FR2725342A1 - SPERM DILUTOR COMPRISING AN INDOLE DERIVATIVE - Google Patents

Abstract

The invention relates to the use of indole derivatives for the preparation of sperm diluters in order to improve the preservation of spermatozoa.

Description

 SPERM DILUTOR COMPRISING AN INDOLE DERIVATIVE.

 The present invention relates to the use of indole derivatives to enhance sperm survival and fertilization ability for artificial insemination or other assisted reproductive practice.

 The techniques of artificial insemination which are more and more used today, pose problems of conservation of the sperm waiting for the insemination.

 In some species, these problems are partially solved by freezing sperm. However, in other species, this freezing is difficult, if not impossible, insofar as it causes the destruction or inactivation of too many spermatozoa. Even in species where freezing is possible, there is always a noticeable loss in the percentage of motile spermatozoa.

 To remedy this situation, it has been proposed to act on the composition of the medium in which the sperm is diluted following its removal, and prior to its use or its freezing (this medium will be hereinafter referred to as "diluter") .

Various sperm dilators have been advocated, with the aim of improving the conservation and the fertilizing capacity of the spermatozoa. All these diluents must take into account a number of physico-chemical requirements such as
- have isotonic osmotic pressure,
contain buffer substances capable of maintaining the pH close to neutrality,
- contain substances necessary for the metabolism of the spermatozoon,
- be free of bacterial agents.

For example, the following compositions are the most commonly used
1) Diluers used in swine species
Many diluents have been used for the storage of boar semen in the liquid state.

Their composition is often qualitatively similar, but there are significant differences in concentrations, resulting in significant variations in pH and osmotic pressure.

In general, these diluents are composed of the following elements:
- Glucose, better assimilated by spermatozoa than fructose, is the best source of energy. It also contributes to maintaining the osmotic balance of the medium.

 - Sodium citrate, sodium bicarbonate, and Tris (trihydroxymethylaminomethane), are the buffers preferentially used for boar semen.

 EDTA, contained in most diluents, would play a role in stabilizing cell volume [BREDDERMAN and FOOT, Expl. Cell. Res., Vol. 66, p. 458-464 (1971)]. In addition, by its chelating properties, it would prevent the peroxidation of membrane phospholipids, harmful for maintaining the fertilizing power [SHANON and CORSON, J. Dairy Sci., Vol.

5, p. 614-620, (1992)].

 All these elements are dissolved in distilled water.

 In order to assess the effectiveness of the various diluents, the determination of the percentage of motile spermatozoa and their fertilizing capacity is conventionally carried out after storage for a given period in the diluent under consideration. Currently, the so-called BTS diluent, the composition of which is shown below, is the one that has given the best results for the 15-C dilution and storage of swine semen, whether taken on the farm or in the a reproduction center. It can be used until the 3rd day after harvest without a significant drop in fertility or prolificacy.

Composition of the BTS
Glucose 37 g
Sodium citrate, 2H20 6.0 g
Sodium bicarbonate 1.25 g
EDTA 1.25 g
Potassium chloride 0.75 g
Distilled water qs 1000 ml
2) Diluers used in goat species
Two types of diluents were mainly studied:
- diluers composed exclusively of mineral salts
- Thinners based on milk or egg yolk.

 Egg yolk would protect sperm from thermal shock. This action would be related to the lipoprotein and lipid compounds of the egg [KAUPSCHMIDT, J. Dairy Sci., Vol. 34, p. 44-51, (1953)].

Milk, rich in protein, fat and sugars, is a nutrient of choice for spermatozoa provided it has been preheated to 56 C for 15 minutes to destroy some of the spermicidal proteins it contains. However, recent studies seem to show the existence of an interaction between seminal plasma triglyceride lipase and milk triglycerides, resulting in decreased motility of spermatozoa [PELICER
RUBIO and COMBARNOUS, Communication 7th days
International Animal Reproduction; Murcia
July 1994].

The comparison of the different diluents, by the evaluation of the percentage of motile spermatozoa and by their fertilizing capacity after conservation, led to the following findings:
Saline diluers, in general, give poor results [DAUZIER, International
Encyclopedia, Vet. Medicine, p. 269-271, (1966)].

 Yolk-based diluers give very poor results. The seminal plasma of the goat species has been shown to contain a phospholipase A which hydrolyzes lecithin from the egg yolk to lysolecithin and fatty acids, which have a spermicidal effect.

 The best results have been obtained with the milk diluter, whose composition is indicated below, and which is currently used conventionally for shelf life of up to 12 hours, without there being a drop in prolificacy or fertility.

Composition of goat diluter made from milk
Reconstituted skimmed milk 10 g
Glucose 194 mg
Distilled water qs 100 ml
The mixture is boiled for 15 minutes before use.

3) Thinner used in sheep species
The conservation studies carried out on sheep semen are essentially the same as those conducted in the goat species.

 The composition of the diluent conventionally used for sheep, indicated below, is similar to that of the goat dilator, and further comprises antibiotics. This diluent makes it possible to preserve the fertilizing capacity of the ovine sperm during 8 hours at 15-C.

Composition of the sheep dilator
100 ml distilled water heated to 60 ° C
. 0.33 g of sulfonamides
cool to 20-25 C
11.1 g of reconstituted skimmed milk
water bath 15 min at 60'C
cool to room temperature
+ 0.11 g of streptomycin and 100000 IU
penicillin.

4) Thinner used in the bovine species
The most commonly used diluter in cattle is a skim milk diluent reconstituted from milk powder, which is marketed under the name LAICIPHOS Nt. Milk has an increased protective effect if it is supplemented with 10% chicken egg yolk and antibiotics.

 This diluter allows seed storage up to 72 hours at 5 ° C.

5) Thinner used in rabbits
The diluent used by the majority of breeders is a saline diluter marketed by IMV under the name of DYLAP 2000.

 This diluter allows conservation of rabbit semen for 6 to 8 hours.

6) Diluers used in fish (salmonids)
A mineral solution simulating the composition of seminal trout fluid (called MMLS, and whose composition is indicated below) constitutes a medium that allows a satisfactory conservation, at a temperature of 0 C, of the fertility of arc trout gametes. -in sky. The fertilization of the spermatozoa subsists without diminution during 30 minutes.

Composition of MMLS
NaCl 110 mmol / l
KCl 28.3 mmol / l
MgSO 4 (7H 2 O) 1.1 mmol / l
CaCl2 (2H2O) 1.8 mmol / l.

 This conservation glow allows good survival of gametes both before and after insemination.

However, in this medium, the spermatozoa remain motionless, especially because of the high concentrations of potassium ions [SCHLENK and KAEMANN,
Biochem. Z., vol. 295, p. 283-301, (1937); BILLARD and
JALABERT, Ann. Biol. Anim. Bioch. Biophys., Vol. 14, p. 601-610, (1974)].

 To allow a return of the motility of the spermatozoa during the addition of the eggs, it is preferable to use an insemination diluter consisting of a solution of NaCl (posm 250, pH 9 to 9.5). This is added to semen (previously diluted in MMLS) and to eggs during insemination.

 This technique, which involves two successive dilutions, makes it possible to obtain a maximum percentage of fertilization.

7) Diluers used in poultry
The sperm of domestic birds remains only a few minutes (guinea fowl) to a few tens of minutes (cock) at the optimum of its fertilizing capacity if it is stored in pure form and at room temperature. The cooling of the sperm at a temperature between 2 and 12-C makes it possible to increase this delay, provided that, in addition, diluents capable of maintaining this fertilizing capacity are used. Their composition (pH, osmotic pressure, chemical species) obviously has a primordial role, but could only be defined empirically.

 The currently existing solutions (diluents used at a rate of 1 to 2 volumes per volume of sperm, with or without aeration of the mixture) allow satisfactory storage for a period not exceeding 6 hours. Beyond that, there is most often a decrease of 5 to 25 points or more in the rate of fertilization of eggs, which can in most cases be offset by a greater number of inseminated spermatozoa.

Two types of diluents have been advocated
diluents comprising numerous cations (Na +, K +, Mg ++, etc.) and organic anions (acetate, glucose, glutamate), such as the diluent called BPSE, the composition of which is given below.

Composition of the BPSE
potassium citrate 0.64 g
potassium glutamate 8.67 g
sodium acetate 4.3 g
0.34 g magnesium chloride
fructose 5.0 g
dipotassium hydrogen phosphate 12.7 g
potassium monophosphate 0.65 g
TES 2.5 g
Distilled water qs 1000 ml.

 simpler diluents such as a solution of TES buffer (Tris-EDTA-Saline), the composition of which is indicated below.

Composition of the TES buffer
TES 6.56 g
. NaOH 7.00 ml
distilled water qs 100 ml.

 Whatever their composition, the known diluents allow a conservation of the fertilizing power of the spermatozoa of rooster only for a duration not exceeding a few hours at 4 C.

 The inventors have now discovered that the addition of indole derivatives in the sperm dilution medium makes it possible to increase very significantly, compared to the diluents known in the prior art, both the shelf life of the sperm, sperm motility, and / or the rate of fertilization observed. The diluents thus obtained are also effective in a large number of species.

 The present invention relates to a sperm dilator comprising the usual constituents suitable for the dilution of sperm of a given species, and characterized in that it further comprises an indole derivative.

According to a preferred embodiment of the present invention, said indole derivative corresponds to the general formula (I)

in which
R1 represents
a hydrogen atom, an acetamide group or a C 1 -C 4 acyl group, which may be substituted in the 2-position by an amine group or by a ketone group, a C 4 primary aliphatic alcohol group or a C 14 primary alkylamine group; ,
R2 represents
a hydrogen atom, a methyl group, a hydroxyl group or a methoxy group,
R3 represents
a hydrogen atom.

 According to a preferred arrangement of this embodiment, said indole derivative is chosen from the group consisting of indol-3-carboxylic acid, indol-3-pyruvic acid, indol-3-butyric acid, indol-3-propionic acid, indol-3-glyoxalic acid, and indol-3-acetic acid.

 Preferably, said indole derivative is indol-3-acetic acid.

 According to another preferred embodiment of a sperm diluter according to the invention, said indole derivative is present at a concentration of between 1 μg / ml and 1 μg / ml, preferably between 1 and 100 μg / ml.

 The present invention finally relates to a method for preparing sperm for preservation, characterized in that said sperm is diluted in a solution comprising, in addition to the usual constituents of the solutions used to dilute the sperm of the species in question, a indole derivative as defined above.

 The implementation of the invention significantly improves the conservation of semen for artificial insemination or any other assisted reproduction practice.

 Sperm prepared according to the invention can be stored either at positive temperatures (the storage temperature depends in this case on the species), or by freezing.

 The present invention will be better understood using the additional description which follows, which refers to examples of use of a diluter according to the invention in different species.

 It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention, of which they in no way constitute a limitation.

EXAMPLE 1 - OVINE SPECIES: EFFECT OF INDOL-3 ACID
ACETIC ON THE CONSERVATION OF SEMENCE OF BELIER A
LIQUID STATE FOR 8 HOURS
1. Experimental protocol
a) Animals
A group of Lacaune ewes (dairy breed) divided into 4 batches of 105 to 136 animals each, was used for this experiment.

 The experiment was carried out over a period of 2 months (June to August).

b) Treatment of the seed
The semen is diluted to a final concentration of 1.4 x 109 spermatozoa / ml
- or in skimmed milk reconstituted from milk powder
or in skimmed milk supplemented with indol-3-acetic acid (AlA), at a concentration of 10 ng / ml.

 The diluted sperm is stored at a temperature of +15 ° C for 8 hours.

2. Results at farrowing
For each of the two groups tested, the number of females inseminated (N1), the percentage of completed pregnancies (number of whelps = n), and the number of born lambs (N2), are recorded, allowing the calculation of prolificacy, fertility and fertility, according to the following formulas - fertility rate = 100 x (n / N1) - prolificacy rate = 100 x N2 / n) - fertility rate = 100 x N2 / N1)
The results are shown in Table I below
TABLE I:

<tb><SEP> Milk <SEP> Control <SEP> Milk <SEP> + <SEP> AIA <SEP> Effect
<tb> 8h <SEP> âh <SEP>
<tb> Females <SEP>
<tb> inseminated <SEP>: <SEP> 132 <SEP> 136
<tb> Mises <SEP> Low <SEP> 84 <SEP> 92
<tb><SEP> Lamb <SEP> born: <SEP> 144 <SEP> 159
<tb><SEP> Fertility <SEP> (%) <SEP> 63.6 <SEP> 67.6 <SEP> + <SEP> 6.3%
<tb><SEP> Prolificacy <SEP> (%) <SEP> 171.3 <SEP> 172.6 <SEP> +
<tb> Fecundity <SEP> (%) <SEP> 109.1 <SEP> 116.9 <SEP> + <SEP> 7.3 <SEP>
<Tb>
These experiments show that when insemination was performed with sperm preserved in the presence of AIA, the fertility of the ewes was increased by 6.3% and the prolificacy by 0.7.

 In total, the cumulative effects on these two parameters lead to a 7.3 increase in fertility (number of lambs born per inseminated female).

EXAMPLE 2 - CAPRINE SPECIES: EFFECT OF INDOL-3 ACID
ACETIC ON THE POWERFUL POWER OF THE SPERM OF BOUC ON
FIFTEEN HOURS.

 To obtain a high fertilization rate of donor female oocytes in the goat species, insemination must be performed in utero by endoscopy approximately 24 hours after the observed onset of oestrus.

 The variability of the time of onset of estrus after superovulation treatment being important, the optimal time for artificial insemination can sometimes extend over 12 to 15 hours. It is therefore important to be able to keep the spermatozoa at least during this period of use. However, when they are kept at +4 C in a conventional diluter based on milk, there is a decrease in the sperm fertility beyond 4 hours of storage.

A) TESTING OF ARTIFICIAL INSEMINATION
1. Exnimental protocol
a) Seed
Ejaculates of Alpine goats produced at the end of the sexual season by males subjected to a favorable photoperiodic treatment are used.

After harvest, each ejaculate is divided into 2 parts, which are diluted at + 30'C:
- one with diluent based on reconstituted milk from powdered milk
the other with the milk diluent reconstituted from milk powder, supplemented with AIA at a concentration of 1 Wg / ml.

 The seed is flaked (0.2 ml) containing 100 x 106 total spermatozoa and stored at 4 ° C before fertilization.

(b) Insemination
In utero insemination under endoscopic control is carried out on 3 lots of 18 alpine goats and
Saanen (100 x 106 spermatozoa per uterine horn), 22 to 26 hours after the observed onset of estrus.

 The insemination of the goats is carried out alternately, in the order of oestrus, with the seed preserved in the presence of AIA, or with that preserved in the milk.

(c) Collection of spray
Collection is performed surgically at 57 or D8. after insemination. Only embryos judged to be of good quality were frozen to be stored in liquid nitrogen.

2. Results
The sperm fertility is determined by the ratio of the number of fertilized oocytes (early embryos) to the total number of structures collected (embryos + unfertilized oocytes).

The results are shown in Table II below
TABLE II

<tb><SEP> MILK <SEP> MILK <SEP> + <SEP> AIS
<tb><SEP> Time <SEP> of <SEP> Retention <SEP>% <SEP> Oocytes <SEP>% <SEP> Oocytes
<tb><SEP> of <SEP> the <SEP> Seed <SEP> Fertilized <SEP> Fertilized
<tb> 1 <SEP> to <SEP> 6 <SEP> hours <SEP> 72 <SEP> t <SEP> 36 <SEP> 66 <SEP> + <SEP> 42
<tb><SEP> (7) <SEP> (11)
<tb> 8.5 <SEP> to <SEP> 14.5 <SEP> hours <SEP> 58 <SEP> + <SEP> 45 <SEP> 71 <SEP> t <SEP> 35
<tb><SEP> (16) <SEP> (12)
<Tb>
Numbers in parentheses indicate the number of females in each batch.

The interest of the addition of ale to the traditional diluter used for the insemination of goats appears for rather long storage times of the seed (8.5 to 14.5 hours) where, (in spite of a great variability), it is observed that the rate of fertilized structures with respect to the rate of structures collected is greater by more than 10% in the case of conservation in the presence of AIA.
B) STUDY OF THE CONSERVATION OF SPEP! ATOZOIDS
IN VITRO
1. Exoerimental procole
Saanen and Alpine goats are used. The collection of the semen is done by an operator using a "go-in" goat and an artificial vagina kept at 37 C.

Different media are tried
Milk alone (skimmed reconstituted from commercial milk powder)
Milk + AIA at 1 Ag / ml (5.7 x 10-6 M)
Milk + AIA at 1 ng / ml (5.7 x 10-9 M)
Milk + AIA at 1 μg / ml (5.7 x 10-12 M)
Immediately after harvest, 100 of sperm are diluted in 2 ml of each of the above media, previously brought to 37 ° C. The whole is cooled and kept at 4'C in a cold room.

2. Results
The seed is examined to determine the percentage of motile spermatozoa and individual motility, immediately after dilution, and after 3, 24, 48, and 72 hours of storage at +4 C.

 For each sample, the spermatozoa are observed in a volume of 41 deposited between slide and coverslip on the heating stage of the microscope.

For each lot of spermatozoa, the following characteristics are determined
Percentage of live sperm,
Motility before artificial insemination.

For this parameter, the speed and quality of sperm movement are evaluated by a numerical score system of 0 to 5 [BISHOP et al., J. Scale].

Agric. Science, vol. 44, p. 127, (1954)] according to the following appreciations
0: no movement, all sperm are immobile
1 the spermatozoon trembles but does not move; a flagellar movement exists but does not allow the displacement
2 the spermatozoon trembles and moves over very small distances without crossing the microscopic field;
3: the spermatozoon is still shaking and moves on a circular path in the microscopic field
4: the spermatozoon does not tremble any more and moves on a rectilinear trajectory
5: the spermatozoon moves in a flickering way on the microscopic field.

The results are illustrated by Figures 1 and 2
Figure 1 represents the effect of different AIA concentrations on the percentage of motile spermatozoa, as a function of time, during storage at 4 ° C for 72 hours of goat spermatozoa (white circle, milk without AIA; white milk + lmg / l of AIA, white triangle milk + lng / ml of AIA inverted white triangle: milk + lpg / ml of AIA).

 Figure 2 shows the effect of different AIA concentrations on motility, as a function of time, during storage at 4 ° C for 72 hours, goat spermatozoa (white circle milk without AIA, white square milk + AIA lmg / Al: white triangle: AIA milk + lng / ml, inverted white triangle: milk + lpg / ml AIA).

 With regard to the evolution of the percentage of motile spermatozoa, the regular decrease of this parameter is identical until 24 hours, whatever the AIA concentrations present in the media.

 Motility decreases in a comparable manner between batches during the 72 hours of storage regardless of the conservation environment.

 However, there are significant differences between the media used (p <0.01). In the presence of AIA, there is a better 24-hour maintenance of the motility, the concentration of 1 ng / ml being the most effective.

EXAMPLE 3 Porcine Species: Effect of Indol-3 Acid
ACETIC ON THE CONSERVATION OF THE SEED OF VERRAT
A. - TESTS IN FRESH SPERM
1) Exodimental protocol
The trial is carried out on three lots of multiparous sows, in double insemination a first insemination after 5 days of storage (D5), and a second insemination the next day. Experimentation is done in shared ejaculates.

BTS double dose diluent control group (D) = 6 x spermatozoa by insemination.

Lot 1 Thinner BTS + AIA double dose (D) = 6 x 109 sperm per insemination
Lot 2 Thinner BTS + AIA single dose (S) = 3 x 109 spermatozoa by insemination.

2. Results
The results after insemination, either with the spermatozoa diluted in the conventional diluent BTS, or with the spermatozoa diluted in the diluter according to the invention, are presented in the Table
III.

TABLE III

<tb><SEP> Lot <SEP> witness <SEP> Lot <SEP> 1 <SEP> Lot <SEP> 2
<tb><SEP> BTS (D) <SEP> BTS + AIA <SEP> (D) <SEP> BTS + AIA <SEP> (S)
<tb><SEP> J5 <SEP> J5 <SEP> J5
<tb><SEP> Total <SEP> inseminated <SEP> 53 <SEP> 86 <SEP> 79 <SEP>
<tb><SEP> Total <SEP> set <SEP> low <SEP> 37 <SEP> 66 <SEP> 65
<tb><SEP>%<SEP> Mises <SEP> Low <SEP> 69.8 <SEP> 76.7 <SE> 82.5
<tb><SEP> Piglets <SEP> born
<tb> I <SEP> Mean / sow <SEP> 11.3 <SEP> 12.2 <SEP> 11.2
<tb><SEP> having <SEP> set <SEP> down
<tb><SEP> Born <SEP> alive
<tb> Mean / Sow <SEP> 10.3 <SEP> 11.2 <SEP>: <SEP> 10.7
<tb> I <SEP> having <SEP> set <SEP> down
<tb><SEP> Born <SEP> alive
<tb><SEP> Average / Sow <SEP> 7.2 <SEP> 8.6 <SEP> 8.8
<tb><SEP> inseminated
<Tb>
FIG. 3 represents the fertility rate of sows inseminated with sperm preserved for 5 days in BTS diluent, in the absence of AIA, and with a double dose of spermatozoa (cross-hatched rectangle), or in the presence of AIA, and single (horizontally hatched rectangle) or double dose (vertically hatched rectangle) of spermatozoa.

 FIG. 4 represents the prolificacy rate of sows inseminated with sperm preserved for 5 days, in BTS diluent, in the absence of AIA and with a double dose of spermatozoa (crossed-hatched rectangle), or in the presence of AIA, and single (horizontally hatched rectangle) or double dose (vertically hatched rectangle) of spermatozoa.

 The cumulative increases in the number of litablas and the number of live pigs per litter in the batches inseminated on day 5 by semen kept in the presence of AIA (lot 1 compared to the control group) lead to a very substantial increase in the final number of piglets. live piglets per sow inseminated (1.4 piglets, ie + 19%).

The results with a single dose of sperm (lot 2) with AIA are as good as those obtained with a double dose (lot 1), and therefore better than those observed with doubledose in absence of AIA (control) ;
B. FROZEN SPERM TESTING
1) Experimental protocol
The 5-boar semen is frozen according to the PAQUIGNON method (PAQUIGNON, 1974, Boar semen freezing technology, Swine Research Day, 6, 71-74). It is stored in the BTS diluent, to which the AIA is added, either only at the beginning of the freezing process, or at the beginning of the freezing process and also at the time of the thaw (concentration of 10 ng / ml). The doses thus prepared were used on gilts.

 The detection of heat is carried out precisely thanks to a male "blower". The time of ovulation is determined a posteriori by the profile of the plasma progesterone concentration curve.

 The gilts are inseminated with the frozen semen (2.5 to 3.0 x 109 spz), in double insemination at 12 hours of interval (father insemination, 24 hours after the beginning of the heat).

 Ultrasounds are performed 21 days after the first insemination and gilts are slaughtered at about 30 days of gestation to count the embryos and corpus luteum.

This experiment is conducted on 4 lots of 25 females each
Lot 1: BTS (-)
Lot 2: BTS + AIA (10 ng / ml) thawing (- +)
Lot 3: BTS + AIA (10 ng / ml) on freezing (±)
Lot 4: BTS + AIA (10 ng / ml) for freezing and thawing (++)
2. Results
The results are shown in the Table
IV below
TABLE IV

<tb><SEP> Lot <SEP> 1 <SEP>: <SEP> Lot <SEP> 2 <SEP>: <SEP> Lot <SEP> 3 <SEP>: <SEP> Lot <SEP>: <SEP> 4
<tb><SEP> ++ <SEP>
<tb> Females
<tb> inseminated <SEP> 23 <SEP> 21 <SEP> 22 <SEP> 20
<tb> Females
<tb> pregnant <SEP> 12 <SEP> 9 <SEP> 13 <SEP> 13
<tb>% <SEP> females
<tb> pregnant <SEP> 52 <SEP> 49 <SEP> 59 <SEP> 65
<tb> (Average) <SEP> (50.5) <SEP> (62)
<tb> Piglets /
<tb> female <SEP> (avg) <SEP> 8.6 + 3.2 <SEP> 9.8 + 3.2 <SEP> 10.4t3.0 <SEP> 10.5 + 3.2 <September>
<tb> Piglets
<tb> live / female <SEP> 8.0 + 2.6 <SEP> 9.3 + 2.7 <SEP> 10.1 + 2.8 <SEP> 10.2 + 3.3
<tb> (Percentage) <SEP> (93 <SEP>%) <SEP> (95 <SEP>%) <SEP> (97 <SEP>%) <SEP> (97 <SEP>%)
<tb> Body <SEP> yellow /
<tb> female <SEP> (avg) <SEP> 15.6 + 3.0 <SEP> 15.2 + 2.2 <SEP> 16.0 + 3.4 <SEP> 15.0 + 3, 1 <SEP>
<tb> Total <SEP> alive /
<tb> total <SEP> females
<tb> inseminated <SEP> 4.2 <SEP> 4.0 <SEP> 6.0 <SEQ> 6.6
<tb> (Average) <SEP> (4.1) <SEP> (6.3)
<Tb>
These results show that when the AIA is present in the diluent at the first stage of the freezing process (lots 3 and 4), an increase in reproduction performance is observed compared to batches where the AIA is still absent (lot 1 ) or introduced only on thawing (lot 2).

More precisely, we observe
1. An increase in the percentage of pregnant females from 50.5% (average of lots 1 and 2) to 62% (average of lots 3 and 4). These favorable results are not due to ovulation rates of sows since the numbers of yellow bodies are identical for the females of the four lots. The effect highlighted is therefore due to the treatment of the seed.

 2. An increase in litter size from 9.2 (lots 1 and 2) to 10.4 (lots 3 and 4) or 1.2 additional piglets per litter (+ 13%).

 3. An increase in the percentages of live pigs from 94% (average of lots 1 and 2) to 978 (lots 3 and 4) is an increase of more than 3%.

 All these cumulative beneficial effects lead to an average of 4.1 live pigs per sow inseminated for lots 1 + 2 (without AIA before freezing), compared to 6.3 live pigs per sow inseminated for lots 3 + 4 ( presence of 10 ng / ml AIA in the freezer diluent).

 This is a 65% increase in the number of live pigs per female inseminated with frozen sperm which is thus observed when AIA is present from the beginning of the freezing procedure.

EXAMPLE 4: POULTRY: EFFECT OF A DILUER CONTAINING
INDOLE-3-ACETIC ACID ON THE CONSERVATION OF SPERM
POULTRY
1. - Experimental conditions
The tests are carried out under standard breeding and insemination conditions, in particular
Duration of illumination: 16 hours (from 1 am to 5 pm)
Period favorable to insemination: the afternoon, after 13 hours
Insemination carried out between 14 and 15 hours, once a week and per hen; eggs collected from 2nd to 9th day after insemination
Number of hens per lot: 40
Standardized number of spermatozoa per dose (100 +15) x 106.

 2 experiments were performed. 550 to 665 eggs per batch were harvested.

 Dilution of sperm immediately after harvest, 1/2 (V / V) in diluents tested.

 Preservation: 24 hours at 4 C.

Lot 1: TES
Lot 2: TES + AIA
2. Results
The results after insemination either with the spermatozoa diluted in the conventional diluent BTS, or with the spermatozoa diluted in the diluter according to the invention, are presented in Table V
TABLE V

<tb><SEP> TES <SEP> TES <SEP> + <SEP> AIA
<tb><SEP> 24h <SEP> / <SEP> 4 <SEP> C <SEP> 24h <SEP> / <SEP> 4 <SEP> C <SEP>
<tb> 1st <SEP> experiment <SEP> 67.0+ <SEP> 1.9 <SEP> 70.0f <SEP> 1.9
<tb> (% <SEP> fertilization)
<tb> 2nd <SEP> experience <SEP> 65.0+ <SEP> 1.9 <SEP> 77.0+ <SEP> 1.7
<tb> (% <SEP> fertilization)
<tb> average <SEP> 66.0% <SEP> 73.5% <SEP>
<tb> (% <SEP> fertilization)
<Tb>
In industrial poultry farming, it is desirable to postpone the timing of sperm collection from that of placement in females. The desired time may be a few hours only, so that the only time of day that is most conducive to the success of inseminations can be devoted to sperm placement alone. But this delay can reach and exceed 24 hours. Therefore, there is a growing demand for preservation methods in view of the fertilizing power of the sperm of poultry.

 The results reported above show a favorable effect (+ 11%) of the AIA on the fertilization rates of chicken eggs, after 24 hours storage of rooster semen.

EXAMPLE 5 RABBITS: EFFECT OF INDOL-3-ACETIC ACID
ON THE RABBIT SEMEN
AIA trials were conducted on a herd of Hyper rabbits. At first, it was verified that AIA did not negatively affect rabbit sperm.

Experimental protocol
The available animals are divided into 2 comparable lots (physiological stage, age).

- Males: After harvest, each ejaculate is kept in a water bath at 30 ° C. Those selected after biological control, are split into 2 equal parts and placed in 2 tubes containing
- one: the diluter without AIA
- the other diluter with AIA (at a concentration of 1 Ag / ml).

 In both cases, the dilution is performed in the 6th: 5 volumes of ejaculate / l volume of diluent.

 The diluents were previously placed in the water bath (30 C).

 Immediately after gentle homogenization, the diluted semen is placed at room temperature while waiting for the moment of insemination.

 - Females The rabbits of the control group are inseminated with 0.5 ml of seed diluted in the control diluent (IMV Dilap 2000). The females in lot AIA are inseminated with 0.5 ml of diluted semen in the same diluent containing 1 μg / ml AIA. Before insemination, the color of the rabbit of the rabbits of both lots was systematically recorded. Insemination is carried out according to the classical method, ovulation being induced by the injection of 0.2 ml of RECEPTAL.

2) Results
The results relate to the color of the vulva, the size of litters at birth and weaning, and the weight at weaning. They are indicated in the
Table VI below.

TABLE VI

<tb><SEP> Number <SEP> Mounts <SEP> Born <SEP> Born <SEP> Born
<tb><SEP> Female <SEP> low <SEP> total / <SEP> alive / <SEP> alive /
<tb><SEP> Scope <SEP> Scope <SEP> Female
<tb> inseminated
<tb><SEP> Dilap <SEP> 14
<tb><SEP> 2000 <SEP> 23 <SEP> (610 <SEP> 8.2 <SEP> 7.1 <SEP> 4.3
<tb><SEP> Dilap <SEP> 19
<tb><SEP> 2000 <SEP> + <SEP> 24 <SEP> (790 <SE> 7.4 <SEP> 7.1 <SE> 5.6
<tb> AlA
<Tb>
The effect of AIA is mainly in the percentage of pregnant rabbits, which in both cases give birth to an average of 7.1 live young. As a result, the total number of live rabbits relative to the number of females inseminated is 30% higher in the lot with AIA compared to the control group.

 This experiment has made it possible to demonstrate a favorable effect of AIA under the classic conditions of insemination in rabbits.

Claims (6)

 1) Sperm diluent, comprising the usual constituents suitable for the dilution of sperm, and characterized in that it further comprises an indole derivative.  2) sperm diluent according to claim 1, characterized in that said indole derivative corresponds to the general formula (I)

 - a hydrogen atom  R3 represents  a hydrogen atom, a methyl group, a hydroxyl group or a methoxy group,  R2 represents  a hydrogen atom, an acetamide group or a C 1 -C 4 acyl group, which may be substituted in the 2-position by an amine group or by a ketone group, a C 1 -C 4 primary aliphatic alcohol group or a primary alkylamine group; in C1-4,  in which R1 represents  3) sperm diluent according to claim 1, characterized in that said indole derivative is selected from the group consisting of indol-3 carboxylic acid, indol-3-pyruvic acid, indol-3-butyric acid, indol-3-propionic acid, indol-3glyoxalic acid, and indol-3-acetic acid.  4) sperm diluent according to any one of claims 1 to 3, characterized in that said indole derivative is present at a concentration of between 1 ssg / ml and 1 μg / ml.  5) sperm diluent according to claim 4, characterized in that said indole derivative is present at a concentration of between 1 and 100 ng / ml.  6) Process for preparing sperm for preservation, characterized in that said sperm is diluted in a diluter according to any one of claims 1 to 5.