Process and composition for low dose insemination

Abstract

The present invention relates to a composition for use in low dose artificial insemination and to a process for artificial insemination of a female mammal using a dose of sperm originating from a male mammal of the same species that is significantly lower than necessary in the absence of the composition. The preferred female mammal is a sow (Sus scrofa), and preferably the composition is for use in artificial uterine insemination, and the process for artificial insemination preferably is uterine insemination.

Claims

1. A composition for enhancing the fertility of an artificial insemination porcine sperm dose, comprising at least one binding agent, which is a lectin or antibody, the binding agent is not modified by a polyethylene glycol (PEG) moiety, the binding agent having affinity for N-acetyl glucosamine or affinity for sialic acid.

2. The composition according to claim 1, containing porcine sperm in a dose which is lower by a factor of at least 5 compared to the dose for use in conventional artificial insemination, which dose for use in conventional artificial insemination for fresh porcine sperm is a dose of 1 to 310.sup.9 for fresh sperm, and which dose for use in conventional artificial insemination for frozen porcine sperm is a dose of 510.sup.9.

3. The composition according to claim 1, wherein the composition is formulated for administration to the genital tract of a gilt or sow prior to or concurrent to the introduction of sperm into the genital tract of the gilt or sow.

4. The composition according claim 1, wherein the composition is formulated for application to the uterus of a gilt or sow.

5. The composition according claim 1, wherein the composition is formulated for insemination of a gilt or sow.

6. A method for providing porcine sperm for use in artificial insemination, comprising contacting the sperm with at least one binding agent, which is a lectin or antibody, the binding agent is not modified by a PEG-moiety, the binding agent having affinity for N-acetyl glucosamine or affinity for sialic acid prior to or concurrent to artificial insemination.

7. The method according to claim 6, wherein the porcine sperm is separated from the at least one binding agent and prior to artificial insemination.

8. The method for preparing a gilt or sow for artificial insemination, comprising administering a composition into the genital tract of the gilt or sow prior to or concurrent to introduction of porcine sperm, the composition comprising at least one binding agent having affinity for N-acetyl glucosamine and/or affinity for sialic acid, which binding agent is a lectin or antibody, the binding agent is not modified by a PEG moiety.

9. The method according to claim 6, wherein introduction of sperm is into the uterus, and wherein concurrent to or following administration of the composition, porcine sperm is introduced into the uterus at a dose of at maximum 0.410.sup.9 for fresh sperm, or at a dose of at maximum 110.sup.9 for frozen sperm.

10. A method for artificial insemination of a gilt or sow with porcine sperm, comprising contacting the sperm with at least one binding agent having affinity for N-acetyl glucosamine and/or affinity for sialic acid, which binding agent is a lectin or antibody, the binding agent is not modified by a PEG moiety, prior to or concurrent to insemination.

11. The method according to claim 10, wherein the porcine sperm is in contact with the binding agent and is introduced into the uterus of the female mammal.

12. The method according to claim 10, wherein the sperm is porcine sperm in a dose of at maximum 0.410.sup.9 fresh sperm, or at a dose of at maximum 110.sup.9 for frozen sperm.

13. An artificial insemination sperm dose containing porcine sperm, comprising the composition according to claim 1.

14. The artificial insemination sperm dose according to claim 13, wherein the porcine sperm is sex-chromosome specific sorted porcine sperm.

15. The insemination sperm dose according to claim 13, wherein the porcine sperm is contained in a dose which is lower by a factor of at least 5 compared to the dose for conventional artificial insemination, which dose for conventional artificial insemination for fresh porcine sperm is a dose of 1 to 310.sup.9 for fresh sperm, and which dose for conventional artificial insemination for frozen porcine sperm is a dose of 510.sup.9.

16. The artificial insemination sperm dose according to claim 15, wherein the porcine sperm is contained in a dose which is lower by a factor of at least 10, compared to the dose for use in conventional artificial insemination.

17. The artificial insemination sperm dose according to claim 15, wherein the porcine sperm is contained in a dose which is lower by a factor of at least 100, compared to the dose for use in conventional artificial insemination.

18. The method according to claim 12, wherein the sperm is porcine sperm in a dose of at maximum 0.210.sup.9 fresh sperm, or at a dose of at maximum 0.510.sup.9 for frozen sperm.

19. The method according to claim 9, wherein the sperm is porcine sperm in a dose of at maximum 0.210.sup.9 fresh sperm, or at a dose of at maximum 0.510.sup.9 for frozen sperm.

Description

(1) The invention is now described in greater detail by way of examples with reference to the figures, which show in

(2) FIG. 1 a confocal micrograph of porcine sperm after 10 min co-incubation with a cultivated UEC monolayer,

(3) FIG. 2A a confocal micrograph of porcine sperm after pre-treatment with ConA after 10 min co-incubation with a cultivated UEC monolayer,

(4) FIG. 2B a confocal micrograph of porcine sperm after pre-treatment with WGA after 10 min co-incubation with a cultivated UEC monolayer, and

(5) FIG. 3A a confocal micrograph of untreated porcine sperm after 10 min co-incubation with cultivated porcine foetal fibroblasts (porc. foet. F),

(6) in FIG. 3B a confocal micrograph of untreated procine sperm after 10 min co-incubation with porcine aortal endothelial cells (pAEC),

(7) in FIG. 4A a confocal micrograph of UEC after pre-treatment with sWGA after 10 min co-incubation with porcine sperm, and

(8) FIG. 4B a confocal micrograph of UEC after pre-treatment with WGA after 10 min co-incubation with porcine sperm.

EXAMPLE 1: ENHANCING FERTILITY BY BLOCKING BINDING SITES ON SPERM

(9) According to the first variant, binding sites of sperm participating in the binding to uterine epithelial cells (UEC) were identified and blocked, then sperm was co-incubated with cultivated UEC, showing reduced binding of sperm to UEC.

(10) Confluent UEC were grown on glass cover slips form primary cell cultures. In total, uteri from 78 primiparous German Landrace or German Edelschwein gilts aged 8-10 months and with live weights of over 110 kg, were retrieved to harvest primary cells. All animals were maintained and handled according to the German regulations for animal welfare. Gilts were monitored for natural oestrus and slaughtered according to standing heat, i.e. at the time when artificial insemination would have been performed. Gilts were stunned electrically and subsequently slaughtered by exsanguination. Three minutes after bleeding the abdomen was opened and the uterus removed in toto. Further, the ovaries, oviducts and the mesometrium were removed by cutting with sterile scissors without damaging the myometrium. The uterine horns were ligated with stitching thread between and a section of 20-25 cm was cut off. The sections were placed in sterile phosphate buffered saline (PBS) without Ca.sup.++ and Mg.sup.++ (Karl Roth, Karlsruhe, Germany) containing 2% Penicillin/Streptomycin (P/S; PAA, Pasching, Austria) in a glass bottle and kept at 5 C. for 45 min. After 45 min at 5 C. the uterine sections were removed from the bottle and placed on cellulose tissue under a sterile laminar flow system. The stitching material was removed. Each horn was fixed with sterile artery clamps ensuring open ends and the lumen was then rinsed three times with 10 ml sterile PBS containing 2% P/S using a 10 ml sterile serological pipette. One end was then shut by a clamp and 10 ml ethylene diamine tetraacetic acid and Trypsin (EDTA/Try; 10% (PAA, Pasching, Austria) in PBS without Ca.sup.++/Mg.sup.++) were inserted via a 10 ml sterile serological pipette into the horn and the remaining end equally closed with a clamp. Subtle movement of the horn ensured equal distribution throughout the lumen. Incubation took place in fresh 20 ml plain PBS containing 2% P/S at 37 C. for 15 min. After enzymatic digestion 10 ml of PBS were added, the horn moved subtly and the liquid caught in a 50 ml centrifuge tube containing 5 ml of warm cell culture medium (D20, 77% DMEM, 20% FBS, 1% Na-pyruvate, 1% amino acids, 1% P/S). The cell suspension was centrifuged for 4 min at 209 g and RT. This procedure was performed three times per horn with a difference in digestion time of ten instead of 15 min for the second and third repeat. After centrifugation the supernatant was removed by aspiration and the cell pellet was gently resuspended in 500 l of 37 C. warm D20 medium. Cells from both horns were pooled and disseminated onto the glass coverslips coated with collagen in a 6-well culture dish and cultured in an incubator at 5% CO.sub.2 saturation at 37 C. with humidified atmosphere. Glass cover slips (22 mm diameter, Karl Roth, Karlsruhe, Germany) were thinly coated with rat tail collagen type-I (Becton Dickinson Biosciences, Heidelberg, Germany) diluted to 50 g/ml in 0.02 M acetic acid in sterile PBS (without Ca.sup.++ and Mg.sup.++). One cover slip was placed in each well of the six-well dish and 600 l collagen solution were carefully pipetted onto each coverslip to form a convex meniscus and incubated at room temperature (RT) for one hour. Remaining liquid was then removed by aspiration and the matrices were used for dissemination of cells. Uterine epithelial cells were harvested, disseminated and cultured in cell culture medium (D20) containing modified whole Dulbecco's modified Eagle's medium DMEM (containing 2 mmol L-Glutamine (Applichem, Darmstadt, Germany) and 0.1 mmol -mercapto ethanol (Sigma Aldrich, Darmstadt, Germany) supplemented with 20% heat-inactivated fetal bovine serum, 1% Modified Eagle's Medium (MEM) non-essential amino acids, 1% P/S (all PAA, Pasching, Austria) and 1% sodium pyruvate (Sigma Aldrich, Darmstadt, Germany). For dissemination of the cells 15 g/ml endothelial cell growth factor (ECGF, ReliaTech, Wolfsburg, Germany) were added. After two days 2 ml fresh D20 medium (containing no ECGF) were added to the cells without removing the old media. This ensured complete adhesion of cells and no removal by aspiration of floating cells. After five days the old media was removed completely and replaced by 2 ml per well of fresh medium every three days.

(11) For identification of epithelial cells, cell culture medium was removed from confluent UEC and the cells were washed with plain PBS and fixed with 1 ml iced methanol (MeOH; 80%; Karl Roth, Karlsruhe, Germany) per well for 10 min. Methanol was removed and 1 ml blocking solution (2% donkey serum in plain PBS) per well was added and incubated at room temperature for 15 min. The cells were washed twice subsequently for 5 min with plain PBS., immune-fluorescence antibody staining was made using an epithelial cell-specific monoclonal rat antibody (Troma III-s; rat anti-cytokeratin-19; Developmental Studies Hybridoma Bank, Iowa, USA) as a primary antibody, specific for cytokeratin-19 (KRT-19), which is an intermediate filament protein responsible for the structural integrity of epithelial cells. The primary antibody was applied at dilutions of 1:100, 1:200 and 1:500 in PBS and Triton (10; Merck, Darmstadt, Germany) to the fixed cells and incubated for 24 h in a moist chamber at 5 C. Unbound antibody was removed by washing the cells with 1 ml plain PBS per well three times. As a secondary antibody, goat anti-Mouse IgG (H+L), AlexaFluor 555 conjugate, MoBiTec, Gttingen, Germany) was applied at 1:2000 dilution and incubated for 60 min at 37 C. The secondary antibody was removed by washing the cells twice with 1 ml of plain PBS per well and for the third rinse 1 ml bisbenzimide H 33342 trichydrochloride (HOECHST-33342; 0.1 mg/ml in H.sub.2O; Sigma Aldrich, Steinheim) was applied and incubated for 10 min at room temperature. Subsequently, the cells were fixed one more time with iced MeOH (80%). For detection with a fluorescent 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 were placed on microscopic slides upside down onto mounting media (VectaShield, Vector Laboratories, California, USA) and fixed with clear nail varnish along the outer edge. For detection UV light and a rhodamine filter (555-565 nm) as well as bright field were used. This analysis confirmed that cultivated cells were UEC.

(12) Confluent UEC grown on glass cover slips, as described above, were used. For comparison, confluent porcine aortal endothelial cells (PAEC) as well as porcine foetal fibroblasts (foet. F) were used. The binding specificity of porcine spermatozoa to the porcine endometrium was confirmed by the reduced binding to the comparative cells. The fibroblasts where used as an inter-species, but non-surface cell type, to prove whether sperm bind to any kind of cell or tissue in the same intensity as to porcine UEC. Porcine aortal endothelia represent a lumen cell from a non-reproductive organ. These cell types are isolated as described by Boquest et al., Biol. Reprod 60, 1013-1019 (1999).

(13) Sperm was collected from four verifiably fertile boars (German Landrace and German Edelschwein). To ensure constant semen quality, the service boars were collected for semen regularly twice a week with two to three days interval. The sperm-rich fraction was collected by the gloved-hand method and carefully extended with same parts with warm D20 medium. Sperm concentration was measured using a NukleoCounter NC-100 (ChemoMetec A/S, Allerod, Denmark), and the sample was examined for motility, membrane integrity and morphological changes.

(14) The sperm concentration was determined using a NukleoCounter NC-100 (ChemoMetec A/S, Allerd, Denmark) and membrane integrity was measured flow-cytometrically using a FACScan using propidium iodide staining. Motility was determined using an IVOS-sperm-analysis system (Hamilton Thorne Biosciences, Beverly, Mass., USA). Ejaculates with 70% motile spermatozoa were dismissed. Semen was then extended to a concentration of 10010.sup.6 sperm cells/ml and washed twice by centrifugation (10 min, 800 g, RT) to remove the seminal plasma. The supernatant was discarded and the pellet was resuspended in D20 medium.

(15) To identify possible seminal plasma effects, UEC were also incubated with epididymal sperm of four (German Edelschwein) known fertile boars. For epididymal sperm, the testes were removed by castration and the seminiferous tubules were dissected from the testes and the caudal epididymes were flushed with warm D20 medium and epididymal sperm were extended to 10010.sup.6/ml, respectively. It could therefore be excluded that seminal plasma components, already attached to the sperm surface, have influence on binding to UEC. Semen was diluted to 10010.sup.6/ml in D20 medium and incubated with one of the following lectins WGA, sWGA, or ConA by incubation with a dilution of 1 l of the lectin in 200 l PBS (without Ca.sup.++ and Mg.sup.++) to gain a concentration of 10 g/ml. Fifteen microliters of this lectin dilution were added to 100 l of sperm and incubated for 15 min at 37 C. in an incubator. Unbound lectin was removed by washing (4 min, 800 g, RT) and resuspending the pellet in D20.

(16) As a control for binding, ejaculated porcine sperm was labelled with FITC-labelled lectins WGA, sWGA, ConA or RCA120, using flow-cytometer tubes (Greiner bio-one, Frickenhausen, Germany) prepared with 480 l PBS (without Ca.sup.++ and Mg.sup.++) and 3 l PI each. After completed incubation, 20 l sperm-lectin solution were added and incubated for further ten minutes at RT. As a control one aliquot of the sperm suspension was treated identically without a lectin. Strong binding was observed in FACS analysis as given below, wherein the glycan ligand is listed, for which the lectin has predominant affinity:

(17) TABLE-US-00001 fluorescence intensity lectin glycan ligand (mean standard deviation) WGA N-acetyl-glucosamine 917.27 332.74 sialic acid sWGA N-acetyl-glucosamine 553.46 153.99 ConA mannose/glucose 260.25 122.15 RCA120 -D-Gal-D-galactosamine 151.56 71.18

(18) These results show that these lectins have strong binding to the sperm cells, indicating the presence of N-acetyl-glucosamine, sialic acid, mannose and glucose, and of -D-Gal-D-galactosamine on ejaculated porcine sperm.

(19) The semen that was pre-incubated with one of the lectins was added to confluent UEC and analysed by confocal microscopy.

(20) For the co-incubation with UEC, 500 l of lectin pre-incubated sperm were released onto a UEC monolayer and the binding activity observed under a phase contrast microscope (Olympus BX 60, Olympus, Hamburg, Germany) equipped with a high resolution digital camera (Olympus DL 70, Olympus, Hamburg, Germany). The binding density was quantified by area under view and compared to results from the control incubation with untreated sperm. Images (2 repeats/boar and lectin) were divided into fields of 61.6 m.sup.2 and the fields with and without sperm were counted.

(21) FIG. 1 shows the microscopic picture of control sperm (not treated, in D20 medium) incubated with UEC, showing strong binding of the sperm cells to UEC. Results are shown in FIG. 2A for sperm after pre-incubation with ConA, in FIG. 2B after pre-incubation with WGA. For control, sperm was treated in parallel but without lectin. Analysis of sperm binding to the cultivated cell layer was by a manual area-under-view method, wherein images were taken at 200 magnification and graded into squares of 61.6 m.sup.2 size. The area covered with and without sperm was quantified. Five images per boar were taken and evaluated. The area evaluation was performed by the same person throughout all experiments. Control sperm bound at 18050.255520.06 m.sup.2, WGA-pre-treated sperm bound at 2362.87248.61 m.sup.2 and sWGA-pre-treated sperm bound at 1684.83107.94 m.sup.2, showing a significant reduction in binding to UEC by the pre-treatment with WGA and sWGA. Sperm pre-treated with ConA (affine for mannose/glucose) bound at 12718.391999.52 m.sup.2, showing a significant reduction in binding, although reduced to a smaller extent than by pre-incubation with WGA or sWGA.

(22) The binding of untreated sperm was repeated using epididymal sperm instead of ejaculated sperm (control). The binding intensity was found to be equivalent.

(23) For analysis of sperm binding to other cells than UEC, cell culture medium was removed from the confluent monolayers of UEC porcine foetal fibroblasts or porcine aortal endothelial cells, each growing on collagen coated coverslips, and 500 l sperm suspension (10010.sup.6/ml) of either ejaculated or caudal epididymal sperm were applied to each well. Co-incubation took place for up to 60 min in an incubator (37 C., 8% CO.sub.2), preferably at ten minutes of incubation as this period was identified to be sufficient. Subsequently, remaining sperm were removed carefully by aspiration and the monolayer was washed gently with warm D20 cell culture medium. The coverslip was mounted onto a microscopic slide with the cells and sperm facing upwards and a 200 l droplet of D20 was pipetted onto the cover slip to protect the cells from drying out. Sperm binding was viewed under a phase contrast microscope (Olympus GX 60, Olympus, Hamburg, Germany) connected to a high resolution digital camera (Olympus DP71, Olympus, Hamburg, Germany). The image and video documentation was performed with the CellP software (Version 1.0, Olympus, Hamburg, Germany). The result is shown in FIG. 3A for porcine foetal fibroblasts (porc. foet. F), in FIG. 3B (sperm heads stained with Hoechst-33342) for porcine aortal endothelial cells (pAEC). Analysis of binding intensity showed a significantly (p=0.002) lower binding intensity of untreated sperm to fibroblasts (3018.4638.1 m.sup.2) compared to UEC (15923.62657.9 m.sup.2), and a significantly lower binding intensity to pAEC (2797.8593.4 m.sup.2). This shows that the binding of sperm to UEC is cell-type specific.

(24) These results show that blocking the binding sites on the sperm by a binding agent having affinity for N-acetyl glucosamine and/or affinity for sialic acid as exemplified by the lectin WGA and/or for mannose/glucose as exemplified by the lectin ConA reduces the binding of sperm to the endometrium, and hence increases the number of sperm available for fertilisation, e.g. at the oviduct or ampulla.

EXAMPLE 2: ENHANCING FERTILITY BY BLOCKING BINDING SITES FOR SPERM ON UEC

(25) Confluent UEC were washed twice with 1 ml PBS (without Ca++ and Mg++) and 45 l lectin suspension (10 g/ml) of one of the four selected lectins (WGA, sWGA, PNA, ConA) and incubated for 15 min at 37 C. at 8% CO.sub.2 in an incubator. Subsequently, the lectin solution was aspirated and cells washed gently with 1 ml PBS (without Ca++ and Mg++) and 500 l of sperm (10010.sup.6 sperm/ml) were released onto the UEC monolayer and incubated for 10 min. Binding activity was observed under a phase contrast microscope (Olympus, BX 60, Olympus, Hamburg, Germany) equipped with a high resolution digital camera (Olympus DL 70, Olympus, Hamburg, Germany) and the density was estimated.

(26) FIG. 4A shows a micrograph of UEC pre-incubated with sWGA after incubation with porcine sperm cells diluted in D20 medium, FIG. 4B a micrograph of UEC pre-incubated with WGA after incubation with porcine sperm cells diluted in D20 medium. Sperm binding density was significantly (p<0.05) lower on UEC pre-incubated with WGA (affinity for Glc-NAc/sialic acid; 5961309.18 m.sup.2) compared to untreated control UEC cells (17426.81.44653.58 m.sup.2). Furthermore, treatment with sWGA (having affinity for Glc-NAc) and ConA (having affinity for mannose/glucose) did not significantly impair sperm binding.

(27) This result shows that blocking the sialic acid on UEC, e.g. by an agent having affinity for sialic acid, or removal of the sialic acid ligand from UEC reduces the binding of sperm to the endometrium, and hence increases the number of sperm available for fertilisation, e.g. at the oviduct or ampulla.

(28) After pre-incubation of UEC with PNA (affinity for -D-(1-3)-D-galactosamine) some areas showed massive sperm binding as seen with untreated UEC, whereas others were not populated at all, similar to WGA-treated UEC.

EXAMPLE 3: ENHANCING FERTILITY BY BLOCKING BINDING SITES

(29) For artificial fertilisation sows were uses as an example of a female mammal Generally, sows were inseminated at standing heat, and again 12 h later, each time using either 5010.sup.6, 10010.sup.6, 50010.sup.6 or 100010.sup.6 freshly diluted sperm for the insemination-. For comparison, a comparison group of sows were inseminated with fresh sperm at a dose of 3 billion sperm in commercially available standard diluent.

(30) Each group comprised 4-6 animals. Administration of all compositions was by standard artificial insemination for deposition at the distal part of the uterine body.

(31) A first group of sows was administered the sperm dose in a composition of 10 g/ml WGA in the standard diluent,

(32) a second group of sows was administered a composition of 100 ml standard diluent containing 10 g/ml WGA, followed after 2 to 20 min by administration of the sperm dose in standard diluent.

(33) Further, sperm from the same boar was sorted into a fraction containing at least 90% X-chromosome bearing sperm using FACS generally according to U.S. Pat. No. 5,135,759 A. The sex-chromosome specific sperm was used without freezing

(34) in a third group of sows for control containing the sperm dose in standard diluent only,

(35) in a fourth group of sows containing the sperm dose in a composition of WGA in the standard diluent,

(36) a fifth group of sows in a composition of 100 ml standard diluent containing 10 g/ml WGA, followed after 2 to 20 min by administration of the sperm dose in standard diluent.

(37) After 36 d, fertilisation was monitored by ultrasound diagnosis.

(38) In the second groups, fertilisation was significantly increased in comparison to the first control group.

(39) In the fourth to fifth groups, fertilisation was significantly increased in comparison to the third control group, also showing a strong bias for female offspring.