POLYPEPTIDE SPERM STABILISER FOR SEMEN USED IN ARTIFICIAL INSEMINATION

Abstract

A polypeptide sperm bio-stabiliser FB-sp having the following sequence of amino acids: RPGLPVFSPL is provided; the use of the polypeptide bio-stabiliser for preserving the gametes of animals; a composition having the polypeptide bio-stabiliser; a conventional or alternative method for artificial insemination involving the use of the composition in conjunction with extender means to be injected into the females of animal species for commercial use.

Claims

1. A spermatic bio-stabiliser polypeptide FB-sp, comprising an amino acid sequence according to SEQ ID NO: 1.

2. The stabilising polypeptide of claim 1, wherein it serves to preserve mammalian gametes.

3. The stabilising polypeptide of claim 1, wherein it serves to prepare a bio-stabiliser composition to preserve mammalian gametes.

4. The stabilising polypeptide of claim 2, wherein mammals are selected from the group consisting of porcine, caprine, bovine, wild boars, horses, canine, felines.

5. The stabilising polypeptide of claim 2, wherein the mammal is human.

6. A sperm bio-stabiliser composition, further comprising: the FB-sp spermatic bio-stabiliser of claim 1 in a concentration of up to 10 M diluted in an extender medium.

7. The composition of claim 6, further comprising a cryopreservant medium.

8. A process to stabilise animal semen, wherein the semen obtained from the ejaculate undergoes a manipulation process that includes the following stages: a) separating the cell fraction (sperm) of the semen, obtained by manipulation of the breeding males, from the liquid fraction consisting of nutrient liquid medium; b) diluting the living cells in physiological medium, wherein short- or long-term extenders are added to form a bio-stabiliser composition; c) adding the FB-sp spermatic stabiliser of claim 1, in the bio-stabiliser composition obtained in step b), in a concentration of up to 10 M, to form a fertilizing solution; d) keeping the fertilizing solution obtained in step c) in a cryopreserved or cold medium, at a temperature in the range from 180 C. to 17 C., stored in an insulated capped flask without contact with the environment until use; e) the fertilizing solution can be stored or transported under these conditions; and e) transporting the fertilizing solution to the place in situ, and raising the temperature to 17 C. and applying the fertilizing solution in the females at the time of use, according to the artificial insemination protocols known in the art.

9. The process according to claim 8, wherein in step d), the temperature range is between 1 C. and 17 C.

10. The process according to claim 8, wherein in step a), the breeding males are selected from the group consisting of porcine, caprine, bovine, wild boars, horses, canine and felines.

11. The process according to claim 8, wherein in step a), the breeding males are human.

12. The process according to claim 8, wherein in step c), a reactive oxygen species (ROS) stabilising complement at a concentration of from 0.001-100 M is added to the fertilizing solution.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0017] FIG. 1: FIG. 1 shows Progressive Gametes viability (%) vs. Time (hours) (.circle-solid.=Using FB-sp bio-stabiliser; .square-solid.=without FB-sp bio-stabiliser), it shows percentage of pig spermatozoa with normal mobility suitable for fertilization, incubated in the presence of FB-sp 10 M at 17 C.

[0018] FIG. 2: Concentration (ug/mL) of FB-sp in Plasma bbki4 vs. Time (hours). It shows plasma FB-sp concentration curve used in a liquid medium for a 90 kg animal.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The present invention describes a spermatic stabiliser, herein named FB-sp having an amino acid sequence, using a three letter nomenclature description, corresponding to: SEQ ID NO 1, [0020] Arg, Pro, Gly, Leu, Pro, Val, Phe, Ser, Pro, Leu,

[0021] also written as one letter nomenclature: [0022] RPGLPVFSPL,

[0023] An embodiment of the present invention is the use of said FB-sp additive to preserve male mammalian gametes, as well as the use of said additive to prepare a mammalian gamete bio-stabiliser composition for mammals such as porcine, caprine, bovine, wild boars, horses, canine, felines, or humans.

[0024] Other embodiment of the present invention is a bio-stabiliser composition comprising said FB-sp spermatic stabiliser used as a lyophilized product, at a concentration of up to 10 M (10 micromol), diluted in any extender medium available in the state of the art, the concentration of the FB-sp may increase up to 10% for treating low-ratio living cells.

[0025] Other embodiment is the previous bio-stabiliser composition optionally added with any cryopreservant available in the state of the art.

[0026] Other embodiment of the present invention consists of a process for preserving mammalian semen comprising the steps of:

[0027] a) Separating by centrifugation, at a value from 35 to 45 g for 5 to 15 minutes, the cell fraction (spermatozoa) of the semen obtained by manipulation of breeding males from the liquid fraction consisting of nutrient liquid medium, which allows settling and separating living cells from inert cell debris that is removed from the process;

[0028] b) Diluting the living cells in physiological medium, wherein short- or long-term extenders are added to form a bio-stabiliser composition;

[0029] c) Adding the sperm stabiliser FB-sp of sequence SEQ ID NO: 1 at a concentration of up to 10 uM to the bio-stabiliser composition of step b) to form a fertilizing solution;

[0030] d) Keeping the fertilizer solution of stage c) in a cryopreservant medium at a temperature in the range from 180 C. to 17 C., stored in an insulated capped flask, without contact with the environment until use, the fertilizing solution can be stored or transported under these conditions, optionally the temperature range is from 1 C. to 17 C.;

[0031] e) Transporting the fertilizing solution to the place in situ and raising its temperature to 17 C. at the time of use and applying the fertilizing solution in the breeding females according to the artificial insemination protocols known in the state of the art;

[0032] f) Optionally in step c) a reactive oxygen species (ROS) stabilising complement is additionally added to the fertilizing solution when the living cell ratio is very small (<20% relative to the average pattern obtained for boards of the porcine farm). The stabiliser supplement may be the same FB-sp spermatic stabiliser increased by 10% in concentration, or the addition of traditional commercial antioxidant molecules such as albumins and others. The concentration will depend on the indications of each antioxidant molecule, considering that xenobiotic molecules that induce a hyperosmolarity shock (greater than 300 mOSM) should never be added;

APPLICATION EXAMPLE

Example 1

[0033] A test of the progressive motility of gametes incubated in fertilizing solution added with the FB-sp bio-stabiliser polypeptide was performed. Progressive gametes are spermatozoa that have normal mobility capacity and are suitable for fertilizing.

[0034] FIG. 1 shows the Progressive Gametes viability (%) percentage vs. Time (hours) (.circle-solid.=using FB-sp bio-stabiliser; .square-solid.=without FB-sp bio-stabiliser)

[0035] The results show the protective effect of FB-sp on the progressive motility of porcine spermatozoa stored for 72 h at 17 C. It can be seen that the incubation of spermatozoa of porcine boars with FB-sp (.circle-solid.) (10 M) preserves progressive motility up to 85% of spermatozoa for a period of 7 days, while the control (.square-solid.) without stabiliser decreases to a value of 70% on day 7, the difference increases over the days. These results are measured through optical microscopy, as measured by the CASA method for pig spermatozoa evaluation. This result is highly significant in relation to the values obtained when only spermatozoa incubation physiological media known in the art are used, such as MR-A or Androstar.

[0036] It is thus found that the FB-sp bio-stabiliser polypeptide provides a spermatozoa protective effect by extending their viability.

[0037] An analysis in silico made using GastroPlus simulation software provide some features of the FB-sp bio-stabiliser polypeptide: 1279 Da molecular weight, the structure of the 10 amino acids of FB-sp shows that only the conservative amino acid V (valine) and R (arginine) have conditions of active sites, i.e., the external pole Ac has a negative charge and the external pole NH2 (terminal amino acid), is positive, which determines that it is a hydrophilic and polar structure.

[0038] It was also seen that for a dose of 1 mg/mL, the absorption coefficient is 0.079 and the physicochemical dissolution coefficient in aqueous medium is 9.9310.sup.3, depending on the calibration of the equipment in ionic liquid media used as controls, MR-A medium was used in this case. This means that it has a low absorption capacity in biological membranes such as spermatozoa membranes, vaginal mucosa membranes, intrauterine mucosa membrane, but it has a very good dissolution capacity in physiological medium thus forming an extensor solution.

[0039] FIG. 2 shows the plasma concentration curve free of product FB-sp (g/mL) administered in a liquid medium. When simulating the process for an animal weighing 90 kg, it can be seen that its blood plasma, after using the fertilizing solution in a concentration of 1 mg/mL of FB-sp diluted in MR-A nutrient medium (nutritive medium for the preservation of porcine semen), has concentrations after 8 hours on the order of only 1.110.sup.8 g/mL in plasma and that its maximum pick is obtained after 2 hours. Then, the in silica model shows that it has a very low permeability coefficient and low residual effect because it is fully degraded from the organism within the first 8 hours.

[0040] Demonstrating that the applied concentration does not have a cytotoxicity risk.