Methods and compositions for low-dose insemination

Abstract

The present invention relates to a composition for low-dose artificial insemination and a method for artificial insemination of a female mammal using a dose of sperm from a male mammal of the same species which is significantly lower than would be required in the absence of the composition. The preferred female mammal is a sow (wild boar), the preferred composition is for artificial uterine insemination, and the preferred method of artificial insemination is uterine insemination.

Description

[0001] This invention relates to compositions for low-dose artificial insemination and methods for artificially inseminating female mammals using sperm from male mammals of the same species, wherein the dose of sperm is significantly lower than that required in the absence of the composition. Preferred female mammals are sows (wild boars, *Sus scrofa*), and the composition is preferably used for uterine insemination, with uterine insemination being the preferred method of artificial insemination. Background Technology

[0002] For traditional artificial insemination of sows, the dosage of fresh boar sperm used is approximately 1 to 3 x 10^10 in a 100 mL volumetric dilution. 9 One sperm was used, which entered the uterus twice within 24 hours, and the dose of frozen sperm used was approximately 5 x 10^ ... 9 Each boar ejaculate can be divided into 5 to 30 doses of fresh sperm. Typically, this traditional artificial insemination is first performed about 24 hours before ovulation, which is observed during the standing heat period.

[0003] Using invasive methods, such as surgical laparoscopy, sperm doses can be as low as 1 x 10⁻⁶. 7 Frozen and thawed sperm can be directly placed into the sow's fallopian tubes for fertilization.

[0004] US 5135759A describes a FACS apparatus and method for the sex chromosome-specific sorting of sperm cells into sperm portions that primarily contain the X- or Y-chromosome-carrying segments.

[0005] WO2010 / 0149739A1 describes a FACS apparatus and method that uses a laser to deflect a fluid portion containing sperm to sort it into portions that primarily contain sperm carrying either an X-chromosome or a Y-chromosome.

[0006] Green et al., Reproduction 122, 305-315 (2001) described the binding of boar sperm to oviduct epithelial cells mediated by carbohydrate polysaccharides.

[0007] Ekhlasi-Hundrieser et al., Biol Reprod 73, 536-545 (2005) described sperm adhesion factor AQN-1 as a candidate receptor for the formation of seminal vesicles in the oviducts of sows.

[0008] Dostalova et al., Eur J Biochem 230, 329-336 (1995), and Calvete et al., Biol Chem 377, 521-527 (1996) described sperm adhesion factors AQN-1 and AQN-3 as exhibiting AWN-like affinity for oocyte zona pellucida glycoproteins.

[0009] Krueger and Rath, Reprod Fertil Dev. 12, 113-117 describe the administration of very low doses of sperm via laparotomy to the tip of the uterine horn to induce pregnancy.

[0010] Taylor et al., Soc Reprod Fertil Suppl 66, 83-84 (2009), described the binding of pig sperm to uterine epithelial cells.

[0011] Purpose of the invention

[0012] One object of the present invention is to provide a composition for artificial insemination, particularly a composition for uterine insemination, which allows insemination with a significantly lower sperm dose. The artificial insemination process should avoid any surgical steps and should preferably include, or consist of, the administration of a sperm dose by conventional artificial insemination, for example, by administration to the uterus of a sow. Summary of the Invention

[0013] This invention achieves this objective through the features of the claims, particularly in the first variant by contacting sperm with a binder having an affinity for N-acetylglucosamine (Glc-NAc) and / or for sialic acid and / or for mannose, and / or with a reagent that hydrolyzes sialic acid and / or N-acetylglucosamine and / or oligomannose, for example, such contact is performed before or simultaneously with the introduction of sperm into the uterus of a female mammal; and in the second variant, contacting the uterus with a binder having an affinity for sialic acid and / or for β-D(1,3)-galactosamine, and / or with a reagent that hydrolyzes sialic acid, and / or with sialic acid and / or N-acetylglucosamine and / or oligomannose or mannose, for example, such contact is performed before or simultaneously with the introduction of sperm into the uterus of a female mammal. The first and second variants may be performed independently or in combination.

[0014] In the first variant, in the first embodiment, contact between sperm and at least one of these compounds can be achieved by providing a composition (also known as a liquid formulation) for artificial insemination containing sperm, wherein the composition contains at least one of these compounds. Preferably, the composition contains a significantly lower dose of sperm than that used in conventional artificial insemination.

[0015] In the first variant, in the second embodiment, sperm can be contacted with at least one of these compounds, separated from the unbound portions of these compounds, for example, by mixing sperm with at least one of these compounds and washing the sperm, for example by collecting sperm from a suspension containing at least one of these compounds by centrifugation, and transferring the sperm to a liquid composition that does not contain at least one of these compounds, for example, suitable for insemination, for example, as at least one insemination dose.

[0016] In a first variant, in a third embodiment, at least one of these compounds for contacting sperm can be provided as a composition for administration to the female reproductive tract before or simultaneously with the introduction of sperm, the sperm being contained in a separate liquid composition.

[0017] In a second variant, in a fourth embodiment, at least one compound for contacting the uterus may be provided as a composition for application to the female reproductive tract (especially the uterus) before or simultaneously with the introduction of sperm, the sperm being contained in a separate liquid composition.

[0018] In the second variant, in the fifth embodiment, at least one compound for contact with the uterus may be provided in the composition containing a sperm dose.

[0019] In embodiments where the method includes applying the composition prior to introducing sperm into the female, for example, where the composition does not contain sperm, the method is used to prepare the female for artificial insemination. Artificial insemination may occur after the application of the composition, for example, by subsequently introducing sperm into the uterus.

[0020] Preferably, the female mammal is a gilt or sow, and the sperm is fresh or frozen boar sperm. In variations, gender-enriched sperm, such as boar sperm preparations, or sex chromosome-specifically sorted sperm, are used fresh or frozen, preferably thawed from frozen.

[0021] This invention is based on the finding that, at least in pigs, fertile sperm binds largely to the uterine epithelium, particularly the endometrium (using uterine epithelial cells as an example), and that reducing sperm binding to the uterine epithelium results in insemination being performed using lower doses of sperm for artificial insemination. To reduce sperm binding to the uterine epithelium, binding ligands that mediate sperm binding to the endometrium can be masked, for example, by contacting the sperm with a binding agent having an affinity for N-acetylglucosamine (Glc-NAc) and / or for sialic acid, said binding agent being, for example, a lectin or antibody; and / or by contacting the sperm with an enzyme that hydrolyzes sialic acid and / or N-acetylglucosamine (e.g., sialidase that hydrolyzes sialic acid). Alternatively or additionally, the endometrium may be contacted with a binding agent having an affinity for sialic acid and / or for β-D(1,3)-galactosamine, said binding agent being, for example, a lectin or antibody; and / or with a reagent that hydrolyzes sialic acid; and / or with sialic acid and / or N-acetylglucosamine and / or oligomannose or mannose. In another alternative, sperm may be contacted with sialic acid and / or β-D(1,3)-galactosamine. Preferably, the lectin, sialidase, and antibody are natural molecules, for example, unmodified, especially unmodified by PEG.

[0022] When N-acetylglucosamine (Glc-NAc) and / or sialic acid and / or oligomannose or mannose are masked on sperm and / or endometrial cells, or when these molecules are removed from sperm and / or endometrial cells, and / or when sperm and / or the uterus are brought into contact with N-acetylglucosamine (Glc-NAc) and / or sialic acid and / or oligomannose or mannose, a decrease in sperm binding to the endometrium is observed, leading to, for example, saturation of molecules bound to these molecules. These observations can ultimately be explained as follows: at least in pigs, sperm binding to the endometrium is mediated by the interaction of N-acetylglucosamine and / or sialic acid and / or mannose and / or glucose present on sperm, particularly in the sperm head, and also by sialic acid and / or β-D(1,3)-galactosamine present on the endometrium.

[0023] Because the compositions and methods according to the invention allow for the insemination of female mammals, particularly non-human female mammals, preferably sows, with a significantly reduced number of sperm compared to conventional artificial insemination using substances such as: binders having an affinity for N-acetylglucosamine (Glc-NAc) and / or for sialic acid and / or for mannose, and / or reagents for hydrolyzing sialic acid and / or for N-acetylglucosamine and / or for hydrolyzing oligomannose (e.g., before or simultaneously with the introduction of sperm into the uterus of the female mammal); and in a second variant, binders having an affinity for sialic acid and / or for β-D(1,3)-galactosamine and / or reagents for hydrolyzing sialic acid, and / or sialic acid and / or N-acetylglucosamine and / or oligomannose or mannose for contact with the uterus, these compositions act as pharmaceutically active compounds to enhance insemination using a smaller number of sperm compared to conventional artificial insemination. Therefore, the composition is used to enhance the fertility of artificial insemination sperm preparations, particularly for use in the female reproductive tract, such as the uterus, before or simultaneously with the introduction of sperm, and / or by contacting the sperm with the composition, particularly before or simultaneously with the introduction of sperm into the female reproductive tract, for example, into the uterus, especially into the short uterus of a sow.

[0024] Here, the sperm dosage administered relates to the number of live sperm in the dosage. Preferably, compared to the dosage used for conventional artificial insemination, the artificial insemination dosage according to the invention is reduced to at most one-fifth, preferably at most one-tenth, more preferably at most one-twentieth or one-thirtieth, more preferably at most one-fiftieth or one-hundredth, wherein in conventional insemination, the sperm are suspended in a synthetic culture medium free of compounds, and wherein, in both the dosage according to the invention and the dosage used for conventional artificial insemination, the sperm are fresh (i.e., not frozen) or frozen. For example, for sows (wild boars), the dosage for fresh sperm in conventional artificial insemination contains approximately 1 to 3 x 10⁻⁶ sperm. 9 (Colenbrander et al., Reprod DomestAnim 1, 298-333 (1991)), and for frozen sperm contains approximately 5 x 10⁻⁶. 9 (It is usually thawed before insemination). Generally, it is preferred that insemination be performed at least 24 hours before ovulation, for example, when estrus is observed.

[0025] Preferably, the dosage for artificial insemination may contain 0.4 x 10⁻⁶ units. 9 0.2x 10 is preferred. 9 More preferably 100x10 6 Or 66x 10 6 More preferably 40x10 6 Or 20 x 10 610 sperm cells (for fresh sperm), and 1 x 10 9 0.5 x 10 is preferred 9 More preferably 250x10 6 Or 165x 10 6 More preferably 100x10 6 Or 50x 10 6 One sperm (for frozen sperm).

[0026] Fertility obtained by the compositions and methods of the present invention and fertility obtained by conventional artificial insemination are preferably defined as the sum of fertilization rate, litter size, and weaning rate, for example, as described in Vazquez et al., Theriogenology 63, 536-547 (2005).

[0027] Optionally, the sperm are primarily those carrying the X chromosome, obtained, for example, through sex chromosome-specific sorting.

[0028] The dosage may include, for example, a volume of 80 to 100 mL.

[0029] The binder may be included in the dosage in an amount of, for example, 0.1-1 or 1.0-5.0 μg / ml.

[0030] Preferably, the sperm preparation may contain at least one conjugate having an affinity for N-acetylglucosamine (Glc-NAc) and / or an affinity for sialic acid, said conjugate may be, for example, a lectin or antibody, and / or may contain an enzyme that hydrolyzes sialic acid and / or N-acetylglucosamine, such as sialidase that hydrolyzes sialic acid. Optionally, alternatively, the sperm preparation may contain N-acetylglucosamine (Glc-NAc), sialic acid, and / or oligomannose and / or mannose.

[0031] Optionally, the at least one binding agent (which may be, for example, a lectin or antibody) having an affinity for N-acetylglucosamine (Glc-NAc) and / or an affinity for sialic acid and / or an enzyme that hydrolyzes sialic acid and / or N-acetylglucosamine (e.g., sialidase that hydrolyzes sialic acid) may be administered to the female reproductive tract, such as the cervix, preferably to the uterus, prior to the introduction of sperm (e.g., fertilization).

[0032] Most preferably, the compositions and methods of the present invention comprise a conjugate having an affinity for N-acetylglucosamine and / or for sialic acid, particularly a lectin or antibody that binds to N-acetylglucosamine and / or for sialic acid, and / or an enzyme that hydrolyzes sialic acid, such as sialidase. These compositions and methods have the advantage of not interfering with or only interfering with sperm binding to the zona pellucida at an acceptablely low level, which is generally considered to depend at least in part on the binding of mannose-containing ligands to the zona pellucida.

[0033] The invention will now be described in more detail with reference to the accompanying drawings and embodiments.

[0034] - Figure 1 These are confocal micrographs of boar sperm after co-culturing with a cultured UEC monolayer for 10 minutes.

[0035] - Figure 2A These are confocal micrographs of bovine sperm pretreated with ConA after co-culturing with a cultured UEC monolayer for 10 minutes.

[0036] - Figure 2B These are confocal micrographs of porcine sperm pretreated with WGA after co-culturing with a cultured UEC monolayer for 10 minutes, and...

[0037] - Figure 3A These are confocal micrographs of untreated porcine sperm co-cultured with cultured porcine fetal fibroblasts (porc.foet.F) for 10 minutes.

[0038] - Figure 3B These are confocal micrographs of untreated porcine sperm after co-incubation with porcine aortic endothelial cells (pAEC) for 10 minutes.

[0039] - Figure 4A These are confocal micrographs of UEC images after co-incubation with pig semen for 10 minutes and preprocessing with sWGA, and...

[0040] - Figure 4B These are confocal micrographs of UEC after co-incubation with pig sperm for 10 minutes and preprocessing with WGA.

[0041] Example 1: Improving fertility by blocking sperm binding sites

[0042] Based on the first variant, binding sites of sperm involved in binding to uterine epithelial cells (UECs) were identified and blocked. Sperm were then co-incubated with cultured UECs, showing a reduction in sperm binding to UECs.

[0043] The fused UECs were grown on glass coverslips to form primary cell cultures. Uteruses were obtained from a total of 78 primiparous German Landrace or 8-10 month old German Edelschwein gilts (with a live weight exceeding 110 kg) for primary cell collection. All animals were raised and handled in accordance with German animal welfare regulations. The gilts were monitored for natural estrus and slaughtered at the peak of estrus, i.e., when artificial insemination was to be performed. The gilts were stunned with electric shock and then bled to death. Three minutes after bleeding, the abdomen was opened, and the entire uterus was removed. Furthermore, the ovaries, fallopian tubes, and mesentery were removed with sterile scissors without damaging the myometrium. The uterine horns were ligated together with sutures, and 20-25 cm sections were cut. The sections were placed in glass vials containing 2% penicillin / streptomycin (P / S; PAA, Pasching, Austria) and free of calcium. ++ and Mg ++ The uterine smear was placed in sterile phosphate-buffered saline (PBS) (Karl Roth, Karlsruhe, Germany) and incubated at 5°C for 45 minutes. After incubation at 5°C for 45 minutes, the uterine smear was removed from the bottle and placed on the fibrinous tissue under a sterile laminar flow system. The suture material was removed. Each corner was secured with a sterile artery clamp to ensure the open end, and the lumen was then flushed three times with 10 ml of sterile PBS containing 2% P / S using a 10 ml sterile serum pipette. One end was then closed with clamps, and 10 ml of EDTA / Try (10% PAA, Pasching, Austria, Ca) was pipetted through a 10 ml sterile serum pipette. ++ / Mg ++ The uterine horn was inserted into the horn, and the remaining end was closed with clamps. Fine movement of the uterine horn ensured equal distribution throughout the lumen. The cells were incubated for 15 minutes at 37°C in 20 ml of fresh, pure PBS containing 2% P / S. After enzymatic digestion, 10 ml of PBS was added to slightly move the horn, and the liquid was transferred to a 50 ml centrifuge tube containing 5 ml of warm cell culture medium (D20, 77% DMEM, 20% FBS, 1% sodium pyruvate, 1% amino acids, 1% P / S). The cell suspension was centrifuged at 209 × g at room temperature for 4 minutes. This process was repeated three times for each horn, with different digestion times; the second and third repetitions were performed for 10 minutes instead of 15 minutes. After centrifugation, the supernatant was removed by aspiration, and the cell pellet was gently resuspended in 500 μl of warm D20 medium at 37°C. Cells from both corners were pooled and dispersed onto collagen-coated glass coverslips in 6-well culture dishes and incubated at 37°C and 5% CO2 saturation in a humid atmosphere. Cells were diluted to 50 μg / ml with 0.02 M acetic acid in sterile PBS (Ca-free). ++ and Mg++ Type I collagen from rat tails (Becton Dickinson Biosciences, Heidelberg, Germany) was thinly coated onto glass coverslips (22 mm in diameter, Karl Roth, Karlsruhe, Germany). One coverslip was placed in each well of a six-well plate, and 600 μl of collagen solution was carefully pipetted onto each coverslip to form a convex meniscus and incubated at room temperature (RT) for 1 hour. Excess fluid was then removed by aspiration, and cells were dispersed using a matrix. Uterine epithelial cells were harvested and dispersed and cultured in DMEM (modified Eagle's Medium) containing 2 mmol L-glutamine (Applichem, Darmstadt, Germany) and 0.1 mmol β-mercaptoethanol (Sigma Aldrich, Darmstadt, Germany), supplemented with 20% heat-inactivated fetal bovine serum, 1% non-essential amino acids from MEM, 1% P / S (all PAA, Pasching, Austria), and 1% sodium pyruvate (Sigma Aldrich, Darmstadt, Germany). To disperse the cells, 15 μg / ml of endothelial growth factor (ECGF, ReliaTech, Wolfsburg, Germany) was added. After 2 days, 2 ml of fresh D20 medium (without ECGF) was added to the cells without removing the old medium. This ensured complete cell adhesion and prevented removal by aspiration of floating cells. After 5 days, the old medium was completely removed, and 2 ml of fresh medium was replaced per well every 3 days.

[0044] To identify epithelial cells, cell culture medium was removed from the confluent UECs, and cells were washed with pure PBS. Each well was fixed with 1 ml of ice-cold methanol (MeOH; 80%; Karl Roth, Karlsruhe, Germany) for 10 min. After removing the methanol, 1 ml of blocking solution (2% donkey serum in pure PBS) was added to each well and incubated at room temperature for 15 min. Cells were then washed twice with pure PBS for 5 min each time, followed by immunofluorescence staining using an epithelial cell-specific monoclonal rat antibody (TromaIII-s; rat anti-cytokeratin-19; Developmental Studies Hybridoma Bank, Iowa, USA) as the primary antibody. This antibody is specific for cytokeratin-19 (KRT-19), an intermediate filament protein responsible for the structural integrity of epithelial cells. Primary antibodies were applied to fixed cells at dilutions of 1:100, 1:200, and 1:500 in PBS and Triton (10x; Merck, Darmstadt, Germany) and incubated in a humidified chamber at 5°C for 24 hours. Unbound antibodies were removed by washing cells three times with 1 ml of pure PBS per well. As a secondary antibody, goat anti-mouse IgG (H+L) was applied at a dilution of 1:2000. 555 conjugate (MoBiTec, Gttingen, Germany) was incubated at 37°C for 60 min. Secondary antibodies were removed by washing cells twice with 1 ml of pure PBS per well, and a third wash was performed using 1 ml of bis(benzoyl)imide H33342 trihydrochloride (HOECHST-33342; 0.1 mg / ml in H2O; Sigma Aldrich, Steinheim) and incubation at room temperature for 10 min. Cells were then fixed again with ice-cold MeOH (80%). For examination using a fluorescence microscope (Olympus BX 60, Olympus, Hamburg, Germany) equipped with a high-resolution digital camera (Olympus DP 71, Olympus, Hamburg, Germany), coverslips were removed from the wells and placed on mounting media. The cells were placed upside down on a microscope slide and fixed along the outer edge with clear nail polish. Detection was performed using UV light and a rhodamine filter (555-565 nm) with a bright field. This analysis confirmed that the cultured cells were UECs.

[0045] As described above, fused UECs grown on glass coverslips were used. For comparison, fused porcine aortic endothelial cells (PAECs) and porcine fetal fibroblasts (foet.F) were used. The binding specificity of porcine sperm to the porcine endometrium was confirmed by the reduced binding with the comparison cells. Fibroblasts, used as interspecies rather than surface cell types, were used to demonstrate whether sperm binds to any type of cell or tissue with the same strength as porcine UECs. Porcine aortic endothelial cells represent luminal cells from non-reproductive organs. These cell types were isolated according to Boquest et al., Biol. Reprod 60, 1013-1019 (1999).

[0046] Semen was collected from four verified fertile boars (German Landrace and German White). To ensure consistent semen quality, semen was collected from the boars twice a week (every two to three days apart). The sperm-rich fraction was collected using the hand-collecting method and carefully diffused in warm D20 medium. NC-100 TM (ChemoMetec A / S, (Denmark) Sperm concentration was measured, and samples were examined for motility, membrane integrity, and morphological changes.

[0047] use NC-100 TM (ChemoMetec A / S, Sperm concentration was determined in Denmark using propidium iodide staining. Flow cytometry was performed to measure membrane integrity. Sperm motility was determined using the IVOS-sperm analysis system (Hamilton Thorne Biosciences, Beverly, MA, USA). Ejaculate with ≤70% sperm motility was discarded. The semen was then diffused to a concentration of 100 x 10⁻⁶. 6 The sample was collected at 10 sperm cells / ml and washed twice by centrifugation (10 min, 800 × g, RT) to remove seminal plasma. The supernatant was discarded, and the precipitate was resuspended in D20 medium.

[0048] To identify potential seminal plasma effects, UEC was also incubated with epididymal sperm from four known fertile boars (German White Pigs). For the epididymal sperm, the testes were removed by castration, the seminiferous tubules were excised from the testes, and the epididymal tail was rinsed with warm D20 medium. The epididymal sperm were then diffused to 100 x 10⁻⁶ ppm. 6 / ml. Therefore, the influence of seminal plasma components already attached to the sperm surface on UEC binding can be ruled out. The semen was diluted to 100x10 in D20 medium. 6 / ml, and incubated with one of the following lectins: WGA, sWGA, or ConA, which is obtained by incubating 1 μl of lectin in 200 μl of PBS (Ca-free). ++ and Mg ++ The lectin was incubated together with the diluent in D20 to obtain a concentration of 10 μg / ml. 15 μL of this lectin diluent was added to 100 μL of sperm and incubated at 37°C for 15 min. Unbound lectin was removed by washing (4 min, 800 x g, RT), and the precipitate was resuspended in D20.

[0049] As a control, flow cytometry tubes (Greiner bio-one, Frickenhausen, Germany) were used to label ejaculated porcine sperm with FITC-labeled lectins WGA, sWGA, ConA, or RCA120, and 480 μl of PBS (calcium-free) was used. ++ and Mg ++ Each of the sperm and 3 μl of PI was prepared. After incubation, 20 μl of sperm-lectin solution was added, and the mixture was incubated at room temperature for another 10 minutes. As a control, an aliquot of sperm suspension was treated in the same way, but without lectin. Strong binding was observed in FACS analysis as shown below, where the glycan ligand to which lectin has the major affinity is listed:

[0050]

[0051] These results indicate that these lectins bind strongly to sperm cells, suggesting the presence of N-acetyl-glucosamine, sialic acid, mannose, and glucose, as well as β-D-Gal-D-galactosamine, on ejaculated porcine sperm.

[0052] Semen pre-incubated with a lectin was added to the fused UEC and analyzed by confocal microscopy.

[0053] To facilitate co-incubation with UEC, 500 μl of sperm pre-incubated with lectins was released onto a UEC monolayer, and binding activity was observed under a phase-contrast microscope (Olympus BX60, Olympus, Hamburg, Germany) equipped with a high-resolution digital camera (Olympus DL 70, Olympus, Hamburg, Germany). Binding density was quantified by observing the observed region and compared with control results from untreated sperm culture. Images (twice replicates per boar and per lectin) were divided into 61.6 μm segments. 2 The regions where sperm are present and absent are counted.

[0054] Figure 1Microscopic images of control sperm (untreated, in D20 medium) incubated with UEC are shown, demonstrating the strong binding of sperm cells to UEC. Figure 2A The results of sperm pre-incubated with ConA are shown in the image. Figure 2B The results for sperm after pre-incubation with WGA are shown. For control, sperm were treated in parallel but without lectins. Analysis of sperm binding to the cultured cell layer was performed using a manual observation area method, where images were captured at 200x magnification and graded to 61.6 μm. 2 Squares of varying sizes were used. The areas of sperm covered and uncovered were quantified. Five photographs were taken from each boar and evaluated. Area evaluation was performed by the same person throughout the experiment. Control sperm were measured at 18050.25 ± 5520.06 μm. 2 Combined, WGA-pretreated sperm had a size of 2362.87 ± 248.61 μm. 2 Combined and sWGA-pretreated sperm at 1684.83±107.94 μm 2 The results showed that pretreatment with WGA and sWGA significantly reduced binding to UEC. Sperm pretreated with ConA (which has an affinity for mannose / glucose) showed a smaller binding density of 12718.39 ± 1999.52 μm. 2 The binding was significantly reduced, although the reduction was smaller compared to pre-culture via WGA or sWGA.

[0055] The binding of untreated sperm was repeated using epididymal sperm instead of ejaculated sperm (control). The binding strength was found to be the same.

[0056] To analyze sperm that bound to cells other than UEC, the cell culture medium was removed from the fused monolayer of UEC porcine fetal fibroblasts or porcine aortic endothelial cells, and the cells were grown separately on collagen-coated coverslips. 500 μl of sperm suspension (100 x 10⁻⁶) of ejaculated sperm or tail epididymal sperm was applied to each well. 6 / ml). Incubate in an incubator (37°C, 8% CO2) for up to 60 minutes, preferably 10 minutes, as this time is considered sufficient. Subsequently, carefully remove any remaining sperm by aspiration, and gently wash the monolayer with warm D20 cell culture medium. Mount a coverslip onto a microscope slide with cells and sperm facing upwards, and drop 200 μl of D20 solution onto the coverslip to prevent the cells from drying out. Observe sperm binding under a phase-contrast microscope (Olympus GX60, Olympus, Hamburg, Germany) connected to a high-resolution digital camera (Olympus DP71, Olympus, Hamburg, Germany). The software (version 1.0, Olympus, Hamburg, Germany) records images and videos. Figure 3A The results show the porcine fetal fibroblasts (porc.foet.F). Figure 3B (Sperm heads stained with Hoechst-33342) show the results of porcine aortic endothelial cells (pAEC). Compared with UEC (15923.6 ± 2657.9 μm) 2 In comparison, analysis of binding strength showed that the binding strength between untreated sperm and fibroblasts was significantly lower (3018.4 ± 638.1 μm). 2 (p=0.002), its binding strength with pAEC was significantly lower (2797.8±593.4μm). 2 This indicates that sperm binding to UEC is cell type specific.

[0057] These results indicate that blocking binding sites on sperm by binding agents with affinity for N-acetylglucosamine and / or sialic acid (e.g., lectin WGA) and / or mannose / glucose (e.g., lectin ConA) reduces sperm binding to the endometrium, thereby increasing the number of sperm available for fertilization (e.g., in the fallopian tubes or ampulla).

[0058] Example 2: Enhancing fertility by blocking sperm binding sites on UEC

[0059] Fusion-bound UECs were washed twice with 1 ml PBS (without Ca++ and Mg++) and 45 μl of a lectin suspension (10 μg / ml) of one of four optional lectins (WGA, sWGA, PNA, ConA), and incubated at 37°C in an 8% CO2 incubator for 15 min. Subsequently, the lectin solution was aspirated, and the cells were gently washed with 1 ml PBS (without Ca++ and Mg++), and 500 μl of sperm (100 x 10⁻⁶ cells) were added. 6 (Sperm / ml) was released onto a UEC monolayer and incubated for 10 minutes. Binding activity was observed and density was estimated using a phase contrast microscope (Olympus, BX60, Olympus, Hamburg, Germany) equipped with a high-resolution digital camera (Olympus DL70, Olympus, Hamburg, Germany).

[0060] Figure 4A The image shows micrographs of UECs pre-incubated with sWGA and then incubated with porcine sperm cells diluted in D20 medium. Figure 4B These are micrographs of UEC cells pre-incubated with WGA and then incubated with porcine sperm cells diluted in D20 medium. Compared to untreated control UEC cells (17426.8 ± 4653.58 μm).2 Compared to ), UEC pre-incubated with WGA showed an affinity for Glc-NAc / sialic acid; 5961±309.18 μm 2 The sperm binding density on the surface was significantly lower (p<0.05). Furthermore, treatment with sWGA (which has an affinity for Glc-NAc) and ConA (which has an affinity for mannose / glucose) did not significantly impair sperm binding.

[0061] The results show that blocking sialic acid on the UEC, for example by using a reagent with an affinity for sialic acid, or by removing sialic acid ligands from the UEC, reduces the binding of sperm to the endometrium and thus increases the number of sperm available for fertilization (e.g., in the fallopian tubes or ampulla).

[0062] After pre-incubating UECs with PNA (which has an affinity for β-D-(1-3)-D-galactosamine), some regions showed abundant sperm binding as seen in untreated UECs, while other regions were completely absent, similar to WGA-treated UECs.

[0063] Example 3: Improving fertility by blocking or removing the binding site

[0064] Artificial insemination uses sows as an example of female mammals. Typically, sows are inseminated during peak estrus, and then again 12 hours later, using 50 x 10 [units of currency] each time. 6 100x10 6 500x10 6 Or 1000x10 6 Freshly diluted sperm was used for insemination. For comparison, the control group of sows was inseminated with fresh sperm at a dose of 3 billion sperm in a commercially available standard diluent.

[0065] Each group consisted of 4-6 animals. All compositions were administered via standard artificial insemination to deposit in the distal portion of the uterine body.

[0066] The sperm preparation was administered to the first group of sows at a concentration of 10 μg / ml WGA in a standard dilution.

[0067] The sperm preparation was administered to the second group of sows using a composition containing 1 U / ml sialidase (obtained from Vibrio cholerae) in a standard diluent.

[0068] The third group of sows were administered a composition containing 10 μg / ml WGA in 100 ml of a standard diluent, followed by the administration of the sperm preparation from the standard diluent 2 to 20 minutes later; and

[0069] The fourth group of sows were given a composition containing 1 U / ml sialidase (obtained from Vibrio cholerae) in 100 ml of standard diluent, followed by the administration of sperm preparation in the standard diluent 2 to 20 minutes later.

[0070] Furthermore, sperm from the same boar is typically sorted using FACS according to US 5135759A to separate fractions containing at least 90% of the sperm carrying the X chromosome. Sex chromosome-specific sperm are used without freezing.

[0071] In the fifth group of sows used as a control, only the standard diluent contained sperm reagent.

[0072] In group six sows, the composition contained sperm preparation in the standard diluent at ... μg / ml WGA.

[0073] In the seventh group of sows, a sperm preparation was included in the composition containing 1 U / ml sialidase (available from Vibrio cholerae) in a standard diluent.

[0074] Group 8 sows were administered a composition containing 10 μg / ml WGA in 100 ml of standard diluent, followed by the administration of sperm preparation from the standard diluent 2 to 20 minutes later.

[0075] Group 9 sows were given a composition containing 1 U / ml sialidase (obtainable from Vibrio cholerae) in 100 ml of standard diluent, followed by the administration of sperm preparation in the standard diluent 2 to 20 minutes later.

[0076] Insemination was performed 36 days later using ultrasound diagnostic monitoring.

[0077] In the second to fourth groups, fertilization was significantly increased compared to the first control group.

[0078] In groups six through nine, fertilization was significantly increased compared to the fifth control group, and a strong preference for female offspring was observed.

Claims

1. Use of a composition for enhancing the fertility of a sperm dose for artificial insemination of a pig, characterized in that The composition comprises at least one binding agent having affinity for N-acetylglucosamine and / or having affinity for sialic acid, which binding agent is an unchemically modified lectin, and which lectin is sWGA or ConA, wherein the use is not a disease treatment method.

2. Use according to claim 1, wherein the composition comprises a dose of porcine spermatozoa which is 100th to 5th of the dose used for conventional artificial insemination, which for fresh porcine spermatozoa is 1 x 10 9 to 3 x 10 9 spermatozoa and for frozen porcine spermatozoa is 5 x 10 9 spermatozoa.

3. Use according to claim 1, characterized in that The composition is for administration to the reproductive tract of a gilt or sow prior to or simultaneously with the introduction of sperm into the reproductive tract of the gilt or sow.

4. Use according to claim 1, characterized in that The composition is for application to the uterus of a gilt or sow.

5. Use according to claim 1, characterized in that The composition is for insemination of a gilt or sow.

6. A method of providing spermatozoa of a pig for artificial insemination, characterized in that Prior to or simultaneously with artificial insemination, sperm is contacted with at least one binding agent having affinity for N-acetylglucosamine and / or having affinity for sialic acid, which binding agent is an unchemically modified lectin, and which lectin is sWGA or ConA, wherein the method is not a disease treatment method.

7. The method according to claim 6, characterized in that Prior to artificial insemination, the pig sperm is separated from the at least one binding agent.

8. A method for artificial insemination of gilts or sows with porcine spermatozoa, characterized in that Prior to or simultaneously with insemination, sperm is contacted with at least one binding agent having affinity for N-acetylglucosamine and / or having affinity for sialic acid, which binding agent is an unchemically modified lectin, and which lectin is sWGA or ConA, wherein the method is not a disease treatment method.

9. The method according to claim 8, characterized in that The pig sperm is contacted with the binding agent and introduced into the uterus of a gilt or sow.

10. The method according to claim 8 or 9, characterized in that For pig sperm, for fresh sperm, the maximum dose is 0.4 x 10 9 or a maximum of 0.2 x 10 9 or for frozen sperm, the maximum dose is 1 x 10 9 or a maximum of 0.5 x 10 9 .

11. An artificial insemination sperm dose comprising pig sperm, characterized by comprising at least one binding agent having affinity for N-acetylglucosamine and / or having affinity for sialic acid, which binding agent is an unchemically modified lectin, and which lectin is sWGA or ConA.

12. An insemination sperm dose according to claim 11, characterized in that The pig sperm is sex-chromosome specifically sorted sperm.

13. An insemination sperm dose according to claim 11 or 12, characterised in that The sperm dose contained is 100thto 5thof the dose used for conventional artificial insemination, which is 1 x 10 9 to 3 x 10 9 sperm for fresh pig sperm and 5 x 10 9 sperm for frozen pig sperm, used for conventional artificial insemination.

14. An insemination sperm dose according to claim 13, characterized in that The dose of sperm comprised is from 100th to 10th of the dose used for routine artificial insemination.

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