Camel semen extender

Abstract

The invention provides a camel semen extender composition which improves fertility of camel semen diluted with said extender and preserved in it, both when the semen diluted with the extender composition is used shortly after collection, or after being maintained under refrigeration up to 3-4 days. The semen extender comprises the carbohydrates glucose, lactose and galactose, antioxidant proteins such as catalase combined with reduced glutathione or bovine serum albumin, antibiotics in form of gentamycin or the combination of penicillin G and streptomycin and HEPES as buffer compound, and has an osmolarity of 320-370 mOsmol / L; it is easy to prepare, transport and preserve, as it is stable in frozen state and can be lyophilized. The invention provides a method for preparing the extender composition and for diluting and preserving semen in it and its use for artificial insemination, particularly of dromedary camels.

Description

[0001] CAMEL SEMEN EXTENDER

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a semen extender composition, manufacturing method, and its uses in semen dilution, preservation, and insemination in dromedary camels.

[0004] BACKGROUND OF THE INVENTION

[0005] During the second half of the last century, dromedary camels in the Arabian Gulf had a special interest due to the local government's financial support, mainly for racing and beauty contests. The selling price of some elite bulls (adult uncastrated males) reaches up to 40 million UAE dirhams (about 10 million US dollars). In comparison to other domestic animals, some new reproductive technologies such as artificial insemination and semen preservation have limited application till now (Skidmore, 2018). Camel semen has overall low volume, poor quality, and high viscosity, factors that are recognized as hampering the successful retrieval and preservation of good-quality sperm (Skidmore et al., 2013).

[0006] The maintenance of spermatozoa outside the body is customarily carried out in artificial insemination (Al) procedures. Numerous factors must be considered in any Al program; one of the major considerations is the extender (or diluent) used to dilute the semen before insemination either in fresh, or stored form. The use of chilled or frozen-thawed semen helps enable the adoption of Al as a technique of breeding in field conditions and facilitates semen transport domestically and internationally. A proper semen extender can prolong the survival of spermatozoa outside the body and enable a single semen ejaculate to be diluted to be useful to impregnate a large number of females.

[0007] Semen diluents previously used for dromedary camels

[0008] The most widely used extender for dromedary camel semen is Green Buffer (GB, I MV Technologies, France: https: / / www.imv-technologies.fr / ), which is the only one designed for camel semen, but the fertilization rates obtained with fresh semen diluted with frozen- thawed GB as extender were low, particularly when multiple ovulating camels were inseminated with diluted semen (Morton et al. 2011). Also, there are some buffers such as INRA-96 (IMV Technologies, France), and Triladyl® CSS One Step (Minitube, Tiefenbach, Germany: https: / / www.minitube.com / catalog / es / ), which are not designed for camels, which have been also tested to dilute the camel semen with variable results. Up till now, very low pregnancy rates following insemination of chilled and stored semen in dromedary camels were achieved (Skidmore et al., 2013; Morton et al., 2013) despite semen being good when evaluated in vitro (mainly by sperm motility). Liquid storage is affecting the fertilizing capacity of spermatozoa, but the underlying cause is unknown especially when considering in vitro semen quality appears to be high (Al-Bulushi et al.,

[0009] 2019). Only in one study, some fertility of short-term chilled camel semen for 24 hours could be achieved after dilution in GB without glycerol, as a cryoprotectant (Malo et al.,

[0010] 2020). However, the group of the present inventors have tested, in their lab, to repeat these fertilization experiments of Malo et al., 2020 with cooled semen kept for 24 hours diluted in GB (IMV Technologies, France) and yielded very low or even zero pregnancy rate. Also, GB should be used fresh and frozen-thawed GB is not preferable to be used and is associated with very low fertility records compared to using fresh, unfrozen GB with fresh semen (Morton et al., 2011).

[0011] Summarizing, all the extenders available for dromedary camel semen to date showed only some fertility with freshly collected semen. This semen should be used within maximum 2 hours from semen collection. Therefore, semen collection, dilution, and female insemination should be done almost at the same site. Semen transportation is necessary to avoid the stress and danger of transportation of valuable racing and beauty camels. Semen transportation for long distances requires cooling the semen at 4-5 °C to keep the sperm viable for some days. To date, only GB was the best extender to be used only for dilution and insemination using fresh semen (for immediate use in situ). No other specific extenders are designed for dromedary camels to increase the fertility of fresh semen more that obtained with using GB in the immediate use.

[0012] Thus, there is a need of a camel semen extender able to improve the fertility of fresh and short-term preserved cooled camel semen to be used for artificial insemination. Such extender should preferably have a long lifespan, being easy to transport worldwide and also being easy to use in the field. It should be stable in its frozen state and easy to thaw before its use, allowing to obtain high fertilization rates with the semen preserved in such extender, even when the extender has been frozen and thawed before diluting the semen with it. Also preferably, a suitable protocol for the use for artificial insemination of semen having been preserved in the aforementioned camel extender should be also established, so that it is possible to obtain optimal results with such preserved semen.

[0013] The present invention provides a solution to said problems. SUMMARY OF THE INVENTION

[0014] The present invention provides a new extender which facilitates semen processing and improves the fertility of fresh and short-term preserved cooled camel semen, being an extender with a long lifespan, easy to preserve, transport and use, which can even be lyophilized and reconstituted before being used. In particular, the semen extender has the following features:

[0015] This extender can be frozen and thawed, without an effect of the sperm fertility, which is an extra advantage than the using of other semen extenders used for camels, like GB. The newly developed extender is stable in its frozen state and easy to thaw shortly before its use. Therefore, it has a long lifespan and is easy to transport and use in the field.

[0016] Complete lyophilization of the extender is possible for longer storage and ease of shipping and transportation worldwide.

[0017] The newly developed extender can be used for cooling semen to be used for insemination within several days from semen collection. It also solves the problem of animal transportation.

[0018] The newly developed semen extender of the present invention gives rise to fertility rates of > 90 %, 70 %, and 30 % with fresh semen, and semen cooled for 24 and 48 hours, respectively. Thus, the invention allows the use of cooled semen for being preserved in the semen extender of the present invention, maintaining good fertility rates when the semen is used for artificial insemination.

[0019] This semen extender includes a sperm cell-protecting amount of carbohydrates, antioxidants, proteins, antibiotics, and a biological buffer to provide a semen extender use solution having a pH of between 6.9 and 7.5, preferably between 7.0 and 7.2, wherein the use solution exhibits a preferred osmolarity of 320-340 mOsmol / L.

[0020] Methods for manufacturing and using the semen extender composition are also provided. All the steps from semen collection, processing, dilution, cooling, and applying artificial insemination are included.

[0021] Such properties have been achieved thanks to the specific components selected for the semen extender composition and the particular proportions of each one in the compositions and the preparation method of the semen which is also part of the present invention. Thus, in a first aspect, the present invention relates to a camel extender composition characterized in that it comprises a) a buffer compound which is

[0022] HEPES: 100 - 200 mmol / L b) a combination of the following sugars: a. lactose 30 - 90 mmol / L b. glucose 30 - 100 mmol / L c. galactose 10 - 50 mmol / l c) proteins selected from: a. the combination of i) catalase enzyme 20 - 100 lU / mL ii) reduced glutathione 0.5 - 3 mmol / L or b. the combination of i) catalase enzyme 20 - 100 IU ii) bovine serum albumin (BSA) 0.8 - 2.5 g / L d) antibiotics selected from a. the combination of i) penicillin G 500 - 1500 lU / mL ii) streptomycin 250 - 750 pg / mL or b. gentamycin 500 - 2000 pg / mL and wherein the osmolarity is 320 - 370 mOsmol / L, and the pH is 6.9 - 7.5.

[0023] In a second aspect, the invention relates to a method for diluting and preserving camel semen to improve fertility, which comprises the steps of: a) preparing a camel semen extender composition of any one of claims 1 to 3, b) optionally adding clarified egg yolk to reach a concentration of 20% volume / volume with regard of the total volume of the final composition to a sample of the camel semen extender composition of step a), if the prepared semen is to be stored in the chilled form before use for at least 24 hours; c) collecting and selecting camel semen by: c.1. preparing and selecting the female camels to be use for collecting semen; c.2. preparing the perineal region of the selected female camels; c.3. having ready for use a semen collection sac; c.4. introducing a semen collection sac intravaginally in a female camel, c.5. fixating the sac to the internal vaginal wall of the female camel, c.6. securing the outer portion of the collection sac to the collection sac to the female’s perineal area, c.7. taking the female camel to a male pen for natural mating and awaiting until the male is dismounted; c.8. withdrawing the semen collection sac from the vagina, c.9. cleaning the semen collection sac from vaginal secretions and squeezing it to collect the deposited semen within, c.10. discarding semen samples contaminated with urine, faeces or sand, c.11. selecting semen showing oscillatory activity of 2 and / or 3, where, 2 of moderate and 3 of rapid activity; d) transferring semen to a water bath at a temperature of 33°C-37°C; e) diluting the semen 1 :1 volume:volume with the camel extender composition prepared in step a); f) keeping the diluted semen obtained in step d) in the water bath for 45 minutes subjecting it to regular pipetting for homogenizing it and reducing its viscosity; g) determining the sperm cell concentration in a sample of the homogenized semen obtained after step f); h) diluting the homogenized semen obtained in step f) to have a final concentration of maximum 150 x 106sperm / mL with : h.1 the camel semen extender composition prepared in step a), if the prepared diluted semen is to be used within 2 hours for artificial insemination, or h.2 the camel semen extender composition prepared in step b), if the prepared diluted semen is to be stored for 24 hours or up to 3 days, in the chilled form at 4-6°C; i) maintaining in the water bath the diluted semen that is to be used in less than 2 hours or storing the diluted semen in the dark at 4-6°C for up to 3-4 days.

[0024] Also, an additional aspect of the invention relates to the use of a diluted semen prepared in accordance with the method of the second aspect of the invention, for artificial insemination of a female camel.

[0025] The invention will be now explained in more detailed in the following sections. DETAILED DESCRIPTION OF THE INVENTION. PREFERRED EMBODIMENTS.

[0026] As commented in the previous section, the present invention relates to: a composition to be used as semen extender; a method for diluting and preserving camel semen to improve fertility wherein the semen is mixed with the semen extender composition of the present invention and is maintained mixed the composition until use for artificial insemination; and to the use of the semen mixed with the semen extender composition for artificial insemination of a female camel.

[0027] Accordingly with the aforementioned definition of the invention, as used herein, the terms “extender” or “semen extender” refer to the composition used to be mixed with the semen before being used for artificial insemination, either for diluting it or, additionally, also for preserving the semen when it is not used for insemination in a short period after collection but it is the mixture of semen and extender what is cooled and maintained at refrigeration conditions for 3-4 days. Thus, the term extender relates to a composition of the present invention both when it is used simply as a semen diluent and also when it is used also as a preserving composition. In certain points of the present application, the word “diluent” will be preferred instead of extender, when the composition is used simply for semen dilution for determination of sperm motility and / or for artificial insemination of freshly collected semen; in such cases, the composition of the present invention will lack egg yolk. When the term “extender” is used, preferably, it refers to the composition that can contain egg yolk (either fresh yolk or clarified egg yolk), and the semen extender composition could be used for cooling the semen and for the short-term preservation of the mixture of semen and extender, preferably at refrigeration temperature, and preferably for not more than 3-4 days. Nonetheless, for the semen mixed with the semen extender composition, both the terms “diluted semen” and “extended semen” will be used.

[0028] The semen extender composition of the present invention, as commented above, in its basic definition, comprises a biological buffer compound, carbohydrates (sugars), proteins including at least a protein with antioxidant properties, and antibiotics, to provide a semen extender use solution having a pH of between 6.9 and 7.5, preferably between 7.0 and 7.2, wherein the use solution exhibits an osmolarity of 320-370 mOsmol / L. Such composition can be used to dilute freshly collected semen, particularly if such semen, already diluted, is to be used while being still fresh for artificial insemination. In some embodiments, the composition can also comprise clarified egg yolk, being that the preferred embodiment of the composition is to be mixed with semen, particularly when the semen diluted with the extender composition is to be stored for 24 hours, or more, before being used for artificial insemination.

[0029] As used herein, freshly collected semen or fresh semen is semen that has been collected at most 2 hours before. The term can be used either for semen as collected or also for diluted semen that has been mixed with a semen extender. Hereinafter, the term “diluted semen” will be used, preferably, for freshly collected semen that has been mixed with the semen extender composition of the invention having its basic formulation, that is, lacking egg yolk. “Extended semen”, as used herein, will refer to semen that has been mixed or diluted with the semen extender composition of the present invention, either prepared according to its basic formulation or, preferably, also including egg yolk (preferably clarified egg yolk); in some Examples of the present application showing comparative assays, extended semen will be also used to refer to semen mixed or diluted with another semen extender already known in the prior art.

[0030] Each component of the semen extender composition of the present invention has been carefully chosen and is added in a specific range of concentrations to achieve the desired properties of the composition, as explained below.

[0031] - Buffer: HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), at 100 - 200 mmol / L, preferably at 130 - 170 mmol / L, more preferably at 150 mmol / L

[0032] Most mammalian spermatozoa use the anaerobic metabolism which produces lactic acid. Therefore, the proper semen extender should be biologically buffered to maintain the viability of sperm cells for a long period. The use solution can exhibit a pH of between about 6.9 and about 7.5, preferably between 7.0 and 7.2 and more preferably between 7.0 and 7.1. Commonly used biological buffers are available, commonly known under the names TRIS, HEPES, phosphate, citrate, triethanolamine, and TES buffers. The most commonly used buffer for extenders previously used for camels is the TRIS (tris(hydroxymethyl)aminomethane) buffer. Contrary to the prior art, the observations of the inventors of the present invention indicate that TRIS buffer is not suitable for camel sperm during cooling, as it causes sperm clumping or agglutination. After testing all the aforementioned buffers for camel semen, the present inventors concluded that HEPES buffer is the best one, especially during cooling and short-term preservation of extended semen.

[0033] The amount of biological buffer to be provided in the use solution (the composition to be used as diluent or, in general, as semen extender) depends on the strength of the biological buffer and the desired buffering capacity of the use solution. In general, it is expected that the molar concentration of the biological buffer in the use solution is between about 0.05 M and about 0.25 M. Many HEPES concentrations were tested and 100 - 200 mmol / L was found to be a suitable one, being preferable a concentration of 130 - 170 mmol / L and 150 mmol / L being the best one.

[0034] - Carbohydrates (Sugars): A combination of the following sugars: lactose (30 - 90 mmol / L, preferably 65 mmol / L), glucose (30 - 100 mmol / L, preferably 50 mmol / L) and galactose (10 - 50 mmol / L, preferably 30 mmol / L).

[0035] As used herein, the term sugar is used for a carbohydrate composed of a low number of monomeric units, more specifically for mono-saccharides, di-saccharides and trisaccharides.

[0036] Sugars provide energy substrates for sperm cells during incubation, maintain the osmotic pressure of the diluent, and act as a cryoprotectant, in case of semen cryopreservation. Mono- (such as fructose, glucose, galactose), di- (lactose, sucrose, maltose, trehalose), and tri-saccharides (raffinose) are commonly used for animal semen extenders. Determination of the used sugars is species-specific. Also, the concentration range of each sugar should be determined and adjusted.

[0037] Sugars were tested and the best sugars for camel sperm cells were selected and their required concentrations were determined.

[0038] - Proteins with antioxidant properties: Proteins selected from: catalase enzyme (20 - 100 IIJ / mL, preferably 40 - 60 IIJ / mL, more preferably at 50 UI / mL) combined a) preferably with reduced glutathione (0.5 - 3 mmol / L, preferably 1.0 - 2.0 mmol / L, more preferably 1.0 - 20 mmol / L) or b) with bovine serum albumin (BSA) (0.8 - 2.5 g / L, preferably 1.0 - 2.0 g / L).

[0039] Preferably, the semen extender composition includes a sufficient amount of antioxidants to provide the desired benefit. In general, it is expected that the used semen diluent or extender includes at least one antioxidant with up to about 3 wt.% antioxidants.

[0040] In principle, many antioxidants (including some enzymes, vitamins, minerals, proteins, etc) can be used for some extenders. Selection of the proper antioxidant / s and its used dose in the semen extender differs from one species to the other, not being obvious the best one to use for camels. Vitamin A, and C (ascorbic acid) were tested and did not improve the camel sperm motility and fertility. Also, some enzymes such as glutathione reductase and superoxide dismutase (SOD) were tested without any improvement. Catalase enzyme and glutathione protein (a tripeptide compound formed by the condensation of glutamic acid, cysteine, and glycine in the form of peptide bonds) have a positive effect on camel sperm motility and fertility, either in fresh and cooled stored form. Different concentrations of both Catalase E and reduced glutathione were tested, finding as acceptable concentrations those of the range of 20 - 100 lll / mL Catalase and 0.5 - 3 mmol / L reduced glutathione, being more suitable the concentrations of the ranges of 40 - 60 lll / mL Catalase and 1.0-2.0 mmol / L glutathione, being optimal the concentrations of 50 lll / mL catalase and 1.5 mmol / L glutathione.

[0041] Although the combination of catalase enzyme and reduced glutathione is preferred, a possible alternative is using a combination of catalase enzyme and bovine serum albumin (BSA), wherein catalase enzyme concentrations can vary in the same ranges and BSA can be in a concentration of 0.8 - 2.5 g / L, preferably 1.0 - 2.0 g / L.

[0042] - Antibiotics: Selected from a) the combination of penicillin G (500 - 1500 IIJ / mL) and streptomycin (250 - 750 pg / mL) as a preferred option or b) gentamycin (500 - 2000 pg / mL, preferably 700 - 1500 pg / mL, more preferably 1000 pg / mL).

[0043] With such formulation, the osmolarity is expected to have values of 320 - 370 mOsmol / L, being preferred the values of 330 -350 mOsmol / L and being particularly preferred 330 - 340 mOsmol / L.

[0044] As commented above with reference to the buffer, the pH value should be near neutral pH, for instance, 6.9 - 7.5, preferably 7.0 - 7.2, more preferably 7.0 - 7.1 , and most preferably 7.0.

[0045] According to the above information, related to the basic formulation for the camel extender composition (the formulation that is preferred to use the camel extender simply as diluent, when the semen is to be used for artificial insemination as fresh semen, in less than 2 hours from collection), one preferred embodiment of the camel extender composition is the one where the composition comprises: a) HEPES: 130 - 170 mmol / L b) a combination of the following sugars: a. lactose 30 - 90 mmol / L b. glucose 30 - 100 mmol / L c. galactose 10 - 50 mmol / L c) proteins selected from: a. the combination of i. catalase enzyme 40 - 60 IIJ / mL ii. reduced glutathione 1.0 - 2.0 mmol / L or b. the combination of i) catalase enzyme 40 - 60 IIJ / mL ii ) bovine serum albumin (BSA) 1.0 - 2.0 g / L d) antibiotics selected from a. the combination of i. penicillin G 500 - 1500 IIJ / mL ii. streptomycin 250 - 750 pg / mL or b. gentamycin 700 - 1500 pg / mL and wherein the osmolarity is 330 - 350 mOsmol / L, and the pH is 7.0 - 7.2.

[0046] In a more preferred embodiment, which is a particular case of the previous one, the composition comprises: a) HEPES: 150 mmol / L b) a combination of the following sugars: a. lactose 65 mmol / L b. glucose 50 mmol / L c. galactose 30 mmol / L c) proteins: the combination of i. catalase enzyme 50 lU / ml ii. reduced glutathione 1.5 mmol / L d) antibiotics: the combination of i. penicillin G 1000 IIJ / mL ii. streptomycin 500 pg / mL and wherein the osmolarity is 330 - 340 mOsmol / L, and the pH is 7.0 - 7.1.

[0047] The above basic formulation of the camel extender composition can be prepared by: a.1. placing the required amounts of the sugars (lactose, glucose and galactose) and HEPES in a recipient; a.2. adding double distilled water; a.3. dissolving HEPES and the sugars by stirring; a.4. adjusting pH to 6.9 - 7.5 by dropping 200 mmol / L sodium hydroxide solution with continuous stirring, a.5. adding the antibiotics and mixing them with the other added components of the composition by stirring continuous, a.6. readjusting pH to 6.9 - 7.5 (preferably, 7.0 -7.2 or, more preferably 7.0 - 7.1) by dropping 200 mmol / L sodium hydroxide solution, if necessary, a.7. adding the proteins selected from the combination of catalase enzyme and BSA or the combination of catalase enzyme and reduced glutathione, and dissolving them by stirring,

[0048] The mentioned sequence of substeps form a possible embodiment of step a) of preparation of the camel extender composition of the method of the present invention for diluting and preserving camel semen to improve fertility.

[0049] Any of the possible embodiments of the camel extender composition of the invention can also comprise, additionally to the other components, clarified egg yolk, preferably 20% volume / volume clarified egg yolk. That is preferred when the camel extender is to be used for facilitating short term preservation of the semen (more than 2 hours, usually at least 24 hours and up to 3 - 4 days) in the chilled form (refrigeration conditions), instead of using it shortly after its collection.

[0050] Whole egg yolk is a conventional cooling protectant during semen preservation. However, whole egg yolk could represent a potential risk of contamination, inhibit sperm respiration, reduce motility of sperm, and interfere with sperm fertility in camel. Therefore, in the last few years, there have been increasing demands to replace whole egg yolk with its cooling protective components in semen extenders. The main effective component in egg yolk is low-density lipoproteins (LDLs). LDLs act through the stabilization of the sperm membrane, formation of a protective layer over the surface of the sperm, replacement of membrane phospholipids and thereby decrease in the membrane phase transition temperatures. Incorporation of LDLs in semen extenders results in the enhancement of sperm viability during storage in liquid or frozen states. Recently, clarified egg yolk (cEY) was introduced to replace using fresh egg yolk due to its simple extraction and has good results with other animal species such as equine and buffalos. In the present extender, clarified egg yolk is preferably added to the camel semen extended composition when semen is not to be used fresh, shortly after collection, but it is to be stored at temperatures below 10°C before use; clarified egg yolk addition is then preferred for short-term storage (up to 3-4 days at 4-6°C).

[0051] Clarified egg yolk refers to the supernatant resulting from centrifuging, at 10,0000 - 20,000 x g, egg yolk diluted 1 :1 to 1 :3 with a HEPES buffer or, preferably, with a semen extender diluted in water. For the purposes of the present invention, it refers particularly to egg yolk resulting from the following preparative substeps: b.1. breaking fresh chicken eggs (for instance, manually): b.2. discarding albumen; b.3. rolling egg yolk carefully on filter paper to remove albumen and chalazas adhering to the vitelline membrane; b.4. perforating the vitelline membrane and collecting the egg yolk; b.5. diluting egg yolk with the semen extender composition of claims 1-3 and distilled water in a ratio of: Freshly collected egg yolk: semen extender : distilled water 1 : 1.5 : 1.5 (volume : volume : volume) and stirring for 10 minutes, b.6 centrifuging the diluted egg yolk of substep b.6 at 10,000 x g for 30 minutes, b.7 collecting the supernatant, which is the clarified egg yolk, and storing it at - 40°C before use.

[0052] The above sequence of substeps b.1 to b.7 form a possible embodiment of step b) of the method of the present invention, the optional substep of adding clarified egg yolk to reach a concentration of 20% volume / volume with regard of the total volume of the final composition to a sample of the camel semen extender composition prepared in accordance to its basic formulation (preferably prepared following the above mentioned substeps a.1 to a.7).

[0053] In another possible embodiment, the semen extender composition of the present invention can be prepared by mixing three different Parts or lots of compounds, wherein each lot or part is composed of components which have been packed and stored together, in powder or in frozen form. This embodiment has the advantage of having the components of the camel extender already prepared in their necessary proportions, in a form easy to store and transport, divided in three parts or lots packed individually that can be sold, and transport if desired, as individual packs or as a kit of components including the three packs, facilitating the preparation of the semen extender composition when desired. The three Parts can be: - Part A is composed of HEPES, lactose, glucose, galactose, and the selected antibiotics, all of them in powder form and can been stored in a closed sealed pack up to 1 year at room temperature,

[0054] - Part B is composed of catalase enzyme and reduced glutathione, all of them in powder form, and has been stored in a small sealed pack up to 1 year in a frozen state, and

[0055] - Part C is composed of lyophilized clarified egg yolk (preferably prepared by substeps b.1 to b.7 set forth above), that can be stored up to 1 year in a frozen state and has to be thawed until reaching room temperature before being added to the solution with the other components of the camel semen extender composition.

[0056] Preferably, the amounts suitable to be added to prepare 100 mL of the semen extender composition are packed in the small sealed sac corresponding to Part A or Part B. In the case of Part C, as it should be present as 20% volume / volume of the semen extender composition, the pack or small sealed sac of Part C will preferably contain the part to be added to 80 mL of the solution already containing Part A and Part B.

[0057] In this embodiment, the camel extender composition is preferably prepared as follows: i) placing in a recipient the components of Part A, dissolving them in double distilled water and adjusting the pH to 6.9 -7.5 by dropping 200 mmol / L sodium hydroxide solution with continuous stirring, ii) adding the components of Part B to the solution with Part A and dissolving them by stirring, iii) if the semen is to be stored refrigerated (at 4-6°C), adding Part C to the solution with Part A and Part B already dissolved.

[0058] The semen extender composition comprising Part A + Part B, and lacking Part C, is suitable for fresh semen dilution and insemination of females within a very short period from semen collection, for instance within a period of 2 hours. Thus, when a semen extender composition is to be used as diluent, Part B is added and mixed well with the solution where the components of Part A are already dissolved in the distilled water (for instance for having been under magnetic stirring), so that a solution (a semen extender composition of the present invention) with the expected volume (for instance, 100 mL) is prepared at that substep.

[0059] Part C is added only when the semen is to be cooled: preserved under refrigeration (4°C - 6°C) for a short term (up to 3-4 days). When the semen is to be cooled, Part C is taken from the freezer, kept 10 min at room temperature, added to 80 mL of a solution with Part A + Part B.

[0060] The method of the present invention for diluting and preserving camel semen to improve fertility also includes, additionally to the already mentioned steps a) and b) of preparing the semen extender composition, and their alternative embodiments, the steps of: c) collecting and selecting camel semen, d) transferring semen to a water bath at a temperature closer to the camel body temperature (33°C-37°C), e) diluting the semen with the prepared camel extender composition of the present invention (either lacking clarified egg yolk or comprising it, depending on the foreseen elapsed time from semen collection and semen use for artificial insemination, as explained above), f) homogenizing and reducing viscosity of the already diluted semen (preferably by subjecting the diluted semen obtained in step d) to regular pipetting, for instance for 45 minutes, while it is kept in the water bath), g) determining sperm cell concentration in a sample of the homogenized diluted semen, h) diluting the homogenized semen with the semen extender composition of the present invention (either lacking clarified egg yolk or comprising it, depending again on the foreseen elapsed time from semen collection and semen use for artificial insemination) to have a final concentration of maximum 150 x 106sperm / mL) and, depending of the foreseen moment when the prepared semen is to be used, i) maintaining it in the water bath if it is to be used in less than 2 hours or storing the prepared semen (which preferably will have been diluted with the semen extender composition comprising clarified egg yolk) under refrigeration conditions (preferably at 4-6°C) and in the dark for up to 3-4 days.

[0061] The method of the present invention has been designed so that fertility improvement is as much as possible, taking into account which could be the suitable embodiments to carry it out.

[0062] For instance, a possible preferred embodiment for step c) of collecting and selecting camel semen, combinable with all the aforementioned embodiments, comprises the substeps of: c.1. selecting female camels for semen collection if they show a follicular activity of 1.1 - 1.6 cm in diameter, c.2. cleaning, webbing and disinfecting with alcohol the perineal region of the selected female camels, c.3. using as semen collection sac one made of flexible low-density polyethylene, comprising a tubular main part with an end designed to be the outer portion of the collection sac once inserted and with a gradually decreasing diameter until the other end where it bifurcates in two fingers designed to be introduced in the uterus, c.4. introducing the semen collection sac intravaginally in the female camel with the aid of a metal applicator sheathed inside the sac, c.5. fixating the sac to the internal vaginal wall through air insufflated from a pump connected to the distal end of the metal applicator, c.6. fixing the outer portion of the sac to the vaginal wall, after drawing the metal applicator out of the vagina, by a fixation sticker which secures the outer portion of the collection sac to the female’s perineal area from the dorsal and lateral sides of the vulva, c.7. using for natural mating camel males which have not been used for natural mating or semen collection during the previous 3 weeks, c.8. detaching the fixation sticker and withdrawing the sac from the vagina manually, c.9. detaching the sac from vaginal secretions and squeezed it manually, c.10. recording the volume and the color of the collected semen and excluding the semen samples contaminated with urine, faeces or sand, c.11. determining oscillatory semen activity in an undiluted semen sample which is examined under the microscope at 100x magnification.

[0063] In order to select female camels for semen collection (substep c.1), females are preferably examined by transrectal ultrasonography and those showing no genital abnormalities and having at least one ovarian follicle of 11-16 mm in diameter are selected. This is a common practice.

[0064] Preferably, semen (the semen ejaculate of the camel males used for natural mating) is collected by using the Camel Semen Collection Kit (CSCK) (provided in the market by the German Standard Group, GSG: https: / / www.gsgroup.co) as previously described by Dr. Nabil Mansour (Mansour, 2023) and also in the patent documents P6000373 / 2022 (United Arab Emirates), 122430781 (Kingdom of Saudi Arabia) and QA / 202203 / 000186 (Qatar). Said Kit is composed of three parts fulfilling the requirements mentioned in substeps c.3 to c.6:, that is: (1) a semen collection sac made of supersensitive flexible low-density polyethylene, (2) a metal stainless still applicator, designed to introduce the collection sac intravaginally and fixate it to the vaginal wall of the female camel through air insufflation; (3) a fixation sticker with a cushion sheet sticker used to secure the outer portion of the collection sac to the female’s as perineal area. CSCK is preferred because, as reported by Dr. Nabil Mansour in 2022 and 2023, semen collected with said kit showed a longer collection period and higher volume, gross activity, sperm motility and percentage of live spermatozoa and a lower rate of visible contamination compared, for instance, to the use of artificial vaginas.

[0065] Collected semen which, after visual inspection, shows contamination with urine, faeces or sand, is discarded.

[0066] As indicated in substep c.11 , gross activity is determined directly after semen collection by determining the oscillatory activity of spermatozoa in an undiluted semen sample. To carry out such determination, an aliquot of 20-40 pl of semen is pipetted on a pre-warmed slide, covered with a coverslip, and examined under a phase contrast microscope at 100 X magnification, as previously described by Al-Bulushi et al. (Al-Bulushi et al., 2019). Semen showing moderate and rapid oscillatory activity is used, discarding semen with no oscillatory activity or slow oscillatory activity.

[0067] As commented before, once semen has been collected and selected (step c)), semen is quickly transferred to a water bath previously adjusted to 33°C-37°C (preferably 35 ° C) (step d)) and diluted with the prepared camel extender composition of the present invention, preferably 1 :1 volume:volume, semen: diluent (step e)).

[0068] To achieve a homogenous mix and reduction of semen viscosity (step f)), the diluted semen can be subjected, for instance, to regular pipetting, for example every 5 min, for a period enough to obtained the desired homogeneous mix, which will be usually 45 min. At this step, and any other subsequent step previous to use for insemination or cooling for preservation, the semen will be maintained in the water bath, at 33°C -37 °C, preferably at 35°C.

[0069] Before final semen dilution, sperm cell concentration per mL of ejaculate is determined (step g)) in a sample of the already homogenized, diluted semen. To determine sperm cell concentration, a sub-sample of 100 pl of already diluted semen can be further diluted 1 :30 volume:volume with 0.5% formaldehyde solution, and spermatozoa are counted in an appropriate chamber, such as a Burker Turk Counting Chamber (Fisher Scientific: https: / / www.fishersci.se / shop / products / buerker-tuerk-counting-chambers-2 / 10297390).

[0070] Once concentration has been determined, semen is diluted (step h)) to have a final concentration of maximum 150 x 106spermatozoa / mL. In order to obtain such concentration, semen ejaculate is finally diluted 1 :2 to 1 :6 (volume:volume) depending on sperm cell concentration of the semen ejaculate.

[0071] Depending on the foreseen moment when the prepared semen is to be used, the formulation of the composition of the present invention used for diluting the semen both in step e) (when the semen is initially diluted, preferably 1 :1 , for viscosity reduction) and in step h) (when the already diluted semen is further diluted to achieved the desired spermatozoa concentration) will be different. If the semen is to be used shortly after collection (within a period of approximately 2 hours after collection), what will be preferably used in both steps will be the formulation that has been called sometimes the basic formulation, that is, the formulation lacking clarified egg yolk (the one that is obtained by mixing Part A + Part B if the composition is prepared by mixing lots or Parts of components which has been packed and stored by lots), as the extender composition will be used mainly as a diluent that will facilitate handling of semen and insemination. When the semen has to be preserved to be used in more than 2 hours, usually next day or within more than 24 hours, the semen will have to be cooled for storing it, so that embodiment of the extender composition of the present invention to be used will be the one including clarified egg yolk in the formulation (Part C if the preparation by mixing Parts or lots of components is used). If the diluted semen must be stored under refrigeration, cooling will be preferably gradual, for instance as described in Example 2 below: at a cooling rate of 0.2 degree / min in one hour and 0.15 degree / min in the following 2 hours.

[0072] Analogously, If the semen is to be used fresh, shortly after collection, it will be maintained in the bath, at 33°C - 37°C, very preferably at 35°C, just until the moment when it is taken to be used for insemination. When the extended semen has to be preserved to be used in more than 2 hours, so that it has to be cooled for storing and preservation, it will be taking from the bath for cooling, once it has been diluted at the desired concentration (step h)).

[0073] The non used semen extender composition (either in its basic formulation, for instance prepared by mixing Part A + Part B, or in the formulation with clarified egg yolk, that is, the solution resulting after adding Part A + Part B + Part C to distilled water if the composition has been prepared by using Parts or lots of components previously stored as such), can be kept frozen at -20°C and used within 3 months, after thawing.

[0074] The use of the semen diluted with the semen extender composition of the present invention, prepared as above described, for artificial insemination of a female camel, is another aspect of the present invention.

[0075] Preferably, the selected female camels show no genital abnormalities. Similarly to the selection made for obtaining semen, female camels are examined by transrectal ultrasonography and those showing no genital abnormalities and with at least an ovarian follicle of 12-16 mm in diameter are selected. Camel is an induced ovulatory animal (Tibary and Anouassi, 1997; Skidmore et al., 2013; Purohit et al., 2023), what means that, naturally, ovulation occurs 24-36 hour after mating; therefore, to do artificial insemination, a systemic inducing factor or gonadotropins (GnRH) (for instance, 2 mL., intramuscularly) should be administered to the female camel 22-26 hours before insemination. Gonadotropin can be replaced by an analogue of the gonadorelin (the gonadotropin releasing hormone) such as buserelin (administered, for instance, as 20 mg buserelin acetate, 22 - 26 hours before insemination).

[0076] Sperm is deposited by the recto-vaginal technique at the tip of the uterine horn ipsilateral to the developed ovarian follicle. As it is known by those skilled in the art, to perform the recto-vaginal technique, female is sedated by intravenous administration of xylazine (for instance, 0.12 mg / kg, Xylazine 2%, available from Agrar Holland BV, The Netherlands), and bandaging the tail, the vulva, and surrounding perineal area are cleaned with sterilized dry gauze and then disinfected with alcohol 70%. The insemination catheter (for instance, the Universal IUI sterile pipette 65 cm, Minitube-Germany) is introduced through the vulvar opening into the caudal vagina and guided toward the anterior vagina and cervix through the rectum. The catheter is further inserted into the tip of the uterine horn ipsilateral to the ovarian follicle where the semen is deposited.

[0077] When the diluted semen is used fresh, within 2 hours or less from the collection moment, a dose of at least 150 x 106motile sperm (>1 mL of diluted semen) is deposited into the tip of the uterine horn. If the diluted semen is used in more than 2 hours from collection and it has been maintained at 4-6°C, it will be used after warming up to 35 °C and at least a dose of 300 x 106motile sperm (> 2 mL of diluted semen) is deposited at the tip of the uterine horn.

[0078] The camel extender composition of the present invention, as well as the method for diluting and preserving camel semen to improve fertility of the present invention and the use for artificial insemination of the extended / diluted semen obtained by said method, have been designed for camels and can be applied to any camels, including Bactrian camels (Camelus bactrianus), but it is particularly suitable for dromedary camels (Camelus dromedarius), so that it is preferred that the semen is collected from dromedary camels and that it is used, once diluted with the semen extender composition of the present invention, for insemination of female dromedary camels.

[0079] The invention will be now explained in more detailed with the following Examples. EXAMPLES

[0080] The assays were carried out with the following materials and the same methodologies, common for both Examples:

[0081] Used diluents and extenders:

[0082] 1- SHOTOR Extender: Tris-based diluent, (214.6 mM Tris, 64.2 mM CitricAcid, 66.6 mM glucose, 49.9 mM fructose, penicillin G sodium 1000 lU / ml, streptomycin sulphate 1000 mg / ml (Niasari-Naslaji et al. 2006) with addition of 20% fresh egg yolk for chilled preservation of semen. This extender was designed for Bactrian camels.

[0083] 2- Green buffer (IMV Technologies, France) with addition of 20% fresh egg yolk for chilled preservation of semen.

[0084] 3- Mansour Extender: Composition with the basic formulation of the semen extender composition of the present invention, in its preferred embodiment (HEPES: 150 mmol / L; lactose: 65 mmol / L; glucose: 50 mmol / L, galactose: 30 mmol / L, catalase enzyme: 50 lU / ml, reduced glutathione: 1.5 mmol / L; penicillin G: 1000 lU / mL, streptomycin: 500 pg / mL; wherein the osmolarity is 330 - 340 mOsmol / L, and the pH is 7.0), with addition of 20% clarified egg yolk (CEY) for chilled preservation of semen. CEY was prepared by mixing 10 mL fresh egg yolk with 15 mL distilled water and 15 mL basic extender (Part A + Part B), subjecting it to centrifugation at 10,000 x g for 30 min and the supernatant was used.

[0085] Animals and location

[0086] Six male dromedary camels of 9-12 years old with a sound history of fertility, and 80 parous females were used in the current study. Each male was housed in a single pen of 9X9 m. The females were kept in two large pens of 80X80 m. The camels were fed on wheat bran, crushed maize, crushed barley, Rhodes grass and dried alfalfa with a rate of 1.5, 0.8, 0.4, 4.0 and 2.0 Kg / head / day, respectively. Additionally, an oral amount of 30 gram / head / day of mineral and vitamin premix supplement for male dromedary camels which contains vitamins A, D, E, B1 , B2, B6, B12, C, niacin and pantothenic acid and minerals such as calcium, phosphorus, magnesium, iron, copper, cobalt, iodine, manganese, zinc and selenium (NeoMix Super®, Neofarma Co., Italy) was administered.

[0087] Semen collection

[0088] The used dromedary males were not used for natural mating or semen collection for a period of 3 weeks prior to the experiment. Semen was collected twice weekly using the Camel Semen Collection Kit (CSCK) (distributed in the market by the German Standard Group, GSG: https: / / www.gsgroup.co) as previously described by Dr. Nabil Mansour (Mansour, 2023). Briefly, the females used for semen collection were examined in the standing position by trans-rectal ultrasonography and those showing a follicular activity of 1.1 -1.6 cm in diameter, were used. These females were slightly tranquilized by administration of xylazine 2% (Alfasan, The Netherlands) at a dose of 0.1 mg / kg IV. The perineal region was cleaned, webbed and disinfected with 70% alcohol. The metal applicator was first sheathed inside the semen collection sac and then inserted into the vagina of a receptive female. A manual air pump was connected to the distal end of the applicator, air was pumped through the apertures in the head of the applicator to the collection sac to be urged against the vaginal wall of the female camel. The outer portion of the collection sac was fixed to the female’s perineal area from the dorsal and lateral outsides of the vulva by using adhesive tape. Then the female camel was subjected to natural mating, the fixation sticker was manually detached from the perineal region and the semen collection sac was withdrawn from the vagina. Then the outer surface of the collection sac was cleaned from vaginal secretions and squeezed to collect the ejaculated semen in a falcon tube prewarmed to 35 °C. Semen was transferred to a water bath containing three extenders which warmed up to 35 °C. Gross activity was described directly after collection as the oscillatory activity of spermatozoa in an undiluted semen sample. An aliquot of 40 pl of semen was pipetted on a pre-warmed slide, covered with a coverslip and examined under the microscope at 100 X magnification. This parameter was scored according to Al-Bulushi et al. (2019) from 0- 3; whereas 0: no oscillatory activity; 1 : slow oscillatory activity; 2: moderate oscillatory activity and 3: rapid oscillatory activity. Semen samples with oscillatory activity of > 2 were divided into three batches and each batch was diluted 1 :2 with one of three diluents or extenders. To achieve a homogenous mix, the diluted semen was subjected to regular pipetting every 5 min for 45 min in the water bath for reduction of semen viscosity (Wani et al., 2008). A sub-sample of 500 pl semen was kept at 35 °C till complete liquefaction. To determine the sperm cell concentration, an aliquot of 100 pl was diluted 1 :50 with 0.5% formaldehyde solution and sperm cells were counted using a Burker Turk Counting Chamber (Fisher Scientific: https: / / www.fishersci.se / shop / products / buerker-tuerk-counting- chambers-2 / 10297390).

[0089] Pregnancy diagnosis

[0090] Pregnancy diagnosis was carried out on Day 14 post-insemination by using a commercial P4-ELISA assay (Ridgeway Science, Alvington, UK). The progesterone standards ranged from 0 to 20 ng / mL. The standards were prepared by the company by dissolving progesterone in the sera of a one-year-old male camel to minimize the effect of nonspecific interference which may occur between the ELISA-P4 antibody and the camel serum. The females were considered pregnant when blood progesterone (P4) was more than 1 ng / mL (Nagy et al., 2015) and showed tail cocking (erected and coiled tail) when approached by a male camel. The pregnant recipients were re-examined by trans-rectal ultrasonography on Day 60 of pregnancy.

[0091] Statistical analyses

[0092] Differences among treatments of sperm motility were assessed for significance using one-way ANOVA with subsequent Duncan test. Fisher exact test was used to evaluate the difference in the pregnancy rates at Day 14 post insemination and 2 months of pregnancy. The differences were considered significant at a probability level of 0.05 and the results were presented as mean ± SD. All the statistical analyses were done using SPSS® 22 for Windows® (SPSS Worldwide Headquarters, Chicago, IL, USA).

[0093] - Example 1 : Fresh semen

[0094] Assay for testing the sperm motility and fertility of semen samples diluted in the three extenders. Ten semen samples were included in this experiment with three repetitions.

[0095] Sperm motility

[0096] Sperm motility was recorded after 1 , 3, 6, and 12 hours from semen collection when diluted semen kept at 35 °C. Briefly, semen was further diluted 1 :2 with the same diluent (final dilution 1 :5, V:V, semen : diluent). Then 2 pl of diluted semen was pipetted on a 37 °C pre-warmed Leja® Standard Count Slide (Leja Products B.V., The Netherlands) and sperm motility was examined under a hotplate Olympus CX41 RF (Olympus Corporation, Tokyo, Japan). Sperm motility was evaluated by computer assisted sperm assessment (CASA -Sperm Vision® SAR software, Minitube GmbH, Tiefenbach, Germany). Three randomly selected microscopic fields were scanned to have approximately 200 spermatozoa counted and recorded by CASA system. The following sperm motility parameters were measured: spermatozoa with a velocity <5 pm / s were defined as immotile, spermatozoa with a velocity of 5-20 pm / s as locally or non-progressively motile and spermatozoa with a velocity >20 pm / s as progressively motile. Finally, the total sperm motility was determined by summation of locally and progressively motile spermatozoa.

[0097] Sperm fertility Females were examined by transrectal ultrasonography and those having at least a follicle of 12-16 mm in diameter were administered 20 mg Buserelin acetate at 22 - 26 h before insemination. For artificial insemination, females were sedated by intravenous administration of xylazine (0.12 mg / kg, Xylazine 2%, Agrar Holland BV, The Netherlands), and bandaging the tail, the vulva, and surrounding perineal area are cleaned with sterilized dry gauze and then disinfected with alcohol 70%. For insemination, Equine Al Kit (REF.: 17207 / 2065, Minitube, Germany) was used. Two semen doses were tested: 75 x 106and 150 x 106motile sperm of fresh semen, sucked in the syringe and the insemination catheter were introduced through the vulvar opening into the caudal vagina and guided toward the anterior vagina and cervix through the rectum. The catheter was further inserted into the tip of the uterine horn ipsilateral to the ovarian follicle where the semen was deposited. Insemination was done after 60-75 minutes of semen collection. For fertilization experiments, same semen samples diluted in the three extenders were used. Minimum 6 females were used in this fertilization experiment for each treatment.

[0098] Results

[0099] The results related to sperm motility are shown in Table 1 below.

[0100] Table 1. Total sperm motility of dromedary camel semen diluted in three extenders and kept at 35 °C.

[0101] Period in hours SHOTOR Green buffer Mansour Extender

[0102] 1 58.9 ± 10.5a84.3 ± 12.6b89.5 ± 16.4b

[0103] 3 18.2 ± 7.6a36.3 ± 9.5b67.4 ± 11.3C

[0104] 6 0.0a12.5 ± 3.5b35.2 ± 4.5C

[0105] 12 0.0a0.0a7.8 ± 6.1b

[0106] Values within a row with different superscripts are significantly different (P < 0.05)

[0107] As can be seen in said Table 1 , fresh semen kept at 35 °C showed higher sperm motility when diluted in Green buffer and Mansour diluent compared to that diluted in SHOTOR diluent after 1 h. Semen diluted in Mansour extender resulted in higher motility rates (P < 0.05) at 3, 6, and 12 hours compared to that diluted in SHOTOR and Green buffer (Table 1). Semen diluted in Green buffer showed superior motility rate than that diluted in SHOTOR diluent at 1 and 3 hours of incubation at 35 °C. There was no sperm motility at 6 and 12 hours in SHOTOR and Green buffers, respectively.

[0108] The fertility results, in turn, are shown in Table 2 below. Table 2. Fertility of freshly collected dromedary camel semen diluted in different extenders.

[0109] Pregnancy at SHOTOR Green buffer Mansour Extender

[0110] Number (%)

[0111] Insemination dose: 150 x 106motile sperm

[0112] 14 days 2 / 6 (33.3)a4 / 6 (66.6)b5 / 6 (83.3)a

[0113] 2 months 1 / 6 (16.6)a3 / 6 (50.0)a4 / 6 (66.6)a

[0114] Insemination dose: 75 x 106motile sperm

[0115] 14 days 0 / 6 (0.0)a1 / 6 (16.6)b4 / 10 (40.0)c

[0116] 2 months - 0 / 6 (0.0)a4 / 10 (40.0)b

[0117] Values within a row with different superscripts are significantly different (P < 0.05).

[0118] As can be seen in said Table 2, freshly collected semen diluted in all tested extenders and inseminated with a dose of 150 x 106motile sperm resulted in some pregnancies at 14 days post-insemination and 2 months of pregnancy. However, semen diluted in Mansour extender showed the highest (P < 0.05) pregnancy rates (83.3 % and 66.6 %) followed by the Green buffer (66.6 % and 50.0 %) and the SHOTOR diluent (33.3 % and 16.6 %) at 14 days post-insemination and 2 months of pregnancy, respectively (Table 2). With the insemination dose of 75 x 106motile sperm, no pregnancy was recorded with the semen diluted in SHOTOR diluent. Only one camel was diagnosed pregnant only at 14 days post-insemination with the semen diluted in Green buffer. However, insemination of the semen diluted in Mansour extender (75 x 106motile sperm) showed higher (P < 0.05 - 40%) pregnancy rate at both 14 days post insemination and 2 months of pregnancy compared to semen diluted in Green buffer.

[0119] - Example 2: Chilled semen

[0120] Example 2 shows an assay for testing the sperm motility and fertility of semen samples diluted in the three extenders, which assay is analogous to that of Example 1 , but for the fact that the semen samples diluted in the three extenders were cooled and kept chilled (at 5°C) until the moment of insemination, when they were warmed until 35°C before use. Ten semen samples were included in this experiment with three repetitions.

[0121] Cooling

[0122] The pre-diluted semen was further diluted 1 :2 with the same tested extender (Final dilution 1 :5; v:v semen: extender) and mixed by continuous pipetting. After 1 hour from semen collection, gradual cooling of the diluted semen was initiated: firstly, diluted semen was cooled gradually from 35 °C to room temperature (23 °C) in one hour to have a cooling rate of 0.2 degree / min; then, it was cooled from room temperature to 5 °C in two hours: cooling rate 0.15 degree / min. Then the semen was stored in a laboratory fridge at 5 °C.

[0123] After 1 , 2, 3, 5, and 7 days of the storage, the sperm motility was recorded. To record the sperm motility, semen sample was taken out of the fridge, mixed 3 times by pipetting, and 2 pl was pipetted on a 37 °C pre-warmed Leja Slide and motility was recorded as mentioned for Example 1.

[0124] Sperm fertility:

[0125] Fertility experiments were applied for chilled semen kept at 5 °C after 1 and 2 days of storage. Fertility experiments was done as described for Example 1 , except the insemination dose was 300 x 106motile sperm for the chilled semen.

[0126] Results

[0127] The results related to sperm motility after chilling are shown in Table 3 below.

[0128] Table 3. Total sperm motility of dromedary camel semen diluted in three extenders and kept at 5 °C for 7 days.

[0129] Period in SHOTOR Green buffer Mansour Extender days + 20% EY + 20% EY (including 20% CEY)

[0130] 1 11.9 ± 8.5a44.3 ± 12.6b59.5 ± 16.4C

[0131] 2 0.0a10.2 ± 2.6b42.3 ± 12.6C

[0132] 3 0.0a0.0a33.1 ± 11.5b

[0133] 5 0.0a0.0a16.5 ± 6.9b

[0134] 7 0.0a0.0a4.6 ± 3.7b

[0135] Values within a row with different superscripts are significantly different (P < 0.05).

[0136] EY: fresh egg yolk; CEY: clarified egg yolk

[0137] As can be seen in Table 3, sperm motility of chilled semen was low in SHOTOR extender after 1st day of incubation and completely stopped starting from the 2nd day of incubation. While semen diluted in Mansour extender showed higher (P < 0.05) motility rates after 1 and 2 days compared to semen diluted in Green buffer containing 20% egg yolk. There was no sperm motility in the Green buffer starting from the 3rd day of incubation. While, semen diluted in Mansour extender showed a motility throughout the 7 days of the experiment. The results related to fertility obtained with chilled semen, in turn, are shown in Table 4 below.

[0138] Table 4. Fertility of chilled dromedary camel semen (kept at 5 °C for 24 hours or

[0139] 48 hours) diluted in different extenders

[0140] Pregnancy at SHOTOR Green buffer Mansour Extender

[0141] Number (%) + 20% EY + 20% EY (including 20% CEY)

[0142] Time at chilling temperature (50C): 24 hours

[0143] 14 days 0 / 6 (0.0)a1 / 6 (16.6)a4 / 6 (66.6)c

[0144] 2 months - 1 / 6 (16.6)a3 / 6 (50.0)b

[0145] Time at chilling temperature (50C): 48 hours

[0146] 14 days 0 / 6 (0.0)a0 / 6 (0.0)a2 / 6 (33.3)a

[0147] 2 months - - 1 / 6 (16.6)

[0148] Values within a row with different superscripts are significantly different (P < 0.05). EY: fresh egg yolk. CEY: clarified egg yolk

[0149] As can be seen in Table 4, insemination of chilled semen diluted in SHOTOR extender kept for 1 and 2 days resulted in no pregnancies. The chilled semen diluted in the Green buffer containing 20 % egg yolk and kept 1 day resulted in one pregnant case (out of 6 inseminated camels) at 14 days post insemination and 2 months of pregnancy. While, there were 4 and 3 pregnant cases when using a 1-day chilled diluted semen in Mansour extender at 14 days post-insemination and 2 months of pregnancy. After 2 days of incubation at 5 °C, only the insemination of diluted semen in Mansour extender resulted in 2 and 1 (pregnancy rate of 33.3% and 16.6%) pregnant camels at 14 days postinsemination and 2 months of pregnancy, respectively.

[0150] The above results show that Mansour Extender gave rise to the best fertility results both when the semen diluted / extended with such extender was used fresh after collection (having been maintained at 350C) or within a short-term after collection (after having been maintained chilled, under refrigeration conditions, for at least 1 or 2 days and warmed shortly before being used for insemination), in comparison with other extenders used for camels, such as the SHOTOR extender of the Green buffer extender. References

[0151] Al-Bulushi S, Manjunatha BM, Bathgate R, Rickard JP, de Graaf SP. (2019). Artificial insemination with fresh, liquid stored and frozen-thawed semen in dromedary camels. PloS One 14:e0224992

[0152] Malo C, Crichton E, Skidmore JA. (2020). Preservation of the spermatozoa of the dromedary camel (Camelus dromedarius) by chilling and freezing: The effects of cooling time, extender composition and catalase supplementation. Theriogenology 153, 9-18

[0153] Mansour N. (2023). A novel, patented method for semen collection in dromedary camel (Camelus dromedarius). Reproduction in Domestic Animals 58: 238. https: / / doi.org / 10.llll / rda.14280

[0154] Mansour N., El-Ramah A., Silveira M.C., Bernardes L.A.M. (2022). An easy, safe, and practical method for semen collection in the dromedary camel (Camelus dromedarius). Emirates Journal of Food and Agriculture 34(4): 339-345. doi: 10.9755 / ejfa.2022. v34.i4.2850245.

[0155] Morton K, Billah M, Skidmore J. A (2013). Effect of sperm diluent and dose on the pregnancy rate in dromedary camels after artificial insemination with fresh and liquid-stored semen. Journal of Camelid Science. 6:49-62

[0156] Morton KM, Billah M, Skidmore JA. (2011). Effect of green buffer storage on the fertility of fresh camel semen after artificial insemination. Reproduction in Domestic Animals 46(3):554-7. doi: 10.1111 / j.1439-0531.2010.01678.x.

[0157] Nagy, P, Faigl, V., Reiczigel. J., & Juhasz. J. (2015). Effect of pregnancy and embryonic mortality on milk production in dromedary camels (Camelus dromedarius). Journal of Dairy Science, 98, 975-986. DOI: 10.3168 / jds.2014- 8546

[0158] Niasari-Naslaji, A., Mosaferi, S., Bahmani, N., Gharahdaghi, A.A., Abarghani, A., Ghanbari, A., 2006. Effectiveness of a tris-based extender (SHOTOR diluent) for the preservation of Bactrian camel (Camelus bactrianus) semen. Cryobiology 53, 12-21

[0159] Purohit, G. N., Vyas, S., Yadav, V., Nain, S., Chaudhary, A. K., Kumar, A., Dholpuria, S., Saraswat, C.S. (2023). Semen characteristics and artificial insemination in dromedary camels. Small Ruminant Research, 220, 106911 , https: / / doi.Org / 10.1016 / j.smallrumres.2023.106911

[0160] Skidmore J, Morton K, Billah M. (2013). Artificial insemination in dromedary camels. Animal Reproduction Science. 136(3): 178-86. https: / / d0i.0rg / l 0.1016 / j.anireprosci.2012.10.008

[0161] Skidmore JA, Malo CM, Crichton EG, Morrell JM, Pukazhenthi BS (2018). An update on semen collection, preservation, and artificial insemination in the dromedary camel (Camelus dromedarius). Animal Reproduction Science, 194, 11-18. https: / / doi.Org / 10.1016 / j. anire prosci.2018.03.013

[0162] Tibary, A., Anouassi, A., 1997. Theriogenology in Camelidae: anatomy, physiology, pathology and artificial breeding. Chapter VI. Reproductive physiology in the female Camelidae. Rabat, Morocco: Acetes Editions, pp. 169-239 1

[0163] Wani, N.A., Billah, M. & Skidmore, J.A. (2008). Studies on liquefaction and storage of ejaculated dromedary camel (Camelus dromedarius) semen. Animal Reproduction Science, 109, 309-318. https: / / doi.Org / 10.1016 / j.anireprosci.2007.10.011

Claims

CLAIMS1. A camel semen extender composition characterized in that it comprises: a) a buffer compound which isHEPES: 100-200 mmol / L b) a combination of the following sugars: a. lactose 30 - 90 mmol / L b. glucose 30-100 mmol / L c. galactose 10-50 mmol / l c) proteins selected from: a. the combination of i) catalase enzyme 20-100 lU / mL ii) reduced glutathione 0.5 - 3 mmol / L or b. the combination of i) catalase enzyme 20-100 IU ii) bovine serum albumin (BSA) 0.8-2.5g / L d) antibiotics selected from a. the combination of i) penicillin G 500-1500 lU / mL ii) streptomycin 250-750 pg / mL or b. gentamycin 500-2000 pg / mL and wherein the osmolarity is 320 - 370 mOsmol / L, and the pH is 6.9 - 7.5.

2. The camel extender composition according to claim 1, which comprises: a) HEPES: 130- 170mmol / L b) a combination of the following sugars: a. lactose 30 - 90 mmol / L b. glucose 30 - 100 mmol / L c. galactose 10-50 mmol / L c) proteins selected from: a. the combination ofi. catalase enzyme 40 - 60 IIJ / mL ii. reduced glutathione 1.0 - 2.0 mmol / L or b. the combination of i) catalase enzyme 40 - 60 IIJ / mL ii ) bovine serum albumin (BSA) 1.0 - 2.0 g / L d) antibiotics selected from a. the combination of i. penicillin G 500 - 1500 IIJ / mL ii. streptomycin 250 - 750 pg / mL or b. gentamycin 700 - 1500 pg / mL and wherein the osmolarity is 330 - 350 mOsmol / L, and the pH is 7.0 - 7.2.

3. The camel extender composition according to claim 1 or 2, which comprises: a) HEPES: 150 mmol / L b) a combination of the following sugars: a. lactose 65 mmol / L b. glucose 50 mmol / L c. galactose 30 mmol / L c) proteins: the combination of i. catalase enzyme 50 lU / ml ii. reduced glutathione 1.5 mmol / L d) antibiotics: the combination of i. penicillin G 1000 IIJ / mL ii. streptomycin 500 pg / mL and wherein the osmolarity is 330 - 340 mOsmol / L, and the pH is 7.0 - 7.1.

4. The camel extender composition according to any one of the preceding claims, which additionally comprises 20% volume / volume of clarified egg yolk.

5. A method for diluting and preserving camel semen to improve fertility, which comprises the steps of: a) preparing a camel semen extender composition of any one of claims 1 to 3, b) optionally adding clarified egg yolk to reach a concentration of 20% volume / volume with regard of the total volume of the final composition to a sample of the camel semen extender composition of step a), if the prepared semen is to be stored in the chilled form before use for at least 24 hours; c) collecting and selecting camel semen by: c.

1. preparing and selecting the female camels to be use for collecting semen; c.

2. preparing the perineal region of the selected female camels; c.

3. having ready for use a semen collection sac; c.

4. introducing a semen collection sac intravaginally in a female camel, c.

5. fixating the sac to the internal vaginal wall of the female camel, c.

6. securing the outer portion of the collection sac to the collection sac to the female’s perineal area, c.

7. taking the female camel to a male pen for natural mating and awaiting until the male is dismounted; c.

8. withdrawing the semen collection sac from the vagina, c.

9. cleaning the semen collection sac from vaginal secretions and squeezing it to collect the deposited semen within, c.

10. discarding semen samples contaminated with urine, faeces or sand, c.

11. selecting semen showing oscillatory activity of 2 and / or 3, where, 2 of moderate and 3 of rapid activity; d) transferring semen to a water bath at a temperature of 33°C-37°C; e) diluting the semen 1 :1 volume:volume with the camel extender composition prepared in step a); f) keeping the diluted semen obtained in step d) in the water bath for 45 minutes subjecting it to regular pipetting for homogenizing it and reducing its viscosity; g) determining the sperm cell concentration in a sample of the homogenized semen obtained after step f); h) diluting the homogenized semen obtained in step f) to have a final concentration of maximum 150 x 106sperm / mL with :h.1 the camel semen extender composition prepared in step a), if the prepared diluted semen is to be used within 2 hours for artificial insemination, or h.2 the camel semen extender composition prepared in step b), if the prepared diluted semen is to be stored for 24 hours or up to 3 days, in the chilled form at 4-6°C; i) maintaining in the water bath the diluted semen that is to be used in less than 2 hours or storing the diluted semen in the dark at 4-6°C for up to 3 days.

6. The method according to claim 5, wherein the camel extender composition of step a) is prepared by: a.

1. placing the required amounts of the sugars (lactose, glucose and galactose) and HEPES in a recipient; a.

2. adding double distilled water; a.

3. dissolving HEPES and the sugars by stirring; a.

4. adjusting pH to 6.9 - 7.2 by dropping 200 mmol / L sodium hydroxide solution with continuous stirring, a.

5. adding the antibiotics and mixing them with the other added components of the composition by stirring continuous, a.

6. readjusting pH to 6.9 -7.2 by dropping 200 mmol / L sodium hydroxide solution, if necessary, a.

7. adding the proteins selected from the combination of catalase enzyme and BSA or the combination of catalase enzyme and reduced glutathione, and dissolving them by stirring,7. The method according to claim 5, wherein the camel extender composition is prepared by adding and diluting subsequently the components in three different lots or parts, wherein the components of each lot or part have been packed and stored together and wherein the camel extender composition is prepared as follows i) placing in a recipient the components of Part A, dissolving them in double distilled water and adjusting the pH to 6.9 -7.2 by dropping 200 mmol / L sodium hydroxide solution with continuous stirring, ii) adding the components of Part B to the solution with Part A and dissolving them by stirring, iii) if the semen is to be stored refrigerated (at 4-6°C), adding Part C to the solution with Part A and Part B already dissolved,wherein-Part A is composed of HEPES, lactose, glucose, galactose, galactose and the selected antibiotics, all of them in powder form and have been stored in a closed sealed pack up to 1 year at room temperature,-Part B is composed of catalase enzyme and reduced glutathione, all of them in powder form, and has been stored in a small sealed pack up to 1 year in a frozen state, and- Part C is composed of lyophilized clarified egg yolk that has been stored up to 1 year in a frozen state and has been thawed until reaching room temperature before being added to the solution with the other components of the camel semen extender composition.

8. The method according to any one of claims 5 to 7, wherein, in step c): c.

1. female camels are selected for semen collection if they show a follicular activity of 1.1 - 1.6 cm in diameter, c.

2. the perineal region of the selected female camels is cleaned, webbed and disinfected, c.

3. the semen collection sac is made of flexible low-density polyethylene and comprises a tubular main part with an end designed to be the outer portion of the collection sac once inserted and with a gradually decreasing diameter until the other end where it bifurcates in two fingers designed to be introduced in the uterus, c.

4. the semen collection sac is introduced intravaginally in the female camel with the aid of a metal applicator sheathed inside the sac, c.

5. the sac is fixated to the internal vaginal wall through air insufflated from a pump connected to the distal end of the metal applicator, c.

6. the outer portion of the sac is fixed to the vaginal wall, after drawing the metal applicator out of the vagina, by a fixation sticker which secures the outer portion of the collection sac to the female’s perineal area from the dorsal and lateral sides of the vulva, c.

7. the camel males used for natural mating have not been used for natural mating or semen collection during the previous 3 weeks, c.

8. the fixation sticker is detached and the sac is withdrawn from the vagina manually, c.

9. the sac is detached from vaginal secretions and is squeezed manually,c.

10. the volume and the color of the collected semen is recorded and contaminated samples with urine, faeces or sand are discarded, c.

11. oscillatory semen activity is determined in an undiluted semen sample which is examined under the microscope at 100x magnification.

9. The method according to any one of claims 5-7, wherein the prepared camel extender composition of step a) is a composition according to claim 3.

10. The method according to any one of claims 5-9, wherein clarified egg yolk is added in step b), which clarified egg yolk has been prepared by the following sub-steps; b.

1. breaking fresh chicken eggs: b.

2. discarding albumen; b.

3. rolling egg yolk carefully on filter paper to remove albumen and chalazas adhering to the vitelline membrane; b.

4. perforating the vitelline membrane and collecting the egg yolk; b.

5. diluting egg yolk with the semen extender composition of claims 1-3 and distilled water in a ratio of: Freshly collected egg yolk: semen extender : distilled water 1 : 1.5 : 1.5 (volume : volume : volume) and stirring for 10 minutes, b.6 centrifuging the diluted egg yolk of substep b.6 at 10,000 x g for 30 minutes, b.7 collecting the supernatant, which is the clarified egg yolk, and storing it at - 40°C before use.11 . The method according to any one of claim 5-10, wherein the semen donor male camel belongs to the species Camelus dromedarius.

12. Use of a diluted semen prepared according to the method of any one of claims 5-11 for artificial insemination of a female camel.

13. The use according to claim 12, wherein the female camel shows no genital abnormalities, has at least an ovarian follicle of 12-16 mm in diameter and ovulation has been induced by injecting gonadotropin (GnRH) intramuscularly to the female camel 22- 26 hours before insemination.

14. The use according to claim 12 or 13, wherein the diluted semen is used within 2 hours from semen collection with a dose of at least 1 mL of diluted semen containing150 x 106motile sperm and it is deposited by the recto-vaginal technique at the tip of the uterine horn ipsilateral to the developed ovarian follicle.

15. The use according to claim 12 or 13, wherein the diluted semen has been maintained at 4-6°C, it is used after warming up to 35 °C and at least 2 mL of diluted semen containing 300 x 106motile sperm is deposited by the recto-vaginal technique at the tip of the uterine horn ipsilateral to the developed ovarian follicle.

16. The use according to any of the claims 12 - 15, wherein the donor male camel and the inseminated female camel are camels of the species Camelus dromedarius.

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