WO2011143741A1

WO2011143741A1 - Extenders for mammalian sperm processing and preservation

Info

Publication number: WO2011143741A1
Application number: PCT/CA2011/000559
Authority: WO
Inventors: Puttaswamy Manjunath; Annick Bergeron
Original assignee: Hopital Maisonneuve-Rosemont
Priority date: 2010-05-15
Filing date: 2011-05-13
Publication date: 2011-11-24

Abstract

The present invention relates to a composition and a method of use or the use of the composition for preserving sperm mobility, viability, membrane integrity or fertility, or all of these, during washing, diluting, cooling, freezing, preserving or cryopreserving of sperm, for in vitro fertilization, for artificial insemination or for sexing semen. The composition comprises DEAE-dextran in a sperm-friendly physiological buffer.

Description

EXTENDERS FOR MAMMALIAN SPERM PROCESSING AND

PRESERVATION

TECHNICAL FIELD

[0001] The present invention relates to composition for use as extenders of mammalian sperm or semen.

Background of the invention

[0002] The maintenance of sperm outside the body of man and animals is customarily carried out in artificial insemination procedures. There are a variety of materials and procedures that are used in the prior art to prolong the survival of sperm outside the body and/or to enable a single sperm-containing ejaculate to be diluted so as to be useful to impregnate a large number of females. Many of these materials and procedures are limited as to the extent they can prolong survival. Materials more complexes than the ordinary balance solutions are commonly referred to as "extenders".

[0003] There is a particular problem with prolonging the survival of mammalian sperm. In many cases, the survival of washed mammalian sperm is limited to only 2 to 4 hours at physiological conditions. Attempts to freeze sperm by conventional techniques have been relatively unsuccessful. Often recovery of less than 20% of motility of the sperm is obtained. Glycerol, which is often considered essential for successful freezing of bovine sperm, can be highly toxic to other mammalian sperm.

[0004] The admixture of either washed or unwashed sperm with any of a variety of commercially available extenders usually results in two dramatic effects. After only a few hours the sperm show progressively decreasing motility. In addition, they agglutinate or clump to one another or to the particulate material normally present in seminal fluid and which is difficult to remove from the spermatozoa by filtration or differential centrifugation.

[0005] Often extenders, such as egg yolk, used for extending mammalian sperm are only effective to allow the seminal plasma of males to be diluted. Thus an ejaculate from a donor can be extended by some commercial extenders to allow several females to be inseminated. However, the life of the sperm is often not extended by commercially available extenders. Thus often in many species, commercial extenders do not extend the life span of the sperm but only permit survival of diluted sperm equivalent to those of undiluted sperm.

[0006] The cryopreservation of bull semen represents one of the most important achievements in dairy farming after the introduction of artificial insemination. These two approaches have the potential to enhance distribution and preservation of desired genetic lines besides major savings. Egg yolk and skimmed milk are also used as protective agents to freeze mammalian sperm.

[0007] It is difficult to find replacement component for egg yolk or milk because the mechanisms involved in sperm protection by egg yolk and milk against cooling, freezing and storage damages remains unclear.

[0008] It is difficult to retrace all studies that have been done in the area of bull sperm cryopreservation, which has been a starting point for designing cryopreservation protocols for most mammalian species. The cooling/freezing protocols have been continuously revised along with the major components and additives for the cryoprotective media, but the basis of method remains unchanged. To date, the glycerol and egg yolk or milk represent the indispensable compounds of practically all used media for bull sperm cryoprotection.

[0009] The role of glycerol is defined in terms of contribution to sperm integrity conservation; however, the protection afforded by egg yolk is more complex. The egg yolk is known to increase the sperm fertilizing ability when present in extenders for semen storage at ambient temperature and appears to prevent sperm cell damage at cooling and freezing. Various components of egg yolk have been investigated in an attempt to isolate the most active component(s) responsible for the protective effect. The low-density lipoprotein, LDL (previously called low density fraction, LDF) shows a certain level of protective ability but the mechanism by which this protection is provided to sperm has not been determined.

[0010] Seminal plasma, which facilitates the transport of sperm in the female genital tract, also contains factors influencing sperm motility and fertility. In addition, seminal plasma also appears to be detrimental for sperm storage.

[0011] Egg yolk and milk currently used are products of animal origin, and as such represent a potential risk of contamination of semen. Moreover, their composition is not uniform.

[0012] Thus, there is an urgent need of developing novel media, free of products from animal origin, to conserve semen.

BRIEF SUMMARY OF THE INVENTION

[0013] In a first aspect, there is provided a new composition free of product of animal origin to conserve semen.

[0014] In a second aspect, there is now provided a method of preserving or maintaining animal sperm fertility comprising diluting sperm in a solution comprising DEAE-dextran, said DEAE-dextran binding seminal plasma proteins, thereby preserving or maintaining sperm fertility.

[0015] In a third aspect, there is provided a composition for use in preserving or maintaining sperm viability, fertility or both, during washing, diluting, cooling, freezing, preserving or cryopreserving of sperm, for artificial insemination or for in vitro fertilization, said composition comprising DEAE-dextran in a physiologically acceptable buffer.

[0016] In a fourth aspect, there is further provided for the use of a composition as defined herein for preserving sperm viability, fertility or both, during washing, diluting, cooling, freezing, preserving or cryopreserving of sperm, for artificial insemination or for in vitro fertilization. [0017] Another use of the present invention is as a diluent for preservation or cryopreservation of mammalian semen and/or sperm including but not limited to human, mouse and primate semen and/or sperm.

[0018] For the purpose of the present invention the following terms are defined below.

[0019] The term "sperm-friendly physiological" solution or buffer is intended to mean a solution or buffer that will not kill, lyse or otherwise destroy sperm cells.

[0020] The expression "improving or preserving sperm motility" is meant to refer to the properties of a solution or a buffer that will maintain sperm motility near to that at the time of ejaculation, thereby preserving same by opposition to a sperm sample alone, which will loose such motility if not diluted in such solution or buffer.

[0021] The term "motility" is meant to refer to the fraction of sperm that display any type of movement (any direction), by opposition to "progressive motility" which is meant to refer to sperm that swims forward in an essentially straight line.

[0022] Every percentages (%) referred to herein in relation to DEAE-dextran, unless specifically otherwise stated, refer to weight to volume percentages.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figs 1A to 1 D illustrate the effect of various doses of DD-10 extender on bovine sperm motility and progressive motility during 2 hours cooling (A and B) and after freeze thawing (C and D);

[0024] Figs. 2A to 2D illustrate the effect of various doses of DD-20 extender on bovine sperm motility and progressive motility during 2 hours cooling (A and B) and after freeze thawing (C and D); [0025] Figs. 3A to 3D illustrate the effect of various doses of DD-40 extender on bovine sperm motility and progressive motility during 2 hours cooling (A and B) and after freeze thawing (C and D);

[0026] Figs. 4A to 4D illustrate the effect of various doses of DD-70 extender on bovine sperm motility and progressive motility during 2 hours cooling (A and B) and after freeze thawing (C and D);

[0027] Figs. 5A to 5D illustrate the effect of various doses of DD-70 extender on bovine sperm acrosomal integrity and sperm viability during 2 hours cooling (A and B) and after freeze thawing (C and D);

[0028] Figs. 6A to 6D illustrate the effect of various doses of DD-150 extender on bovine sperm motility and progressive motility during 2 hours cooling (A and B) and after freeze thawing (C and D);

[0029] Figs. 7A to 7D illustrate the effect of EY addition to DD-70 extender on bovine sperm motility and progressive motility during 2 hours cooling (A and B) and after freeze thawing (C and D); and

[0030] Figs. 8A to 8D illustrate the effect of EY addition to DD-70 extender on bovine sperm acrosomal integrity and sperm viability during 2 hours cooling (A and B) and after freeze thawing (C and D).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0031] In the course of the studies that led to the present invention, the detrimental effect of seminal plasma on sperm preservation was further investigated. It was found that although binder of sp_erm (BSP) proteins are essential for fertility, they also appear to be detrimental to sperm in the context of sperm storage. Long contact of sperm with seminal plasma, which contains BSP protein, is detrimental to sperm. Therefore in the context of the invention, efforts were made to identify agents that would prevent the binding of BSP to sperm. BSPs and homolog or ortholog proteins are found to be present in seminal plasma of stallion, boar, ram, goat, water buffalo, and bison. Interestingly, extender containing egg yolk is used to preserve sperm from these species as well as from other mammals. Thus it appears that the mechanism of sperm protection by egg yolk could be the same in all these species.

[0032] Furthermore, in the studies that led the inventors to the present invention, it was found that preventing BSP from binding to sperm is the mechanism by which semen could be extended. It was now been found that BSP binds diethylaminoethyl (DEAE)-dextran, making it thus a candidate as sperm extender.

[0033] Accordingly, the invention also relates to a composition for preserving sperm, comprising diethylaminoethyl (DEAE)-Dextran in a sperm-friendly physiological solution, which is useful to inhibit and/or remove seminal plasma proteins deleterious to sperm cells. The preserving solution of the invention is suitable to preserve sperm properties, such as viability and/or motility, during washing, diluting, cooling, freezing, preserving or cryopreserving sperm samples which are suitable for use in clinical and diagnostic research, sorting sexed semen and artificial insemination application. The solution contains DEAE-Dextran for maintaining sperm viability, and/or motility during different processing. Since DEAE-Dextran specifically binds to BSP family proteins, this interaction is useful to isolate BSP or BSP-like proteins from seminal fluids of various mammalian species.

[0034] In one embodiment, the dextran used is of molecular weight between 5 and 1000 kilodaltons (kDa), preferably between 10 and 300 kDa, and most preferably of 70 kDa, so as to have a viscosity suitable for the use, i.e. not too viscous so as to prevent motility, but sufficiently so as to preserve the integrity of the sperm cells. In another embodiment, the degree of coupling of DEAE described herein corresponds to approximately 1-6 DEAE-substituents per 3 glucose units.

[0035] DEAE-Dextran can be used at various concentrations in the context of the present invention. The concentration of DEAE-Dextran useful for such composition to be used as basic diluter for semen storage in ambient or cold temperatures preferably vary from 0.01-50%, 0.1-25%, or 0.1-10% in a sperm friendly acceptable buffers such as in Tris buffers, citrate buffers, having a pH of 6-7. In fact any buffers currently used with extenders commercially available would be acceptable, as these are "sperm friendly".. The same diluter to be used for storing semen in liquid nitrogen is preferably supplemented with 0.5- 10% glycerol.

[0036] According to one embodiment of the invention, there is provided a method for preserving sperm using a composition comprising DEAE-Dextran. The source of DEAE-dextran can be without limitation of natural source, synthetic, synthetic analogs, chemically or biochemically modified or portions thereof.

[0037] In one embodiment, DEAE-dextran is used in a composition for improving at least one sperm property selected from the group consisting viability, motility, membrane integrity, sperm penetration rate, and sperm fertilization rate.

[0038] In another embodiment, there is provided a composition comprising DEAE-dextran for use in diluting semen, preferably diluting between 5-400 times, preferably 5-50 times, and more preferably 8-20 times the normally occurring concentration of sperm in seminal fluid.

[0039] In accordance with one embodiment of the invention, there is provided a composition comprising DEAE-dextran for use in the conservation of the sperm at temperature between 0°C and 40°C, preferably either at 4°C, 18°C, 37°C, or at room temperature, or still at a temperature when frozen in liquid nitrogen or in liquid nitrogen vapors.

[0040] In accordance with the present invention, there is also provided diluents or extenders to conserve in liquid or frozen state semen/sperm from farm animals such as bulls, rams, goats, stallions, buffalos, bison, boars, turkeys etc., domestic animals such as dogs and cats, endangered animals and zoo animals. Such diluents or extenders comprise DEAE-dextran diluted in a sperm-friendly physiological solution. [0041] Another use of the present invention is as a diluent for preservation or cryopreservation of mammalian semen and/or sperm including but not limited to human, mouse and primate semen and/or sperm.

[0042] The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I DEAE-dextran used for sperm storage

[0043] In accordance with the present invention, DEAE-Dextran (Bio Basic Inc., Markham, Ontario, Canada Mol. Wt 500 000) was used in a composition for human and bovin semen storage. As such, DEAE-Dextran was thus tested at various concentrations (Table 1 ) to demonstrate the effect of such compound on sperm after storage or preservation.

[0044] Table 1 shows the effect of DEAE-Dextran at various doses on bovine sperm motility, during 2 hours cooling at 4°C and after a freeze-thaw cycle. For this analysis, four tubes were prepared, one to be used as control, containing only Tris-glycerol (TG: 200 mM Tris, 5.6% glycerol, pH 6.7) (2.84 ml), a second tube containing 0.1 % DEAE-Dextran in TG (150 μΙ DEAE-dextran 2% + 2.69 ml TG), a third tube containing 0.3% DEAE-Dextran in TG (450 μΙ DEAE-dextran 2% + 2.39 ml TG), and a fourth tube containing 1 % DEAE-Dextran in TG (1500 μΙ DEAE 2% + 1.34 ml TG). Then, 160 μΙ of semen (37°C) was added to each of the above tubes preincubated at 37°C which were then incubated at 4°C. Aliquots were withdrawn at specific intervals (Oh, 1 h, and 2h) for motility (a drop) analysis. At the end of 2h, semen was packed in 250 μΙ straws and sealed. The sealed straws were then frozen in temperature programmed automatic semen freezer under liquid nitrogen vapors. The frozen straws were stored under liquid nitrogen overnight. Frozen straws were then thawed at 37°C for 1 minute and used for sperm motility tests. For motility assessment, a drop of semen and a drop of citrate solution were mixed on a warm slide. Motility was then monitored under the light microscope. At 1 % DEAE-dextran concentration, the motility was superior (50-60%) compared to control (40-50%) during the 13-hour cooling (Table 1).

Table 1

Effect of DEAE-Dextran on Bovine Sperm Motility During 13 hours Cooling

EXAMPLE II

Effect of various doses of DEAE-Dextran solution as extender on bovine sperm motility and progressive motility during 2 hours cooling and after freeze thawing

[0045] In addition to the percentage of DEAE-dextran used, it is understood that depending on the molecular weight of the dextran used, the solution of DEAE-dextran will be more or less viscous, which will have an impact on the capacity of the DEAE-dextran to be used as extender or diluter. Accordingly, various molecular weight of dextran are now being tested. The molecular weight of the various DEAE-dextran reported herein refers to the molecular weight of dextran before modification with DEAE. The DEAE-dextrans used in Examples 2-9 (Figs 1-8) were synthesized by Polydex Pharmaceutical Limited (Toronto, Canada).

[0046] In accordance with the present invention, the effect of various doses of DEAE-Dextran 10 kDa (DD-10) as extender on bovine sperm motility and progressive motility during 2 hours cooling and after freeze thawing was tested. Briefly, 580 μΙ of bull semen (37°C) immediately after collection was added to each tubes containing various doses of DD-10 (0.1 %, 0.25% and 0.5%) in TG- buffer (34°C) or 20% egg yolk (EY) as positive control extender (34°C). The diluted semen in different extenders (20 ml, 48 X 10⁶ sperm/ml) was then cooled down at 4°C in a refrigerator. Aliquots were withdrawn at designated intervals (0 h, 1 h, and 2 h) for the analysis of motility and progressive motility (2.5 μΙ). At the end of 2 h, diluted semen was packed in 0.25 ml straws, frozen according to the industry standard under nitrogen vapors and stored in liquid nitrogen. A week later, semen was thawed and incubated at 34°C, analyzed for sperm motility and progressive motility at 0 h and after 2 h. Sperm motility and progressive motility were monitored by a Computer Assisted Sperm Analyzer (CASA; Hamilton-Thorn). In this example or the subsequent ones, no negative control was used, as it was determined that using only Tris buffer, without DEAE-dextran, after a freeze-thawing cycle would not allow significant sperm survival.

[0047] As can be seen from Figs. 1A and 1 B, the sperm motility and the progressive motility in extenders with 0.1 %, 0.25% and 0.5% of DD-10 were similar to EY-control at time 0 h and decreased slightly compared to EY-control during the 2 hours cooling. After freeze thawing, as can be seen from Figs. 1 C and 1 D, the sperm motility and the sperm progressive motility were slightly lower than EY-control for all three doses of DD-10 tested.

EXAMPLE III

Effect of various doses of DD-20 extender on bovine sperm motility and progressive motility during 2 hours cooling and after freeze thawing

[0048] As in the preceding example, 649 μΙ of bull semen (37°C) immediately after collection was added to each tubes containing various doses of DEAE- Dextran 20 kDa (DD-20) (0.1 %, 0.25% and 0.5%) in TG-buffer (34°C) or 20% egg yolk (EY) as control extender (34°C). The diluted semen in different extenders (20 ml, 48 X 10⁶ sperm/ml) was then cooled down at 4°C in a refrigerator. Aliquots were withdrawn at designated intervals (0 h, 1 h, and 2 h) for the analysis of motility and progressive motility (2.5 μΙ). At the end of 2 h, diluted semen was packed in 0.25 ml straws, frozen according to the industry standard under nitrogen vapors and stored in liquid nitrogen. A week later, semen was thawed and incubated at 34°C, analyzed for sperm motility and progressive motility at 0 h and after 2 h. Sperm motility and progressive motility were monitored by a Computer Assisted Sperm Analyzer (CASA; Hamilton- Thorn). [0049] As can be seen in Figs. 2A and 2B, the sperm motility and the sperm progressive motility in extenders with 0.1 %, 0.25% and 0.5% of DD-20 were slightly higher or similar to EY-control during the 2 hours cooling. After freeze thawing, as can be seen in Figs. 2C and 2D, the sperm motility in the presence of DD-20 decreased slightly compared to EY-control and the sperm progressive motility was similar to EY-control for all concentrations of DD-20 used.

EXAMPLE IV

Effect of various doses of DD-40 extender on bovine sperm motility and progressive motility during 2 hours cooling and after freeze thawing

[0050] As in the preceding examples, 583 μΙ of bull semen (37°C) immediately after collection was added to each tubes containing various doses of DD-40 (0.1 %, 0.25% and 0.5%) in TG-buffer (34°C) or 20% EY as control extender (34°C). The diluted semen in different extenders (20 ml, 48 X 10⁶ sperm/ml) was then cooled down at 4°C in a refrigerator. Aliquots were withdrawn at designated intervals (0 h, 1 h, and 2 h) for the analysis of motility and progressive motility (2.5 μΙ). At the end of 2 h, diluted semen was packed in 0.25 ml straws, frozen according to the industry standard under nitrogen vapors and stored in liquid nitrogen. A week later, semen was thawed and incubated at 34°C, analyzed for sperm motility and progressive motility at 0 h and after 2 h. Sperm motility and progressive motility were monitored by a Computer Assisted Sperm Analyzer (CASA; Hamilton-Thorn).

[0051] As can be seen in Figs. 3A and 3B, the sperm motility in extenders with 0.1 %, 0.25% and 0.5% of DD-40 was slightly higher than EY-control during the 2 hours cooling. The sperm progressive motility was higher than control at time 0 h but was similar to EY-control after 1 and 2 h cooling. After freeze thawing (Figs. 3C and 3D), the sperm motility and the sperm progressive motility were slightly lower or similar to EY-control for all three doses of DD-40 tested. EXAMPLE V

Effect of various doses of DD-70 extender on bovine sperm motility and progressive motility during 2 hours cooling and after freeze thawing

[0052] As in the preceding examples, 625 μΙ of bull semen (37°C) immediately after collection was added to each tubes containing various doses of DD-70 (0.1 %, 0.25% and 0.5%) in TG-buffer (34°C) or 20% EY as control extender (34°C). The diluted semen in different extenders (20 ml, 48 X 10⁶ sperm/ml) was then cooled down at 4°C in a refrigerator. Aliquots were withdrawn at designated intervals (0 h, 1 h, and 2 h) for the analysis of motility and progressive motility (2.5 μΙ). At the end of 2 h, diluted semen was packed in 0.25 ml straws, frozen according to the industry standard under nitrogen vapors and stored in liquid nitrogen. A week later, semen was thawed and incubated at 34°C, analyzed for sperm motility and progressive motility at 0 h and after 2 h. Sperm motility and progressive motility were monitored by a Computer Assisted Sperm Analyzer (CASA; Hamilton-Thorn).

[0053] As can be seen in Figs. 4A and 4B, the sperm motility and the progressive motility in extenders with 0.1 %, 0.25% and 0.5% of DD-70 were slightly higher or similar to EY-control during the 2 hours cooling. After freeze thawing (Figs. 4C and 4D), the sperm motility and the sperm progressive motility were slightly lower than EY-control at time 0 h but was similar or slightly higher than EY-control after 2 h for all three doses of DD-70 tested.

EXAMPLE VI

Effect of various doses of DD-70 extender on bovine sperm acrosomal integrity and sperm viability during 2 hours cooling and after freeze thawing

[0054] As in the preceding examples, 625 μΙ of bull semen (37°C) immediately after collection was added to each tubes containing various doses of DD-70 (0.1 %, 0.25% and 0.5%) in TG-buffer (34°C) or 20% EY as control extender (34°C). The diluted semen in different extenders (20 ml, 48 X 10⁶ sperm/ml) was then cooled down at 4°C in a refrigerator. Aliquots were withdrawn at designated intervals (0 h, 1 h, and 2 h) for the determination of the sperm acrosomal integrity and viability (10 μΙ). At the end of 2 h, diluted semen was packed in 0.25 ml straws, frozen according to the industry standard under nitrogen vapors and stored in liquid nitrogen. A week later, semen was thawed and incubated at 34°C, analyzed for sperm acrosomal integrity and sperm viability at Oh and after 2h. For the viability assessment, 10 μΙ of washed sperm sample and 10 μΙ Eosine-Nigrosin dye were mixed and staining was observed under microscope. For the acrosomal integrity assessment, 10 μΙ of washed sperm samples were smeared on slides, dried and stained with naphtol yellow- erythrosin B.

[0055] As can be seen in Figs. 5A and 5B, the sperm acrosomal integrity in extenders with 0.1 %, 0.25% and 0.5% of DD-70 was similar to EY-control during the 2 hours cooling. The sperm viability was slightly lower than EY- control after 2 h for all three doses of DD-70 tested. After freeze thawing (Figs. 5C and 5D), the sperm acrosomal integrity was similar and the sperm viability was slightly lower than EY-control after 2 h for all three doses of DD-70 tested.

EXAMPLE VII

Effect of various doses of DD-150 extender on bovine sperm motility and progressive motility during 2 hours cooling and after freeze thawing

[0056] As in the preceding examples, 588 μΙ of bull semen (37°C) immediately after collection was added to each tubes containing various doses of DD-150 (0.4%, 0.7% and 1 %) in TG-buffer (34°C) or 20% EY as control extender (34°C). The diluted semen in different extenders (20 ml, 48 X 10⁶ sperm/ml) was then cooled down at 4°C in a refrigerator. Aliquots were withdrawn at designated intervals (0 h, 1 h, and 2 h) for the analysis of motility and progressive motility (2.5 μΙ). At the end of 2 h, diluted semen was packed in 0.25 ml straws, frozen according to the industry standard under nitrogen vapors and stored in liquid nitrogen. A week later, semen was thawed and incubated at 34°C, analyzed for sperm motility and progressive motility at 0 h and after 2 h. Sperm motility and progressive motility were monitored by a Computer Assisted Sperm Analyzer (CASA; Hamilton-Thorn).

[0057] As can be seen in Figs. 6A and 6B, the sperm motility in extenders with 0.4%, 0.7% and 1% of DD-150 was similar to EY-control during the 2 hours cooling. The sperm progressive motility was higher or similar to EY-control at the beginning of the incubation at 4°C but decreased slightly compared to EY- control after 2 h incubation when 0.7% and 1 % of DD-150 were used. After freeze thawing (Figs. 6C and 6D), the sperm motility and the sperm progressive motility decreased compared to EY-control for all three doses of DD-150 tested.

EXAMPLE VIII

Effect of EY addition to DD-70 extender on bovine sperm motility and progressive motility during 2 hours cooling and after freeze thawing

[0058] To complement the above examples, the inventors now investigated whether there was synergy, or additive effects of EY combined with DD-70 on sperm motility and progressive motility. As in the preceding examples, 625 μΙ of bull semen (37°C) immediately after collection was added to each tubes containing 0.25% DD-70, with or without 1 % (1/20) or 2% (1/10) (v/v) EY in TG- buffer (34°C), or 20% EY (v/v) as control extender (34°C). Diluted semen in different extenders (20 ml, 48 X 10⁶ sperm/ml) was then cooled down at 4°C in a refrigerator. Aliquots were withdrawn at designated intervals (0 h, 1 h, and 2 h) for the analysis of motility and progressive motility (2.5 μΙ). At the end of 2 h, diluted semen was packed in 0.25 ml straws, frozen according to the industry standard under nitrogen vapors and stored in liquid nitrogen. A week later, semen was thawed and incubated at 34°C, analyzed for sperm motility and progressive motility at 0 h and after 2 h. Sperm motility and progressive motility were monitored by a Computer Assisted Sperm Analyzer (CASA; Hamilton- Thorn).

[0059] As can be seen in Figs. 7A and 7B, the sperm motility in extenders with 0.25% of DD-70 and with or without EY (final concentration 1 % or 2%) was similar to 20% EY-control during the 2 hours cooling. The sperm progressive motility values with EY addition were slightly higher than 0.25% DD-70 alone and slightly higher than the control (20% EY). After freeze thawing (Figs. 7C and 7D), the sperm motility values with EY addition were higher than DD-70 alone and the 20% EY-control after 2 h incubation. The sperm progressive motility with EY addition were slightly higher than 0.25% DD-70 alone and similar to the control (20% EY). EXAMPLE IX

Effect of EY addition to DD-70 extender on bovine sperm acrosomal integrity and sperm viability during 2 hours cooling and after freeze thawing

[0060] As in the preceding example, 625 μΙ of bull semen (37°C) immediately after collection was added to each tube containing 0.25% DD-70, with or without 1% (1/20) or 2% (1/10) EY in TG-buffer (34°C) or 20% EY as control extender (34°C). The diluted semen in different extenders (20 ml, 48 X 10⁶ sperm/ml) was then cooled down at 4°C in a refrigerator. Aliquots were withdrawn at designated intervals (0 h, 1 h, and 2 h) for the determination of the sperm acrosomal integrity and viability (10 μΙ). At the end of 2 h, diluted semen was packed in 0.25 ml straws, frozen according to the industry standard under nitrogen vapors and stored in liquid nitrogen. A week later, semen was thawed and incubated at 34°C, analyzed for sperm acrosomal integrity and sperm viability at 0 h and after 2 h. For viability assessment, 10 μΙ of washed sperm sample and 10 μΙ Eosine-Nigrosin dye were mixed and staining was observed under microscope. For acrosomal integrity assessment, 10 μΙ of washed sperm samples were smeared on slides, dried and stained with naphtol yellow- erythrosin B.

[0061] As can be seen in Figs. 8A and 8B, the sperm acrosomal integrity with 0.25% of DD-70 extender with or without EY (final 1 and 2%) was similar to 20% EY-control during the 2 hours cooling. The sperm viability with EY addition was similar to 0.25% DD-70 alone and slightly lower than the control (20% EY). After freeze thawing (Figs. 8C and 8D), the sperm acrosomal integrity with EY addition was slightly higher than 0.25% DD-70 alone or 20% EY-control and the sperm viability with EY addition was higher than DD-70 alone or 20% EY-control after 2 h.

It is now clear in light of the two preceding examples that EY can be added to DEAE-dextran for slightly improved performance in extending sperm viability. In such a case, smaller quantities of EY were used, such that possible unwanted effects of EY could be minimized. However, with an aim at avoiding using animal material such as EY, it is now clear in light of the data presented herein that DEAE-dextran can now be used as a substitute for EY as extender for sperm.

[0062] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of preserving or improving animal sperm motility ex vivo comprising diluting sperm in a solution comprising DEAE-dextran, said DEAE-dextran binding seminal plasma proteins, thereby preserving or improving sperm motility.

2. The method of claim 1 , wherein the DEAE-dextran has a molecular weight from 5 to 1000 kDa.

3. The method of claim 2, wherein the DEAE-dextran has a molecular weight from 10 to 300 kDa.

4. The method of any one of claims 1 to 3, wherein the solution comprises from 0.01 % to 50% weight to volume of the DEAE-dextran.

5. The method of claim 4, wherein the solution comprises from 0.01 % to 25%, preferably from 0.1 % to 10% weight to volume of the DEAE- dextran.

6. The method of any one of claims 1 to 5, wherein the DEAE-dextran consists of a dextran moiety coupled to DEAE, said DEAE-dextran having a degree of coupling of said DEAE of about 1-6 DEAE molecules per 3 glucose units.

7. A composition for use in preserving or maintaining sperm viability, fertility or both, during washing, diluting, cooling, freezing, preserving or cryopreserving of sperm, for artificial insemination or for in vitro fertilization, said composition comprising DEAE-dextran in a sperm- friendly physiological buffer.

8. The composition of claim 7, wherein the DEAE-dextran has a molecular weight from 5 to 1000 kDa.

9. The composition of claim 8, wherein the DEAE-dextran has a molecular weight from 10 to 300 kDa.

10. The composition of any one of claims 7 to 9, wherein said composition comprises from 0.01% to 50% weight to volume of the DEAE-dextran.

11. The composition of claim 10, wherein said composition comprises from 0.01% to 25%, preferably from 0.1% to 10% weight to volume of the DEAE-dextran.

12. The composition of any one of claims 7 to 11 , wherein the DEAE- dextran has a molecular weight of 70 kDa.

13. Use of a composition as defined in any one of claims 7-12 for preserving sperm viability, fertility or both, during washing, diluting, cooling, freezing, preserving or cryopreserving of sperm.

14. The use of claim 13, wherein the sperm is from bulls, rams, goats, stallions, buffalos, bison, boars, turkeys, dogs, cats, endangered animals or zoo animals.

15. The use of claim 13, wherein the sperm is human sperm, mouse sperm or primate sperm.