WO1996010908A1 - Dilueur de sperme comprenant un derive d'indole - Google Patents

Dilueur de sperme comprenant un derive d'indole Download PDF

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Publication number
WO1996010908A1
WO1996010908A1 PCT/FR1995/001322 FR9501322W WO9610908A1 WO 1996010908 A1 WO1996010908 A1 WO 1996010908A1 FR 9501322 W FR9501322 W FR 9501322W WO 9610908 A1 WO9610908 A1 WO 9610908A1
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WO
WIPO (PCT)
Prior art keywords
sperm
aia
group
indol
diluent
Prior art date
Application number
PCT/FR1995/001322
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English (en)
French (fr)
Inventor
Yves Combarnous
José FERREIRA NUNES
Original Assignee
Institut National De La Recherche Agronomique - I.N.R.A.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut National De La Recherche Agronomique - I.N.R.A. filed Critical Institut National De La Recherche Agronomique - I.N.R.A.
Priority to AU36581/95A priority Critical patent/AU3658195A/en
Priority to BR9509331A priority patent/BR9509331A/pt
Publication of WO1996010908A1 publication Critical patent/WO1996010908A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/10—Preservation of living parts
    • A01N1/12—Chemical aspects of preservation
    • A01N1/122—Preservation or perfusion media
    • A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators

Definitions

  • the present invention relates to the use of indole derivatives to improve the survival of spermatozoa and their ability to fertilize, for the purpose of artificial insemination or any other assisted reproduction practice.
  • these problems are partially solved by freezing the sperm.
  • this freezing is difficult, if not impossible, insofar as it results in the destruction or inactivation of too large a number of spermatozoa.
  • this medium will hereinafter be referred to as "diluent" .
  • Different semen diluents have thus been recommended, with the aim of improving the conservation and the fertilizing capacity of the spermatozoa. All these dilutors must take into account a certain number of physicochemical imperatives such as: - having an isotonic osmotic pressure,
  • compositions which are most commonly used may be mentioned, for example:
  • Diluents used in the porcine species Many diluents have been used for the conservation of boar semen in the liquid state. Their composition is often similar qualitatively, but presents notable differences in terms of concentrations, which leads to significant variations in pH and osmotic pressure. In general, these dilutors are composed of the following elements:
  • Glucose which is better absorbed by sperm than fructose, is the best source of energy. It also contributes to maintaining the osmotic balance of the environment.
  • BTS the composition of which is indicated below, is the one which has given the best results for the dilution and storage at 15 ° C of porcine semen, whether it is collected on the farm or in a breeding center. It can be used until the 3rd day after harvest without significant drop in fertility or prolificacy.
  • composition of the BTS is Composition of the BTS:
  • the egg yolk would protect the sperm from thermal shock. This action would be linked to the lipoprotein and lipid compounds of the egg [KAUPSCHMIDT, J. Dairy Sci., Vol. 34, p. 44-51, (1953)].
  • Egg yolk dilutors give very poor results.
  • the seminal plasma of the goat species has been shown to contain a phospholipase A which hydrolyzes lecithin from egg yolk to lysolecithin and fatty acids, which have a spermicidal effect.
  • composition of the goat milk dilutor the composition of which is indicated below, and which is currently used conventionally for storage times of up to 12 hours, without any drop in the prolificacy or fertility.
  • composition of the dilutor conventionally used for sheep shown below, is similar to that of the goat diluent, and further includes antibiotics. This diluent makes it possible to preserve the fertilizing power of ovine sperm for 8 hours at 15 "C.
  • Composition of the sheep diluter is similar to that of the goat diluent, and further includes antibiotics. This diluent makes it possible to preserve the fertilizing power of ovine sperm for 8 hours at 15 "C.
  • the diluent most conventionally used in cattle is a dilutor based on skimmed milk reconstituted from milk powder, which is marketed under the name of LAICIPHOS ND. Milk has an increased protective effect if it is added with 10% chicken egg yolk and antibiotics.
  • This diluent allows a conservation of the semen up to 72 hours at 5 * C.
  • Diluent used in rabbits The diluent used by the majority of breeders is a saline-based diluent marketed by the company IMV under the name of DYLAP 2000.
  • a mineral solution simulating the composition of the seminal liquid of trout constitutes a medium which allows satisfactory preservation, at a temperature close to 0 * C, of the fertility of the gametes of trout Rainbow. The fertility of the sperm remains without decrease for 30 minutes.
  • the spermatozoa remain immobile, especially due to the high concentrations of potassium ions [SCHLENK and KAHMANN, Biochem. Z., vol. 295, p. 283-301, (1937); BILLARD and JALABERT, Ann. Biol. Anim. Bioch. Biophys. , flight. 14, p. 601-610, (1974)].
  • an insemination diluent consisting of a NaCl solution (posm 250, pH 9 to 9.5). This is added to the sperm (previously diluted in MMLS) and to the ova during insemination.
  • BPSE diluent-diluers comprising many cations (Na +, K +, Mg ++ ...) and organic anions (acetate, glucose, glutamate), such as the diluent called BPSE, the composition of which is given below below.
  • Composition of BPSE potassium citrate 0.64 g. potassium glutamate 8.67 g sodium acetate 4.3 g magnesium chloride 0.34 g fructose 5.0 g dipotassium hydrogen phosphate 12.7 g. potassium monophosphate 0.65 g
  • Tris-EDTA-Saline Tris-EDTA-Saline
  • distilled water qs 100 ml. Whatever their composition, known diluents only allow the fertilizing power of rooster sperm to be preserved for a period not exceeding a few hours at 4 "C.
  • indole derivatives in the semen dilution medium makes it possible to very significantly increase significant, compared to the diluents known in the prior art, both the shelf life of the sperm, the motility of the spermatozoa, and / or the fertilization rate observed.
  • the diluents thus obtained are also effective in a large number of species.
  • the subject of the present invention is a semen diluent comprising the usual constituents suitable for diluting the sperm of a given species, and characterized in that it also comprises an indole derivative.
  • said indole derivative corresponds to the general formula (I)
  • R represents: - a hydrogen atom, an acetamide group, an acyl group C j __4, which may be substituted in the 2 position by a group or a ined by a keto group, a primary aliphatic alcohol moiety 1-. 4, a C alkyl primary alkylamine group. ,
  • R2 represents:
  • R3 represents:
  • said indole derivative is c-.isi in the group consisting of indol-3-carboxylic acid, indol-3-pyruvic acid, indol-3- butyric, indol-3-propionic acid, indol-3-glyoxalic acid, and indol-3-acetic acid.
  • said indole derivative is indol-3-acetic acid.
  • said indole derivative is present at a concentration of between 1 ⁇ g / ml and 1 pg / ml, preferably between 1 and 100 ng / ml.
  • the present invention finally relates to a process for preparing sperm with a view to its conservation, 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 considered, a indole derivative as defined above.
  • the implementation of the invention clearly improves the conservation of sperm for artificial insemination or any other assisted reproduction practice.
  • the sperm prepared in accordance with the invention can be stored either at positive temperatures (the storage temperature depends in this case on the species concerned), or by freezing.
  • Lacaune sheep (dairy breed) divided into 4 batches of 105 to 136 animals each, was used for this experiment.
  • the seed is diluted to a final concentration of 1.4 x 10 ⁇ spermatozoa / ml:
  • skimmed milk reconstituted from milk powder or in skimmed milk supplemented with indol-3-acetic acid (AIA), at a concentration of 10 ng / ml.
  • AIA indol-3-acetic acid
  • the diluted sperm is stored at a temperature of + 15 ° C for 8 hours.
  • insemination must be carried out in utero by endoscopy approximately 24 hours after the observed onset of estrus.
  • each ejaculate is divided into 2 parts, which are diluted to +30 "C: one with the diluent based on milk reconstituted from milk powder;
  • the collection is carried out surgically, on D7 or D8. after insemination. Only embryos judged to be of good quality were frozen to be stored in liquid nitrogen.
  • Sperm fertility is determined by the ratio of the number of fertilized oocytes (early embryos) the total number of structures collected (embryos + unfertilized oocytes).
  • the numbers in parentheses indicate the number of females in each batch.
  • Saanen and Alpine goats are used.
  • the semen collection is carried out by an operator using a goat "bott-en-train” and an artificial vagina maintained at 37 * C.
  • the semen is examined to determine the percentage of mobile sperm and individual motility immediately after dilution, and after 3, 24, 48, and 72 hours of storage at +4 "C.
  • the spermatozoa are observed in a volume of 7 ⁇ l deposited between blade and coverslip on the heating stage of the microscope.
  • the sperm is still trembling and moves on a circular path in the microscopic field
  • FIG. 1 represents the effect of different concentrations of AIA on the percentage of mobile spermatozoa, as a function of time, during the storage at 4 "C for 72 hours of the spermatozoa goats (white circle: milk without AIA; white square: 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 concentrations of AIA on motility, as a function of time, during storage at 4 ° C for 72 hours, goat spermatozoa (milk white circle without AIA; white square: milk + lmg / ⁇ l of AIA; white triangle: milk + lng / ml of AIA; inverted white triangle: milk + lpg / ml of AIA).
  • the regular decrease in this parameter is identical up to 24 hours, regardless of the concentrations of AIA present in the media.
  • the motilite decreases in a comparable manner between batches during the 72 hours of storage regardless of the storage medium.
  • EXAMPLE 3 PORCINE SPECIES: EFFECT OF INDOL-3- ACETIC ACID ON THE CONSERVATION OF VERRAT SEED
  • FIG. 3 represents the fertility rate of inseminated sows with sperm stored for 5 days in a BTS dilutor, in the absence of AIA, and with a double dose of spermatozoa (rectangle hatched in braces), or in the presence of AIA, and with single (horizontally hatched rectangle) or double dose (vertically hatched rectangle) of sperm.
  • FIG. 4 represents the prolificacy rate of inseminated sows with sperm stored for 5 days, in a BTS diluter, in the absence of AIA and with a double dose of sperm (rectangle hatched in braces), or in the presence of AIA, and with single (horizontally hatched rectangle) or double dose (vertically hatched rectangle) of sperm.
  • Heat detection is carried out precisely using 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 10 9 spz), in double insemination at 12 hour intervals (1st insemination, 24 hours after the start of the heat).
  • Lot 1 Lot 2: Lot 3: Lot: 4
  • Body yellow / female (avg.) 15.6 ⁇ 3.0 15.2 ⁇ 2.2 16.0 ⁇ 3.4 15.0 ⁇ 3.1
  • EXAMPLE 4 POULTRY: EFFECT OF A THINNER CONTAINING
  • the tests are carried out under standardized breeding and insemination conditions, in particular:
  • Duration of lighting 16 hours (from 1 am to 5 pm); .
  • Period favorable to insemination afternoon, after 1 pm; Insemination carried out between 2 and 3 p.m., once a week and per hen; eggs collected from the 2nd to the 9th day after insemination; .
  • Standardized number of sperm per dose (100 ⁇ 15) x 10 6 .
  • AIA tests were carried out on a herd of Hyper rabbits. First, it was verified that the AIA did not have a negative impact on rabbit sperm. Experimental protocol
  • the available animals are divided into 2 comparable lots (physiological stage, age).
  • each ejaculate is kept in a water bath at 30 ° C.
  • Those selected after biological control, are divided into 2 equal parts and placed in tubes containing 2:
  • the diluent without AIA the other: the diluent with AIA (at a concentration of 1 ⁇ g / ml).
  • the dilution is carried out in the 6th: 5 volumes of ejaculate / 1 volume of diluent.
  • the dilutors were previously placed in the water bath (30 "C).
  • the diluted semen is placed at room temperature while waiting for the time of insemination.
  • the rabbits in the control batch are inseminated with 0.5 ml of semen diluted in the control diluent (IMV Dilap 2000).
  • the females of the AIA batch are inseminated with 0.5 ml of semen diluted in the same diluent containing 1 ⁇ g / ml of AIA.
  • the color of the vulva of the rabbits of the two batches was systematically recorded.
  • the insemination is carried out according to the conventional method, ovulation being induced by the injection of 0.2 ml of RECEPTAL.
  • the results relate to the color of the vulva, the size of the litters at birth and at weaning as well as the weight at weaning. They are shown in Table VI below.
  • the effect of AIA is mainly manifested in the percentage of rabbits giving birth, which give birth in both cases, to litters of 7.1 live young on average.
  • the total number of live rabbits relative to the number of inseminated females is 30% higher in the lot with AIA compared to the control lot.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Dentistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Environmental Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
PCT/FR1995/001322 1994-10-11 1995-10-11 Dilueur de sperme comprenant un derive d'indole WO1996010908A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU36581/95A AU3658195A (en) 1994-10-11 1995-10-11 Sperm diluter comprising an indole derivative
BR9509331A BR9509331A (pt) 1995-10-11 1995-10-11 Diluente de esperma compreendendo um derivado de indol

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
FR9412122A FR2725342A1 (fr) 1994-10-11 1994-10-11 Dilueur de sperme comprenant un derive d'indole
FR94/12122 1994-10-11

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WO1996010908A1 true WO1996010908A1 (fr) 1996-04-18

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Application Number Title Priority Date Filing Date
PCT/FR1995/001322 WO1996010908A1 (fr) 1994-10-11 1995-10-11 Dilueur de sperme comprenant un derive d'indole

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AU (1) AU3658195A (enrdf_load_stackoverflow)
FR (1) FR2725342A1 (enrdf_load_stackoverflow)
WO (1) WO1996010908A1 (enrdf_load_stackoverflow)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116616276A (zh) * 2023-05-30 2023-08-22 青岛农业大学 Ma-5的新用途及猪精液常温保存稀释液

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR3072246B1 (fr) 2017-10-13 2020-06-12 Biodesiv Efnium Polymere antimicrobien pour semences animales

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU399237A1 (ru) * 1972-04-15 1973-10-03 Всесоюзный ордена Трудового Красного Знамени научно исследовательский институт животноводства Среда для разбавления спермы
SU1274652A1 (ru) * 1985-01-14 1986-12-07 Тихоокеанский научно-исследовательский институт рыбного хозяйства и океанографии Способ повышени жизнестойкости оплодотворенной икры рыб

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SU399237A1 (ru) * 1972-04-15 1973-10-03 Всесоюзный ордена Трудового Красного Знамени научно исследовательский институт животноводства Среда для разбавления спермы
SU1274652A1 (ru) * 1985-01-14 1986-12-07 Тихоокеанский научно-исследовательский институт рыбного хозяйства и океанографии Способ повышени жизнестойкости оплодотворенной икры рыб

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Week 7429, Derwent World Patents Index; AN 74-53183V [29] *
DATABASE WPI Week 8731, Derwent World Patents Index; AN 87-219981 *
DATABASE WPI Week 9423, Derwent World Patents Index; AN 94-189797 [23] *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116616276A (zh) * 2023-05-30 2023-08-22 青岛农业大学 Ma-5的新用途及猪精液常温保存稀释液
CN116616276B (zh) * 2023-05-30 2025-06-24 青岛农业大学 Ma-5的新用途及猪精液常温保存稀释液

Also Published As

Publication number Publication date
FR2725342B1 (enrdf_load_stackoverflow) 1997-02-21
FR2725342A1 (fr) 1996-04-12
AU3658195A (en) 1996-05-02

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