DESCRIPTION

INVENTION FIELD The present invention is in the field of the extenders for the artificial insemination aimed to maintain and/ or extend the vitality of productive livestock and pets' sperm cells.

KNOWN ART

The artificial insemination is the most common technique for the productive livestock breeding. This technique has several advantages on the direct mating because it allows inseminate multiple females with the semen of a single male, therefore guaranteeing a careful genetic selection of the desired features and a better repeatability. In the last years, the success and resulting increase of artificial insemination, in terms of both insemination quantity and efficiency, is attributable firstly to the constant improvement of the collected semen preservation techniques, and particularly to the semen extension and cryopreservation.

The use of frozen semen has several advantages, because it guarantees the shelf life extension of the extended semen and the possibility of transport the semen itself even on long distances, facilitating the spreading of assisted insemination (D. Waberski et al. Theriogenology 70 (2008) 1346-1351 ; M. Yoshida, Animal reproduction science 60-61 (2000) 349-355). For productive livestock, the preferred semen preservation choice is based on freezing thereof since collection until use; however, it is proved that for most mammals, semen cryopreservation causes a significant fertility reduction: such an unwanted effect is particularly pronounced for boar semen. The considerable vitality decline of boar semen after thawing is attributed to multiple concurrent factors among which the low cholesterol/ phospholipids ratio of boar spermatozoa membrane and the production of reactive oxygen species: this causes a reduced cooling resistance, membrane instability and an earlier spermatozoa capacitation resulting in a severe reduction of inseminating potential as a whole (W.M. Maxwell and L.A. Johnson, Theriogenology 48 (1997) 209-219; L.A. Johnson et al., Animal Reproduction Science 62 (2000) 143- 172; S. Cerolini et al., Anim. Reprod. Sci. 58 (2000) 99-1 1 1). Moreover, Fraser and Strzezek in 2007 demonstrated that cryopreservation causes a chromatin structure destabilization in boar sperm (L. Fraser, J. Strzezek, Theriogenology 68 (2007) 248-257). For these reasons, freezing is not the preferred technique for boar sperm preservation; the preservation of fresh extended semen is instead preferred, at a temperature of about 16 °C, in menstrual fluids able to preserve its vitality, in the short- or long-term.

In a similar way, in rabbit farming, cooled sperm preservation is preferred to the semen freezing, though it is still possible. As a matter of facts, for this species too, the spermatozoa freezing process causes a reduction in the percentage of alive and moving spermatozoa and of cells with intact acrosomes and early capacitation (E. Moce, J.S. Vicente, Animal Reproduction Science 1 10 (2009) 1-24); this encourages to prefer the semen preserved at 15- 18 °C for up to four days after collection. In the canine species, the cooled semen is preferred over the frozen one. In fact, the cooled semen guarantees a higher successful insemination percentage (about 80% of pregnancies compared to 70% obtained using frozen semen). The use of cooled semen is simpler and more common among professionals in the sector, especially from the semen preparation perspective. Therefore, liquid preservation at "cooling" temperatures guarantees better sperm cells survival compared to cryopreservation in certain species and, at the same time, it offers an adequate period to allow inseminating a high number of females and transporting the collected semen to different locations.

Several extenders are known on the market, whose common purpose is to increase the sample volume, as well as to extend as much as possible the spermatozoa vitality. Their effectiveness is based on the capacity to supply the energy sources needed to sustain the metabolic activity of sperm cells, and the protective substances able to control pH and osmotic pressure of the solution and to inhibit the microbial growth. This allows sufficient time useful for distance transport, to perform qualitative tests on the semen before use and makes the artificial insemination more flexible. The extended sperm vitality reflects in a higher insemination success, as spermatozoa will survive longer also in the female genital system, therefore covering a longer timespan during ovulation; this aspect is particularly relevant for species having multiple ovulation. For example, the sow estrus lasts 50-60 hours, but the maturation of ovarian follicles starts at about 30-35 hours after the start of the same; the broodmare estrus instead generally lasts 3-8 days; in the bitch the insemination must be carried out within 48 hours from ovulation, which does not happen at the same time for all the ovarian follicles: for this reason the possibility to have a wider period of sperm vitality translates into a higher rate of insemination success.

The semen vitality extension firstly depends on the availability of a proper energy source. Sperm cells metabolism are metabolically able to exploit various energy sources with different efficiencies, mainly simple sugars such as fructose, glucosemannose, sorbitol, ribose, galactose as well as non-monosaccharide energy sources such as lactate, pyruvate, citrate, glycerol, glycerol-3-phosphate and short-chain fatty acids (A.R. Jones and L.A. Chantrill, Reproduction, Fertility and Development 1 ( 1989) 357- 367;A.R. Jones and WA, Journal of reproduction and fertility 1 19 (2000) 129- 135). Most products currently on the market or covered by patent, intended to the short- or long-term semen preservation, include glucose, cf. e.g. the patent applications KR 100807643, CN102599147A, CN101803595A, CN101543208A, US2009325144A1 , US2012197068A1 , JP2000247801A, CN101731 1999A and WO02060252A1 ; less frequently they include fructose (patent applications US2008199846A1 , CN101263806A) or lactose (patent application CN101595866A) as energy source; cf. also the publications Gaeda, Spanish Journal of Agricultural Research, 2003, 1 (2), 17-27 or Ponglowhapan et al., Theriogenology, 62 (2004) 1498- 1517 or the patent applications ES-A 1-2106686, WO9927884, all disclosing semen extenders containing glucose and/or fructose.

Until today, there is no indication that spermatozoa may use complex sugars, nor that these cells have membrane transporters suitable to internalize such molecules or the hydrolytic enzymes needed to obtain energy therefrom. For this reason, disaccharides such as sucrose or trehalose are present in extenders formulation for semen freezing mainly as non-permeating cryoprotective agents, (Cf. e.g. Yidiz et al., Theriogenology, 200, p.579-585; C. Malo, Cryobiology 61 (2010) 17-21 ; J. Gomez-Fernandez, Animal Reproduction Science 133 (2012) 109-1 16). The patent applications CN 102986647 and CN 101554152 report extenders compositions for boar semen freezing including sucrose as cryoprotective agent. In fact, Akcay et al. (E Akcay, Fertilizing ability of turkey semen diluted with simple sugar-based extenders after cooled storage, XII European Poultry Conference 2006), reports that the use of sucrose as energy source in turkey semen extender causes a quick decline in semen vitality, making it unusable for insemination within the first 24 hours from collection.

The formulation of both frozen and non-frozen boar semen extenders further include proteins of various origins, some with enzymatic activity, having different purposes. Among the proteins known to play a role in semen preservation, there are: egg yolk or skimmed milk derived proteins, normally used as cryoprotective agents; bovine serum albumin (BSA) used to increase or maintain cell vitality and to stabilize pH. The patent application WO20081 17026A1 concerns the preparation of semen extenders including protein Hsc70. Formulations with added enzymes are instead quite uncommon: in the patent application CN101554152A a semen freezing extender is reported to which an enzyme having antioxidant activity was added; the patent application GB1352709A reports the addition of β-glucuronidase to preparations for the bovine semen preservation, with the purpose of improving spermatozoa motility and inseminating capacity. The hydrolysis of sucrose by invertase is per se known: for example, in J.Am.Chem.Soc, 50(8), 1928, Nelson et al. have prepared acqueous solutions of sucrose and invertase, and measured the rate of hydrolysis of sucrose.

In spite of the wide use of the invertase enzyme at industrial level, no disclosure exists wherein the saccharolytic enzymes, and particularly the invertase enzyme, are somehow associated to the preparation of formulations for animal semen extenders.

SUMMARY

The present inventors have devised an improved composition for extenders useful for the fresh and long-term preservation of vital semen of productive livestock or pets. The composition is characterized by the presence of polysaccharides and/or oligosaccharides, particularly D-(+)- saccharose, and an enzyme able to hydrolyze said polysaccharides and/ or oligosaccharides into energy-giving substrates usable by the spermatozoa. A preferred embodiment uses D-(+)-saccharose as oligosaccharide and invertase as enzyme; said enzyme, in aqueous environment, hydrolyzes the D-(+)-saccharose originating D-(+)-glucopyranose and D-(-)-fructose in an equimolar ratio. The extender according to the invention can include also other additives, for example salts and molecules suitable to give a proper osmotic concentration and pH, one or more antibiotics, etc. The composition is particularly effective in long-term preservation of non- frozen semen, maintaining its progressive motility above 70% for at least 5 days. DETAILED DESCRIPTION OF THE INVENTION

In the present application, with "extender" it is meant an aqueous solution that allows increasing the semen volume, preserving the functional characteristics of the sperm cells, maintaining a proper fertility level and performing artificial insemination in multiple females using the same ejaculate.

With "short-term preservation", it is meant the sperm preservation at a cooling temperature between 4 and 18°C, up to three days (72 hours) since collection.

With "long-term preservation", it is meant the sperm preservation at a cooling temperature between 4 and 18°C, for a period equal to or greater than five days since collection.

With the term "effective preservation" it is meant that the seminal material maintain the inseminating capacity, being closely correlated to the progressive motility and membrane integrity of the spermatozoa. With "artificial insemination", it is meant the set of techniques suitable to artificially insert the male semen into the female genital system. With the terms "sperm", "seminal material" or "semen" it is meant the ensemble of spermatozoa and fluid substances produced by animal male genital organs.

With "extension of sperm vitality" it is meant the capacity to maintain the spermatozoa vitality as close as possible to the one evaluated upon ejaculation for the longest possible period and in any case for a period longer than the standard life of control spermatozoa.

For "controlled temperature", when related to the semen preservation conditions, it is meant a temperature generally between 4 and 18 °C; such temperature is selectable case by case according to criteria known in the art, depending on the semen type; for example canine or equine semen can be preserved at 4-8°C, boar semen can be preserved at 14- 18°C, etc.

A first object of the present invention is a composition for extenders, suitable for long-term temperature-controlled preservation of non-human semen meant for artificial insemination.

The extender composition object of the present invention includes: (a) a polysaccharide and/or an oligosaccharide and (b) a hydrolytic enzyme.

A further object of the invention is the extender itself, prepared by adding water to the above-mentioned composition, until reaching a preset target volume.

With "target volume", it is meant the liquid quantity (water or aqueous solution) in which the semen is preserved: such volume depends on the quantity and type of the semen to be preserved and is generally between 2 mL and 15 mL per mL of semen.

Consequently, in the present text the expression "quantity of substance x suitable to obtain a concentration y" means the quantity of substance x able to obtain the concentration y in the target volume.

In the present invention, the polysaccharide and/ or oligosaccharide works as energy source precursor for the semen. Examples of polysaccharides and/ or oligosaccharides that can be used are sucrose, dextran, glycogen, pullulan, starch, amylopectin, amylose, fructan, levan, inulin, dextrin, cyclodextrins, maltodextrins, maltose, isomaltose, maltulose, isomaltulose, cellobiose, panose, isopanose, raffinose, trehalose, maltotriose, isomaltotriose, isomaltopentose; preferably non-reducing poly/ oligosaccharides are used, particularly the oligosaccharide D-(+)- saccharose (or sucrose); the latter is used in a quantity to obtain a concentration generally between 10 and 60 g/L, preferably between 20 and 50 g/L and more preferably between 30 and 40 g/L. The use of non- reducing sugars, particularly sucrose, gives an additional advantage to the formulation quality: in fact these molecules, being exempt from free aldehyde or ketone groups, cannot act as reducing groups in respect of other components of the formulation; this prevents browning or other physical alterations of the powders, guaranteeing an extended formulation stability.

In the present invention, the hydrolytic enzyme, in presence of water and at a proper working temperature, hydrolyzes the polysaccharide and/ or oligosaccharide in the solution, gradually making available the corresponding monosaccharides as semen nutrition sources. Examples of enzymes that can be used are: EC 3.2.1.2 β-amylase, EC 3.2. 1.7 inulinase, EC 3.2.1.10 oligo- l ,6-glucosidase, EC 3.2.1.1 1 dextranase, EC 3.2.1.26 β-fructofuranosidase, EC 3.2.1.33 amylo-a- l ,6-glucosidase, EC 3.2.1.41 pullulanase, EC 3.2.1.48 sucrose-a-glucosidase, EC 3.2.1.54 cyclomaltodextrinase, EC 3.2.1.57 isopullulanase, EC 3.2.1.68 isoamylase, EC 3.2. 1.91 cellulose 1 ,4-b-cellobiosidase, EC 3.2.1.94 glucan 1 ,6-a-isomaltosidase, EC 3.2.1.95 dextran 1 ,6 a-isomaltotriosidase, EC 3.2.1.1 15 exo- l ,2-a-glucosidase, EC 3.2.1.1 16 glucan 1 ,4-a- maltotriohydrolase, EC 3.2.1.133 glucan 1 ,4-a-maltohydrolase, EC 3.2.1.135 neopullulanase, EC 3.2.1.142 limit dextrinase, EC 3.2.1.150 oligoxyloglucan reducing-end-specific cellobiohydrolase, EC 3.2.1.176 cellulose l ,4- -cellobiosidase. The preferred enzyme is invertase (other names: saccharase, β-fructofuranosidase, β-D-fructofuranoside fructo hydrolase, EC 3.2.1.26): it is used in a quantity suitable to obtain a concentration between 0.001 and 45000 enzymatic units per liter (U/L), preferably between 1000 and 30000 U/L, more preferably between 1500 and 7000 U/L, where the enzymatic unit (U) is the enzyme quantity which catalyzes the transformation of 1 micromole of substrate per minute at standard conditions of pH, temperature and concentration. In the invertase case, 1 enzymatic unit corresponds to the enzyme quantity, which hydrolyze 1 micromole of sucrose to inverted sugar per minute at pH 4.5 at 55 °C. The invertase enzymatic activity can be also expressed as Sumner Unit (SU), defined as the enzyme quantity needed to form 1 mg of inverted sugar in 5 minutes, starting from 6 ml of a 5.4% sucrose solution at pH 4.5 at 20°C. For the scope of this patent application, it is assumed that 1 U = 3.125 SU. The composition of the invention can additionally include various optional components, such as salts, one or more antibiotics and one or more buffers in order to obtain a solution having a pH preferably between 6.8 and 7.5 and having an osmolarity preferably between 160 and 400 mOsm/L. The composition can further include chlorides of alkaline metals, particularly potassium chloride in a quantity to obtain a concentration for example between 0.4 and 1 g/L, preferably between 0.7 and 0.9 g/L.

The composition can further include carbonic acid salts, particularly carbonic acid sodium salts and more particularly sodium bicarbonate in a quantity to obtain a concentration for example between 1 and 2 g/L, preferably between 1.3 and 1.4 g/L.

The composition can further include ethylenediaminetetraacetic acid salts and particularly sodium salts and more particularly ethylenediaminetetraacetic acid disodium salt dihydrate in a quantity to obtain a concentration for example between 1 and 2 g/L, preferably between 1.3 and 1.4 g/L.

The composition can further include organic acid salts, particularly citric acid sodium salts and further citric acid trisodium salt dihydrate, in a quantity to obtain a concentration between 4 and 10 g/L, preferably between 5 and 7 g/L.

The composition can further include pharmacologically active substances, for example an antimicrobial agent and, particularly, an antibiotic or salt therof. By way of example, the antibiotic can belong to the aminoglycoside class and can be (6 mg/L) amikacin (amikacin sulfate), apramycin, dibekacin, gentamicin (gentamicin sulfate), kanamycin, (neomycin), spectinomycin, streptomycin (500-1000 μg/ml), aminosidine, (tobramycin); the antibiotic can belong to the macrolide class and can be tylosin, amphotericin B, (erythromycin, oleandomycin, spiramycin, avermectins, clarithromycin, azithromycin, flurithromycin); the antibiotic can belong to the cephalosporin class and can be cefoxitin, cefapirin, cefazolin, cefoperazone, ceftiofur, ceftriaxone, cefquinome, (cefadroxil, cefalexin, cefradine, cefaclor, cefamandole, cefuroxime, cefonicid, loracarbef, cefmetazole, cefotetan cefalotin, cefuroxime cefprozil, loracarbef, cefotaxime, ceftriaxone, cefixime, ceftizoxime, cefpodoxime, ceftazidime, cefdinir, cefepime, cefpirome); the antibiotic can belong to the quinolone class and can include nalidixic acid, pipemidic acid, flumeqine; the antibiotic can belong to the fluoroquinolone class and can be danofloxacin, enrofloxacin, marbofloxacin, (ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, norfloxacin, oloxacin, trovafloxacin); the antibiotic can belong to the nitrofuran class and can be (nitrofurantoin, nitrofurazone, furazolidone, nifuroxime, nifuratel, fuiylfuramide); the antibiotic can belong to the penicillin class and can be amoxicillin, ampicillin, penicillin, ticarcillin, piperacillin (bacampicillin, oxacillin, cloxacicillin, dicloxacicillin, nafcillin, flucloxacillin, carbenicillin, mezlocillin, piperacillin, azlocillin, apalcillin). The antibiotic can belong to the tetracycline class and can be tetracycline, doxycycline, minocycline, and oxy tetracycline. The antibiotic can belong to the glycopeptide class and can be colistin, (vancomycin, teicoplanin, bacitracin, bleomycin; the antibiotic can belong to the lincosamide class and can be lincomycin, (clindamycin); the antibiotic can belong to the monobactam class and can be aztreonam; the antibiotic can belong to the rifamycine class and can be rifaximin. The antimicrobial can belong to the sulfonamide class and can be sulfamethoxazole, sulfadimethoxine, sulfamethazine, succinylsulfathiazole, (acetazolamide, diclofenamide, ethoxzolamide, methazolamide, sulfanilamide, sulfamerazine) . For example, the antimicrobial can belong to the Diaminopyrimidines class and can be, for example, trimethoprim. Preferably, the antibiotic is amikacin, gentamicin and/ or corresponding salts thereof, or marbofloxacin; more preferably, the antibiotic is gentamicin sulfate, in a quantity to obtain a concentration between 0.5 and 2 g/L and preferably between 1.1 and 1.3 g/L.

The extender composition object of the invention is usually provided and stored as dry material, to be diluted in a proper target volume of water in order to obtain the solution ready to use. Alternatively, it is possible to store the composition pre-diluted in the target volume (or in a portion thereof), at a temperature lower than the enzyme activation one, usually less than 2°C: the hydrolysis enzymatic reaction is thus inhibited until the composition is brought to the enzyme working temperature (typically between 4°C and 18°C). Typically in the present invention, the enzyme activation takes place extemporaneously, namely at the time of use, just before the semen addition; particularly, once the target volume of water has been added and, where applicable, the solution has been brought to the enzyme working temperature, the extender is added with the animal semen, previously prewashed. The extenders object of the invention are suitable for the preservation of semen from any animal species, typically from productive livestock or pets, for example, pig, horse, sheep, goat, rabbit, turkey, dog and cat, etc. The semen to be preserved is preferably fresh semen, namely collected from the animal within a short time, preferably within 15 minutes and however not over 25 minutes, without undergoing pretreatments such as cryopreservation that can reduce its vitality. The present extenders are particularly useful in the conservation of the semen, in particular fresh semen, wherein said conservation does not involve freezing.

In the composition object of the present invention, the presence of polysaccharides and/ or oligosaccharides as energy source precursors, together with invertase, allows the long-term survival of the sperm; particularly, in aqueous solution, and at temperatures between 4 and 18 °C, over time the enzyme gradually makes the monosaccharide energy- giving substrates available, such as D-(+)-glucopyranose and D-(-)-fructose derived from the hydrolysis of D-(+)-saccharose; this guarantees a high vitality of the sperm cells; moreover, the gradual release causes an improvement over the conventional compositions, wherein the sperm cells are initially in contact with an excess of monosaccharide, followed by a progressive consumption until deficit of the same: such monosaccharide quantity variations are potentially detrimental to the life duration of the above-mentioned cells and therefore to the semen quality.

A further object of the invention is the above defined composition for extenders, packaged in association with written instructions directing to its use as an extender for animal semen. For example, said written instructions may be contained within the package (e.g. in the form of a leaflet), be part of the package itself (e.g. printed thereupon), be attached to the package, etc.

A further object of the invention is a kit comprising the above defined composition for extenders in the dry state, the kit including written instructions directing to its use as extender for animal semen, and further including an aliquot of water or water solution to be mixed with said dry composition at the moment of use.

A further object of the invention is the above defined composition "in use", i.e. containing the animal semen extended therein. In summary, the composition object of this patent application is useful to realize effective extenders for non-human animal semen able to preserve it in the long-term without resorting to freezing. The utility of said composition is not confined to a surprising retention of overall sperm motility over time, but also involves a higher semen effectiveness in artificial insemination practice, causing a higher offspring number per parturition compared to reference the extender.

By way of non-limitative example, some preparation examples are reported in the following.

EXPERIMENTAL PART Experiment design

Formulations were developed for boar and rabbit semen extension; the formulations included D-(+) saccharose, invertase and gentamicin. In both cases, said formulations were used as semen extenders and furthermore, control comparative tests were carried out using a reference extender. In a first series of experiments, in order to evaluate the quality of semen under evaluation, namely the vitality of spermatozoa contained therein, the sperm progressive motility was studied: in all cases, a retention of the sperm progressive motility higher than 70% was ensured for at least four days, reaching even 10 days from the collection time, namely six more days compared to the control formulation. Moreover, it was observed that the preparation object of this patent application allows preserving the sperm under evaluation for at least two more days compared to the formulation used as reference. In addition, several tests were carried out for the same male sperm donor, using extender preparations having the same antibiotic and sucrose concentrations, but different invertase concentration: the sperm progressive motility resulted non directly proportional to the enzyme concentration increase.

In a second series of experiments, it was chosen to evaluate the effectiveness of the composition object of this patent application by calculating the pregnancies percentage, as well as the average number of young rabbits per parturition after carrying out some artificial insemination attempts on rabbits. By comparing a known commercial extender to the one under evaluation, it was possible to evaluate that the percentage of pregnancies compared to the number of artificially impregnated breeding does was higher for the present extender; the same applied to the average number of young rabbits per parturition. Example 1

It is described the preparation of a boar sperm extender composition, dissolved one hour before use. For that purpose, 0.172 g of potassium chloride, 0.2752 g of gentamicin sulfate, 0.2924 g of sodium bicarbonate, 0.2924 g of disodium EDTA, 1.31 15 g of sodium citrate dihydrate and 8.600 g of sucrose were weighed. The powders were mixed in an orbital mixer of type Turbula (Bachofen AG) for 1 hour at 32 rpm. The obtained mixture was dissolved in purified water until reaching a 215 mL volume (solution 1). Therefore, the gentamicin concentration in solution 1 was equal to 1.28 g/L, while the sucrose one was equal to 40 g/L. Aside, 15 mg of invertase enzyme (14504, >300.000 U/g, Sigma-Aldrich) were weighed and dissolved in 15 mL of solution 1 to give solution 2. Therefore, the invertase enzyme concentration in solution 2 was greater than 300.000 U/L. The remaining solution 1 was divided in different shares added with different invertase volumes. Particularly, 8 formulations were prepared, being so composed: Formulation Volume of Volume of Invertase number solution 1 solution 2 concentration

1 9.9 mL 0. 1 mL

> 3.000 U/L

2 9.82 mL 0. 18 mL

>5.400 U/L

3 9.75 mL 0.25 mL

>7.500 U/L

4 9.65 mL 0.35 mL

> 10.500 U/L

5 9.5 mL 0.5 mL

> 15.000 U/L

6 9.4 mL 0.6 mL

> 18.000 U/L

7 9.25 mL 0.75 mL

>22.50() U/L

8 9. 1 mL 0.9 mL

> 27.000 U/L

Table 1; example 1, preparation of the va riuos formula tions used as extenders

Two replicas of the solutions were prepared 1 hour before use. In the collection site (a boar center with certified breeding subjects), the two selected boars belonging to hybrid genetic lines (boar A and boar B) were carefully cleaned; the sperm was collected by trained staff using the gloved hand technique, using single-use non-spermiotoxic nit rile gloves and a dummy. The semen was collected in an open plastic container being preheated at 38 °C and having a 500 mL capacity, over which a single-use filtering gauze was placed in order to separate the gel components. The pre-spermatic fraction and the sperm-poor fraction were discarded, while the spermatozoa-rich fraction was processed. Immediately after collection, the ejaculate was evaluated for color, smell, and weight /volume rat io. The spermatic concentration was calculated by using a Jenway LTD 60-51® spectrophotometer (Bibby Scientific Equipment Division, Staffordshire, UK). The collection quality was evaluated in the following way: 10 μΐ of non-extended semen were distributed on microscope slides being preheated at 37 °C, covered with a cover slip and the motility was observed using a phase-contrast optical microscope with a 200 X magnification in at least 4 slide fields: the motility was estimated considering the average of the counts in each field.

Then the semen was extended in the formulations 1 , 2, 3, 4, 5, 6, 7, 8 and in a commercial extender used as a reference ( Vitasem LD, Magapor, Saragozza, Spain) in order to have a concentration of 30 X 10 ⁶ spermatozoa /mL. The extended samples were transferred to the laboratory within 40 minutes from collection in a thermostat at 23-24 °C, and then slowly cooled for 1 hour until reaching the 16 °C temperature. Each sample was analyzed every 24 hours in the following days evaluating the progressive motility and aggregation phenomena by using an optical microscope with a 100 X magnification and until the motility was less t han 40% . The progressive motility was evaluated as described above for the ejaculate. The checks were always carried out by the same operator. The sperm whose progressive motility at the insemination time was equal to or greater that 70% was deemed useful for artificial insemination. The so measured progressive motility resulted as follows:

Table 2; example 1, boar A, average motility test. Formulation Progressive motility %

Time from collection (days)

0.3 2 3 4 5 6 7 8 9 10

Reference 75 75 70 70 65 60 60 60 40

1 80 80 80 80 75 75 75 70 70 70

2 80 80 80 75 70 70 75 70 70 50

3 80 80 80 75 70 70 75 60 50 50

4 80 80 80 80 75 80 75 60 60

5 85 85 85 80 80 80 75 65 60 40

6 80 80 80 80 70 70 65 60 60 40

7 80 85 85 75 70 70 70 60 60

8 75 75 75 75 70 70 65 65 40

Table 3; example 1, hoar B, average motility test.

As it is clear from the tables above, independently from the sperm quality at the ejaculat ion time, the ejaculate extension using the formulation object of this patent application allows the retention of the sperm progressive motility above 70% for at least two days more compared to the reference extender.

Example 2

It is described the preparation of an extender composition for rabbit semen, to be dissolved upon use. For that purpose 250 mg of invertase (Maxinvert 200000, >200.000 SU/g, [equivalent to > 64.000 U/g] Chimab), 8 g of potassium chloride, 12.8 g of gentamicin sulfate, 13.6 g od sodium bicarbonate, 13.6 g of disodium EDTA, 61 g of sodium citrate dihydrate and 400 g of sucrose were weighed. The powders were mixed in an orbital mixer of Turbula type (Bachofen AG) for 1 hour at 32 rpm. The obtained mixture was divided in 51 g shares. Each share was diluted in 1 L of purified water in order to prepare 1 liter of semen extender.

The gentamicin concentration was equal to 1.28 g/L, the sucrose concentration was equal to 40 g/L, while the invertase one was greater than 5000 SU/L (equivalent to > 1640 U/L). The rabbit semen was collected and extended, then preserved at 18°C for 18 hours before insemination. Table 3 reports the young rabbits birth data on a total of 1767 inseminations, 867 of which being carried out using the semen extended with extender of this patent application and 900 being carried out using a reference extender [Lepus, Medinova, Reggio Emilia).

Extender Lepus

Percent (invention) (reference)

increase

No. of artificial

inseminations

(AI) 867 900

Palpations No. of impregnated

breeding does

746 753

% pregnancies/ IA + 2.75 %

86.0 83.7

Parturitions No. of parturition

714 71 1

% parturition/ IA 82.4 79.0 + 4.30 %

Young rabbits No. of young

being born rabbits 6623 6348 Average No. of + 4.5 % young

rabbits / parturition 9.3 8.9

Young rabbits No. of young

being born rabbits 6505 6103

alive Average No. of +5.81% young

rabbits / parturition 9.1 8.6

Table 3; example 2, artificial inseminations results

As evident from data shown in table 3, the use of the semen extended with the formulation object of this patent application led to a higher pregnancies percentage compared to those obtained using the Lepus extender. The higher pregnancies percentage was also followed by a higher parturitions percentage and a higher number of young rabbits. The number of young rabbits being born alive on average was 9.1 per parturition, compared to an average of 8.6 when Lepus was used, therefore with a significant increase, being greater than 5.81%. Example 3

It is described the preparation of an extender composition for dog semen, to be dissolved upon use. For that purpose 25 mg of invertase (Maxinvert 200000, >200.000 SU/g, [equivalent to > 64.000 U/g] Chimab), 0.8 g of potassium chloride, 1.28 g of gentamicin sulfate, 1.36 g of sodium bicarbonate, 1.36 g of disodium EDTA, 6.1 g of sodium citrate dihydrate and 40 g of sucrose were weighed. The powders were mixed in an orbital mixer of Turbula type (Bachofen AG) for 1 hour at 32 rpm. The obtained mixture was divided in 0.51 g shares.

Three extender test tubes were reconstituted (powder + twice-distilled water a 37°C) for a total of 10 ml each, 30 minutes before collection. Gentamicin concentration was equal to 1.28 g/L, sucrose concentration was equal to 40 g/L, while the invertase one was greater than 5000 SU/L (> 1640 U/L).

In the collection site, an English bulldog male dog, 3 years old, whose fertility was proved, was washed using neutral soap and dried at foreskin level in order to remove secretions, then the semen was collected in the presence of a bitch in her est rus period . The procedure was carried out by a veterinary by manual collection, using a glass artificial vagina, being previously sterilized and using single-use non-spermiotoxic gloves. The first ejaculate fraction, without cells, was not collected as well as the last one, while the second one forming the fraction rich of spermatozoa was collected keeping the semen at 37°. The ejaculate was immediately evaluated for color, smell and volume. The spermatic concentration was calculated by using the manual count with a h e m o cy t o meter. The collection quality was evaluated in the following way: 10 ill of non- extended semen were dist ributed on microscope slides being pre-heated at 37 °C, covered with a cover slip and the motility was observed using a phase-contrast optical microscope with a 200 X magnification in at least 4 slide fields: the motility was estimated considering the average of the counts in each field and was found to be equal to 90% . The spermatozoa count was equal to 250 x 10 ⁶ per mL.

Then the semen was extended in the formulation at a 1 to 3, 1 to 5 and 1 to 7 ratio. The extended samples were t ransferred t o the laboratory within 40 minutes from collection in a thermostat at 23-24 °C, and then slowly cooled for 1 hour until reaching the 16 °C temperature. Each sample was analyzed every 24 hours in the following days evaluating the progressive motility and aggregation phenomena by using an optical microscope with a 100 X magnification and until the motility was less than 40%. The progressive motility was evaluated as described above for the ejaculate. The checks were always carried out by the same operator. The sperm whose progressive motility at the insemination time was equal to or greater that 70% was deemed useful for artificial insemination. The so measured progressive motility was as follows: Formulation Progressive motility %

Time from collection (days)

1 2 3 4 5 6 7

1 to 3 80 80 70 70 70 60 20

1 to 5 85 80 70 70 70 70 60

1 to 7 75 70 70 60 50

Table 5, example 3, average motility test