analyzer (Hamilton Thorne®).

Sperm viability (percentage of living sperm) Efe ^"In some ressâîs Tnesurée ^~ by ^~ ^ cytoτnétrie eτrf lux eir utilisantuπe ^method" of vital staining sperm using two dyes (SYBR 14 and propidium iodide). In other tests, sperm viability was measured by vital staining (Hoechst staining). After glutaraldehyde fixation and incubation in bisbenzimide 33258, the spermatozoa were examined under a microscope with interferential phase contrast. Dead cells whose membranes are damaged and pass the dye appear blue and are therefore easily quantifiable.

The quality of the movement is evaluated visually by observation under a phase contrast microscope (x 200) of a drop of sample deposited between a slide and a slide. The quality of the movement is expressed by a score of 1 to 5.

In the case of sorted seed, the fertilization ability was verified using fertilization manipulations and in vitro embryo production according to the techniques developed and in force in the laboratory of the Department of Research and Development of the UNCEIA. These techniques are described in "Evaluation of bull semen fertility by homologous in vitro fertilization tests", Marquant Leguienne B, Humblot P, Thibier M & Thibault C. 1990, in Reproduction Nutrition Development, 30: 259-266.

First series of tests

In a first series of tests (Tables 1 to 4), the in vitro protective activity of certain molecules was evaluated by testing the resistance of spermatozoa with respect to oxidation by hydrogen peroxide (H ₂ O ₂ ). . The presence of oxygenated water makes it possible to alter the membranes of the spermatozoa by oxidation, and thus to simulate the conditions encountered by the spermatozoa during freezing of the semen. Spermatozoa were incubated for one hour in a Tris-type single-saline buffer supplemented with increasing dose oxygenated water with and without antioxidant ("sample" and "control").

The percentage of motile spermatozoa was measured as well as the quality of movement (control: no antioxidant, sample: with antioxidant). Table 1

Table 2

Table 3

Table 4

The tables above demonstrate that positive effects are obtained with the following molecules: β-mercaptoethanol, L-cysteine, dithiotreitol, reduced glutathione. All these compounds, belonging to the family of thiols, effectively protect the spermatozoa against oxidation in vitro by hydrogen peroxide with a dose effect observed between 1 and 5 mM. Table 1 shows that the addition of β-mercaptoethanol is advantageous from 0.2 mM and maximum efficiency to 5 mM.

The addition of L-cysteine (Table 2) is effective on all doses tested (from 50 μM to 5 mM), and all the more effective as the dose is increasing. It is the same for the dithiotreitol, tested from 0.1 to 5 mM.

With the other compounds of the above nomenclature list, not belonging to the thiol family, no protective activity was found at the doses studied.

Second series of tests In a second series of tests, the effect of antioxidants on the quality of frozen semen was studied.

Molecules with in vitro antioxidant properties for sperm, identified by the first series of trials, were then added to commercial freezing diluents to improve the quality of the frozen semen.

The semen of ten Holstein bulls was collected and frozen in a protective medium containing the identified antioxidant molecules and a commercially available diluent. A diluent based on Tris and egg yolk marketed by the company Biovet, France, under the name Optidyl®, and a diluter without egg yell marketed under the name of Bioxcell® by the company IMV, France, have have been tested. The production and conditioning of the semen was carried out in approved insemination centers according to the procedures conventionally used.

In addition to sperm mobility and viability, curvilinear velocity

(VCL), and lateral head amplitude (ALH), descriptive of sperm movement, were also measured. The materiality threshold was lower than

0.05. "T" refers to tests carried out with a control diluent, not supplemented with antioxidant.

Table 5 shows that the mobility, the viability and the speed of movement of the spermatozoa are significantly improved when the concentration of beta-mercaptoethanol (N-molecule) in the diluent is optidyl.RTM.

5 mM. The other molecules (L, Z, E) tested under the same conditions had no effect. Table 5

Table 6 summarizes the results of tests carried out with the addition, to Optidyl® diluent, of 1 mM carnitine (AE), 5 mM L-cysteine (AC) and a mixture of 1 mM L-cysteine and of 1 mM carnitine (ACE).

Table 6

Table 6 shows that the addition of carnitine provides no significant advantage over the control diluent (T). This addition, however, does not alter the efficacy of L-cysteine, especially on the quality of sperm movement.

The following Table 7 demonstrates that the sperm movement speed is also significantly improved, by additions of reduced glutathione molecules or β-mercaptoethanol in an optidyl® dimer or a Bioxcell® diluent.

Table 7

Third series of tests

In a third series of tests, the effect of antioxidants on the quality of sexed semen was studied.

Sorting spërmatozόïâes according to the ^'gender, or "sexing sperm," is by the flow cytometry technique. This method leads to a decreased seed quality after the sorting protocol, particularly due to mechanical stress on the spermatozoa.

The trials first focused on a seed diluted in a protective medium containing Optidyl® alone (control = T) or in a test protective medium containing Optidyl® and an antioxidant (N = β-mercaptoethanol). 5 mM). After freezing, each seed was first thawed and then centrifuged.

The seed was then stained with Hoechst 33342® by incubation for one hour in an incubator diluter. The incubation diluents used were control (T) or test (N) control medium. In Table 8, the seed diluted before freezing in a control protective medium and then diluted in a test medium during staining is denoted "TN". The seed diluted before freezing in a test protective medium and then diluted in a control protective medium during staining is denoted "NT". Finally, seeds diluted twice with the control medium or twice with the test medium are labeled "TT" and "NN", respectively.

After staining, the seeds are sexed.

Mobility, quality of movement and viability are measured throughout the sexing process: after thawing, after centrifugation washing of the thawed seed, after staining with Hoechst 33342®, that is just before sorting , and finally after sorting by cytometry.

The results are summarized in Table 8.

Table 8

The mobility, quality of movement and viability of frozen spermatozoa with antioxidant N are higher than those of control T, both before and after sorting, with a stronger effect on the ability to resist sorting. In addition, the addition of the antioxidant N during the sorting process improves the quality of the seed after sorting whatever the freezing conditions (T or N).

The tests then related to a fresh seed preserved in Bioxcell® with an antioxidant (N = β-mercaptoethanol 5 mM) or without (control = T). The results are summarized in Table 9.

The semen of ten bulls was collected, diluted in Bioxcell® (IMV) supplemented or not with 5mM β-mercaptoethanol (N), stored for 5 hours at 4 ° C and subjected to the spermatozoa sorting process. The viability and sperm motility were measured before and after sorting in both conditions expénmejτtaje_s (ayec_ou saαs _^ _^ βj: meLcaptoéth.aπol 5.mM.toutau iαng_du process.). The 5mM β-mercaptoethanol antioxidant improves the mobility, viability and quality of movement of fresh spermatozoa stored in a Bioxcell® diluent at 4 ° C before and after cytometric sorting.

Table 9

In addition, the use of 5 mM β-mercaptoethanol antioxidant significantly improved fertilization and embryo development rates during in vitro production (Table 10). In these preliminary trials, development rates up to the blastocyst stage of 17% were obtained after placing the seed with the antioxidant before sorting, whereas this rate is only 7% with the sorted seed. witness ".

Table 10

Fourth series of tests

In a fourth series of tests, the effect of antioxidants on the spermatozoa ability of a fresh seed to support conservation by simple refrigeration at 4 ° C was evaluated.

The use of fresh seed in the seed production policy is indeed of strategic importance for some breeding units. With the same fertilizing power, the fresh semen of a bull can contain three to five times less spermatozoa than the frozen semen of that same bull. The number of flakes of fresh seed that can be made from a bull ejaculate is therefore three to five times larger than the number of frozen seed flakes. For the owner of an elite bull in particular, the use of fresh seed is thus of considerable economic importance.

Seed from seven adult bulls housed in a production center was collected for three consecutive weeks at a weekly ejaculate per animal. The sampling took place on Monday (OJ) in order to have the whole week to carry out the semen exams until Friday (D4). All ejaculates were collected using artificial vaginas using standard techniques.

Each of the 21 ejaculates collected was divided into three equivalent fractions. Each seed fraction was diluted to room temperature in an Erlenmeyer flask identified to give a final concentration of 5 million total spermatozoa per straw. The three fractions of each of the 21 ejaculates were each diluted in a different diluent, respectively POptidyl®

("Control group"), a mixture of Optidyl® and β-mercaptoethanol at a concentration of 1 mM ("batch 1"), and a mixture of Optidyl® and β-mercaptoethanol at a concentration of 5 mM ("batch 2), respectively.

Each batch was then placed in a refrigerated showcase at 4 ° C to reach this temperature in about one hour. An equilibration time of at least two hours was then respected. After equilibration, the seed lots were then flaked in the refrigerated showcase and then kept horizontally to avoid any risk of sedimentation of the spermatozoa.

A letter-code inscribed on the sequins following the reference "ejaculate" made it possible to identify the batches and to carry out the blind examinations.

All fractions of the same ejaculate were treated in exactly the same way.

The evolution of seed quality was monitored from Monday (OJ) to Friday (D4) during the three weeks of the test.

Evaluation of the percentage of motile spermatozoa. ^"" Crïaqϋe ^{^"week"} p ^~ endanfTës ^~ Trots ^~ s ^ mâiήe ^ ^~ dê ^{~ "tëlsf} _I ^~ of ^{^" ~} mê ^ ^'res ^~ ¥ Lr percentage of motile sperm were performed on samples obtained from collected ejaculates every Monday (JO) on the seven bulls. For a lot and a determined bull, a measurement was made on Monday (OJ), Tuesday (D1), Wednesday (D2), Thursday (D3) and Friday (D4). Each day a measurement was thus made on seven samples (one per bull) from the control group, seven samples from batch 1 and seven samples from batch 2. Each sample consisted of a mixture of two straws from the same batch for one sample. same bull.

The average over the three weeks of all the measurements on the seven samples of a batch carried out on a given day D0-D4, corresponds to a point in the attached figure 1. It is observed that in OJ and J1, the percentage of mobile cells is substantially identical regardless of the treatment. The differences appear from J2. At day 4, 40% of the cells still exhibit mobility when they are stored in the presence of 1 mM antioxidant (lot 1). This percentage is only 20% at the concentration of 5mM (lot 2) and drops to 13% in the absence of antioxidant (control group).

At D4, the percentage of mobile cells is still considered acceptable for field use.

Evaluation of the quality of the movement of the spermatozoa.

The quality of the movement follows exactly the same evolution as that of the mobility shown in Figure 1 (data not shown). On D4, the sperm diluted with the 1mM solution still has a score of 2.6 while the control is only 1. At day 4, the quality of the movement is still considered acceptable for use in the field.

Fertility assessment

Fertility was assessed from the 4-24 day non-return rates (TNR4-24) after first artificial insemination (AII) calculated for two bulls during the last month of the fresh seed campaign in one of the weaning cooperatives. in place (CMP) of the Union which participated in the tests.

The 4-24 non-return rate refers to the percentage of cows that, after insemination, do not return to heat between the fourth and twenty-fourth days after insemination. This rate therefore indicates the percentage of cows that are pregnant following insemination.

The concentration of β-mercaptoethanol in the diluent was 1 mM. For practical reasons, it was not possible to work with split ejaculates. For the analysis of the effect of the antioxidant on fertility, the TNR4-24 calculated during the experimental month were compared to those recorded for the same bulls and the same CIvIP during the previous month with the same diluent but without additive. .

Results obtained with fresh seeds inseminated at 0 and 1 (ie after storage at 4 ° C for one day) were compared.

Table 11: Difference TNR4-24 to OJ - TNR4-24 to J1

Table 11 demonstrates that the difference between the 4-24 day non-return rate (TNR4-24, in percentage) at 0 and 1, decreases with a protective medium comprising β-mercaptoethanol (1 mM). This is particularly clear for the results of bull 1 which, without the addition of antioxidant, fall by about 14 points from one day to another while the drop is reduced to 3.5 points thanks to the protective medium according to the invention.

The addition of β-mercaptoethanol thus advantageously results in a reduction in the loss of fertility of the seed between OJ and J1.

Table 12 provides the raw fertility data (TNR4-24) and the number of corresponding raw artificial insemination (NIAP) per day of seed use for the two bulls concerned by presence ("treated" lines). or not ("control" lines) of β mercaptoethanol in the diluent. Table 12

The addition of β mercaptoethanol in a diluter seems to result in a degradation of TNR4-24 to OJ. For use of the O diluent, it would be better not to add antioxidant. On day 1, however, the TNR4-24 of the seed diluted in a protective medium according to the invention reaches or even exceeds

TNR4-24 of the seed diluted in the control protective medium. A protective medium according to the invention thus proves to have a better "hold" over time. This is especially true for bulls whose seed undergoes strong oxidative and degenerative phenomena over time (bull 1).

The addition of β-mercaptoethanol to the fresh seed therefore advantageously allows a prolonged storage thereof.

The results obtained with the fresh seed make it possible to envisage an optimization of their use.

Indeed, until today, the sudden fall to D2 of TNR forced users of fresh seeds to work only at OJ and D1. This resulted in significant logistical difficulties in supplying inseminators with very tight deadlines and therefore high costs. In addition, the duration of seed use was also very low.

The addition of an antioxidant according to the invention in a diluent would advantageously maintain fertility until J3 against J1 today. The field of use of fresh seed is thus advantageously considerably extended. Cτnquîèτrιe sérfe ^~ ^~ ^~ d ^* ess ais ^~ In a fifth series of tests, the effect of antioxidants on the spermatozoa ability of a seed to support a freeze / thaw sequence was evaluated.

The excess of available doses compared to the requirements for the fourth freshest test was frozen in a programmable freezer (Digitcool 5300®, IMV Technologies, Aigle, France) to complete the tests. in fresh semen by testing frozen semen from the same animals. After thawing for at least 30 seconds in a water bath at 37 ^° C., the doses of frozen semen were subjected to the same examinations as in the previous test. Sperm viability was assessed by flow cytometry after vital staining with SYBR / IP.

The effect of the β-mercaptoethanol antioxidant is illustrated in the following Table 13.

Table 13

With frozen semen, the most effective dose for the antioxidant is

5 mM. At the dose of 1 mM, no significant difference with the control is shown. In addition, it appears that, on the group of bulls, the dose of 5mM gives more consistent results than the other two fractions over time. These observations are confirmed by the other examinations carried out (mobile sperm percentages, quality of movement and Hoechst staining).

Sixth series of tests

The sixth series of tests below demonstrates a remarkable synergistic effect of a combination of L-cysteine and β-mercaptoethanol (N + AC). Trials were performed on frozen, thawed and ₂ O ₂ semen doses.

In all the tests, the concentration of H ₂ O ₂ is 1M (100OmM in the following tables), this concentration allowing the best externalization of the stress and anti-oxidant power. Two concentrations of preferred antioxidants are 1 and 3 mM, these concentrations providing good protection without being toxic.

Preferably, however, the concentration of antioxidant in the medium is determined as a function of the concentration of free radicals present in the protective medium, which concentration is difficult to know a priori in an ejaculate, the concentration of free radicals may in particular vary in function of the bulls.

For each concentration tested, an antioxidant-free control was compared with a protective medium containing AC, an N-containing protective medium and a protective medium containing AC + N, the latter medium comprising AC and N in concentrations equal to that of Comparison media containing AC and N (eg control, 1mM AC, 1mM N, 1mM AC + 1mM N). The concentration is that of the active molecule of antioxidant in the ready-to-use protective medium, that is to say before adding the seed.

The percentage of motile spermatozoa and the quality of their movements were estimated under a microscope by a technician.

The other measurements were determined using an automated device (IVOS, Hamilton Thorn, marketed by IMV Technologies, France).

The following abbreviations are used:

Word: Percentage of motile sperm in light microscopy (%)

Note: quality of movement noted from 1 to 5 in optical microscopy

VAP: speed recorded on the mean trajectory of the spermatozoon (μm / s)

VSL: linear velocity (μm / s)

VCL: curvilinear velocity (μm / s)

ALH: lateral amplitude of displacement of the head (μm)

BCF: crossing frequency of the mean trajectory by the spermatozoon head

STR: VSL / VAP ratio (%)

LIN: VSLA / CL ratio (%)

SIZE: size of the spermatozoa head, measured at PHamilton (μm)

MOTI: Percentage of motile spermatozoa measured in Hamilton (%)

PROG: Percentage of motile sperm, measured at Hamilton (%)

Table 16

The tables above show that a combination of L-cysteine and β-mercaptoethanol (N + AC) provides a remarkable synergistic effect. Preferably, the total molecular concentration of these two antioxidants in the protective medium is between 0.1 and 10 mM, preferably between 0.5 and 6 mM, preferably between 4 and 6 mM or between 0.1 and 3 mM. more preferably about 5mM when the protective medium is to be frozen or about 1mM otherwise.

More preferably, the protective medium according to the invention has the following concentrations: N: 2.5 mM + AC: 2.5 mM.

As now clearly apparent, the addition, in a protective seed medium, of an antioxidant of the thiol family, in particular of β- mercaptoethanol, L-cysteine, dithiotreitol, or reduced glutathione, improves the fertilizing capacity of this seed in vivo and in vitro and the maintenance of this fertilizing ability over time. The seed, especially the fresh seed, can thus be kept longer, and thus be transmitted to insemination centers far from the place of its production. In addition, fresh seed production may be favored at the expense of the production of frozen semen that has lower fertilizing potential. This results in a considerable economic advantage.

Finally, the invention strengthens the resistance of the seeds to the various treatments that it can undergo, in particular freezing and sexing. Of course, the invention is however not limited to the embodiments described.

In particular, the protective medium may comprise egg yolk or not. Other saline media than Tris are also possible.

The protective medium can also be used to protect the seed during treatments other than freezing and sexing.

Claims

1. Protective medium for fresh or frozen semen containing an antioxidant of the thiol family.

2. Medium according to claim 1, characterized in that said antioxidant is selected from the group consisting of β-mercaptoethanol, L-cysteine, dithiotreitol, reduced glutathione, and mixtures thereof.

3. Protective medium according to one of claims 1 and 2, characterized in that said antioxidant is selected from the group consisting of β-mercaptoethanol, L-cysteine and mixtures thereof.

4. Protective medium according to one of the preceding claims, characterized in that said antioxidant is a mixture of L-cysteine and β-mercaptoethanol.

5. Protective medium according to one of the preceding claims, characterized in that the molecular concentration of said antioxidant is between 0.1 and 10 mM.

6. Protective medium according to claim 5, characterized in that the molecular concentration of said antioxidant is between 0.5 and 6 mM.

7. Protective medium according to claim 6, characterized in that the molecular concentration of said antioxidant is between 4 and 6 mM.

8. Protective medium according to claim 5, characterized in that the molecular concentration of said antioxidant is between 0.1 and 3 mM.

9. Seed floss for artificial insemination comprising a protective medium according to any one of the preceding claims.

10. Process for sorting spermatozoa from a seed intended for fertilization in vitro or in vivo, in particular by artificial insemination, characterized in that, after a possible defrosting, the said seed is diluted to -mθins-dan & a protective medium according to any one of claims 1 to 8 ..

11. Use of a protective medium according to any one of claims 1 to 8 or a straw according to claim 9 for protecting a seed of a mammal or a bird.

12. Use of a protective medium according to any one of claims 1 to 8 or a straw according to claim 9 for protecting a seed of a bovine, an equine, a pig, a sheep, a goat, a rodent or a lagomorph.