ITTO20120984A1 - PROCESS AND DEVICE FOR INSTRUMENTAL INSEMINATION OF BOVIDI, SPECIFICALLY FOR BUFALS - Google Patents

Description

â € œProcess and device for instrumental insemination of bovids, specifically of buffaloesâ €

â € œA process and a device for the artificial insemination of bovids, specifically of buffaloesâ €

DESCRIPTION

Technical field

The present invention refers to a process, and to the relative device, for the instrumental insemination, also known as artificial, of specimens belonging to the family of bovids (Bovidae) and, specifically, to the genus of buffaloes (Bubalus).

In particular, the present invention refers to a process, and to the relative device, for the instrumental insemination of bovids, specifically of buffaloes, in vivo with the use of cryopreserved seminal material.

Even more particularly, the present invention refers to a process, and to the relative device, for the instrumental insemination of bovids, specifically of buffaloes, in which the cryopreserved seminal material is deposited in the uterus in the form of microcapsules.

Note Art

Reproductive technologies and biotechnologies in the veterinary and zootechnical fields are widely used.

Instrumental insemination, specifically in buffaloes, is by now of consolidated use as it allows the improvement of hygienic-sanitary conditions in farms and the dilution of the ejaculate in several doses, thus reducing the number of males in the breeding and producing an economic benefit; it also allows for the control of the genetic patrimony of animal species, specifically those that are valuable and / or endangered.

Among the prized species, the Mediterranean buffalo has recently assumed importance, which is considered the most selected dairy buffalo and also suited to the production of meat; this species has a Herd Book established with the Ministerial Decree of 23 June 1980 and, with the subsequent Ministerial Decree no. 201992 of 5 July 2000, the Ministry for Agricultural, Food and Forestry Policies recognized the buffalo registered in said Herd Book belonging to its own breed: â € œMediterranea Italianaâ €.

Currently (Istat data, 2011) about 361,000 heads of Mediterranean buffalo are bred in Italy which there is interest in increasing numerically, keeping the species pure, for the zootechnical reasons highlighted above.

The genetic selection of bovidae mainly uses instrumental insemination within the company.

In the bovine species, conventional instrumental insemination, ie intrauterine instrumental insemination, is performed trans-cervically and involves the positioning of the seminal material in the uterine body; in order to reduce the number of spermatozoa needed for fertilization; It is essential that the spermatozoa are kept viable for a sufficiently long period of time after insemination.

Biotechnological methods aimed at improving the fertilization rate have been developed and described, for example, by Nebel and collaborators who have conveyed spermatozoa even cryopreserved into the uterine environment of cows, in microcapsules of calcium alginate and protamine, thus setting up matrix systems commonly referred to as â € œbeadsâ € (Nebel RL, Bame JH, Saacke RG and Lim F (1985); â € œMicroencapsulation of bovine spermatozoaâ €; Journal of Animal Science 60 (6): 1631-1639, 1985); able to produce a controlled release of bovine spermatozoa from the microcapsules ensuring an increase in fertility and prolificacy, however still not optimal in the case of cryopreserved seminal material very susceptible to deterioration.

In this context, research is still aimed at optimizing conservation procedures, specifically through cryopreservation.

Schenk JL, Cran DG, Everett RW and Seidel GE Jr's study entitled â € œPregnancy rates in heifers and cows with cryopreserved sexed sperm: effects of sperm numbers per inseminate, sorting pressure and sperm storage before sortingâ € and published in Theriogenology 2009, 71: 717-728, shows that, for cattle, the fertilization data obtained from traditional instrumental insemination procedures carried out with refrigerated semen are similar to those achieved with natural mating (80%), while through the use of cryopreserved sperm fertilization is 60%.

At present, therefore, the cryopreservation and thawing techniques still present some criticalities of use since the spermatozoa do not resist these treatments without altering, in particular without compromising the characteristics of quality, vitality and fertilization capacity.

The US Patent No. US 8,178,130 B2, issued on 15.05.2012 in the name of Kommisrud et al., Describes polymeric matrices, based on alginate, capable of supporting the fertilizing capacities of spermatozoa of various animal species such as, for example, bovine and porcine. The matrix systems set up, of spherical or cylindrical shape, can be used in the instrumental insemination procedure; the inventors also highlight the possibility of adopting polymeric particles only to immobilize, store and cryopreserve the spermatozoa, thus dissolving the matrix systems before insemination.

The aforementioned document solves the problem of improving the fertilization rate by increasing the shelf life and vitality of the collected spermatozoa, both during the storage period, both after thawing in the case of cryopreserved spermatozoa, and after insemination.

The aforementioned document, however, does not address the problem of carrying out instrumental insemination, specifically instrumental uterine insemination, combining the semen freezing technique with that of microencapsulation: and this, in particular, for the buffalo species.

In fact, the buffalo species has some physiological peculiarities which, despite having been the subject of in-depth studies and specific researches, have not up to now provided satisfactory information; the physiological peculiarities of the buffalo species consist in the fact that the buffalo does not show clear signs of the estrus period, which in itself is an obstacle to instrumental insemination, and in the fact that the collection of the seminal material of the buffalo is difficult and late that is, the semen is collected later than the other species).

Specifically, as demonstrated by the study conducted by De Rensis F and Lòpez-Gatius F entitled â € œProtocols for synchronizing estrus and ovulation in buffalo (Bubalus bubalis): a reviewâ € and published in Theriogenology 2007, 67: 209-216, the application of the techniques of instrumental insemination to the buffalo species has not, up to now, allowed to increase its reproductive efficiency.

As in part mentioned above, the factors that influence the low female reproductive efficiency in the buffalo species are the reduced ovarian activity during the summer season, the long time intervals necessary between the births and the late maturity of the females (see, for example, the study by Singh J, Nanda AS and Adams GP titled â € œThe reproductive pattern and efficiency of female buffaloesâ € and published in Animal Reproduction Science 2000, 60-61: 593-604).

Furthermore, factors such as the poor detection of estrus (30-40%), the variable duration of estrus (4-64 h) and the clinical and technological difficulties encountered in predicting the time of ovulation do not allow an easy application of instrumental insemination techniques to buffaloes, in a massive way, as illustrated by Ohashi OM in the study entitled â € œEstrous detection in buffalo cowâ € and published in Buffalo Journal 1994, 10: 61-64.

In follicular dynamics, there is the formation of 2-3 waves of development of the follicles; each follicular wave implies the development of a dominant follicle, the last follicular wave is however the only one that leads to the formation of an ovulatory follicle and to the estrus phase as described by Baruselli PS, Mucciolo R, Visintin GA, Viana VC, Arruda RP and Madureira EH in â € œOvarian follicular dynamics during the estrous cycle in buffalo (Bubalus bubalis) â €, published in Theriogenology 1997, 47: 1531 1547. To facilitate the management of estrus and optimize the moment of Ovulation hormonal protocols for the synchronization of oestrus and ovulation have been put in place, while a suitable solution has not yet been identified to guarantee the prolonged release of spermatozoa to cover the maximum time of the oestrus-ovulation period.

In conclusion, the need for a technological solution that allows a wider diffusion and efficiency of instrumental insemination as an ordinary reproductive practice in the buffalo species is therefore still unsatisfied and that allows, by virtue of the increased zootechnical interest in buffalo Mediterranean, to achieve the genetic improvement of the species and the consequent significant increases in production and quality of milk per head, as well as the reduction of the risk of infection and consanguinity.

Aims and Brief Description of the Invention

The present invention, starting from the information on the drawbacks and shortcomings of the known art, in the field of reproductive technologies and biotechnologies of the buffalo, intends to remedy them.

In particular, the present invention addresses the following problems which are still unsolved or solved in a way that is not yet complete and satisfactory by the previous technical solutions:

- allow the spread of instrumental insemination as an ordinary reproductive practice in the buffalo species;

- achieve the genetic improvement of the species and increase the production and quality of milk per head raised by virtue of the current zootechnical interest in the Mediterranean buffalo;

- increase the reproductive efficiency of the buffalo species, also with the use of cryopreserved seminal material;

- keep the spermatozoa viable for a sufficiently long period of time both after collection and after insemination;

- guarantee the prolonged release of spermatozoa near ovulation.

Therefore, the purpose of the invention is to develop a process, and the relative device, to facilitate, promote and spread instrumental insemination as an ordinary reproductive practice also in the buffalo species.

More precisely, the present invention defines and provides a process, and the relative device, capable of carrying out instrumental insemination, specifically uterine insemination, in buffalo by combining the cryopreservation technique of the semen with that of the controlled release based on microencapsulation. .

Another object of the present invention, achieved in parallel, is to provide a process, and the relative device, suitable for keeping the spermatozoa vital for the entire duration of ovulation, ensuring their reproductive capacity.

More precisely, the present invention provides a process, and the relative device, for instrumental insemination in vivo with the use of cryopreserved seminal material. A not least object of the present invention is to provide a process, and the relative device, suitable for guaranteeing the release of spermatozoa for the entire duration of vitality of the ovulated oocyte, allowing greater operational freedom regarding the precise detection of the best time of insemination.

More precisely, the present invention provides a process, and the relative device, for instrumental insemination in which the cryopreserved seminal material is deposited in the uterine horns of the female reproductive system in the form of microcapsules.

In view of these purposes, the present invention provides a process and the relative device having, respectively, the characteristics of the attached claims 1, 5 and 7, to which reference is made for the sake of brevity; moreover, the present invention also provides for the use of said process and of said device specifically for the instrumental insemination of buffaloes having the characteristics of the attached claims 9 and 10, to which reference is made for the sake of brevity.

Therefore, a first object of the present invention, possibly independent and usable autonomously with respect to the other aspects of the invention, is a process, and the relative device, to allow the diffusion of instrumental insemination as an ordinary reproductive practice in the buffalo species, specifically uterine instrumental insemination, combining the semen freezing technique with that of microencapsulation.

Therefore, another object of the present invention, possibly independent and usable autonomously with respect to the other aspects of the invention, is a process, and the relative device, designed to keep the spermatozoa viable for a sufficiently long period of time both after collection and after insemination, thus ensuring their reproductive capacity, for instrumental insemination in vivo with the use of cryopreserved seminal material.

Therefore, another object of the present invention, possibly independent and usable autonomously with respect to the other aspects of the invention, is the use of a process, and the relative device, suitable for guaranteeing the release of spermatozoa for the entire duration of the ovulated oocyte vitality, allowing greater tolerance in the identification of the moment of insemination, through which the cryopreserved seminal material is deposited in the uterus, in the form of microcapsules.

Therefore, another object of the present invention, possibly independent and usable autonomously with respect to the other aspects of the invention, is the use of a process, and of the relative device, for the instrumental insemination of buffaloes.

Further advantageous detailed technical characteristics of the process and of the relative device according to the present invention are described in the corresponding dependent claims.

The aforementioned claims, defined hereinafter, are intended as an integral part of the present description.

Description of the Figures

The present invention, together with its objects and advantages, will become more evident from the detailed description that follows, relating to preferred embodiments of the process and of the relative device claimed herein exclusively, given by way of example and by way of non-limiting example, with reference to the attached drawing tables, also provided for example only, in which:

- Fig. 1 is a diagram showing the average percentage membrane integrity, and the relative standard deviation, of four treatment groups according to the present invention;

- Fig. 2 is a diagram showing the mean percentage total motility, and the relative standard deviation, of four treatment groups according to the present invention;

- Fig. 3 is a diagram showing the mean percentage progressive motility, and the relative standard deviation, of four treatment groups according to the present invention;

- Fig. 4 is a diagram showing the average path velocity (VAP, Average Path Velocity) of the spermatozoa, and the relative standard deviation, of four treatment groups according to the present invention;

- Fig.5 shows a microcapsule according to the present invention;

- Figs. 6A and 6B show a first and a second variant of â € œpailletteâ € according to the present invention.

Detailed Description of the Invention

While the invention is susceptible of various modifications and alternative implementations, some of its embodiments will be described in detail below, in particular by means of some illustrative examples.

It should be understood, however, that there is no intention of limiting the present invention to the specific forms described but, on the contrary, the invention intends to cover all modifications, alternative and equivalent implementations that fall within the scope of the protection. € ™ invention as defined in the attached claims.

In the following description, therefore, the use of "for example", "etc." and "or" indicates non-exclusive alternatives without limitation, unless otherwise indicated.

The use of "also" means "including, but not limited to," unless otherwise indicated.

A preferred embodiment of the present invention relates to a process for the instrumental insemination of bovids, specifically of buffaloes, comprising the steps of: i. preparing microcapsules, or â € œbeadsâ €, (1) incorporating seminal material, the formulation of said microcapsules (1) being such as to allow their dissolution in vivo;

ii. prepare doses, or â € œpaillettesâ €, (10,100) including one or more of said microcapsules (1);

iii. freezing at least one of said doses (10,100);

iv. introduce at least one of said frozen doses (10,100) into the female reproductive system of the animal, specifically in the uterus of said reproductive system; v. allow the in vivo dissolution of said microcapsules (1) to take place, consequently obtaining fertilization.

Preferably and precisely the seminal material used belongs to the buffalo species.

With reference to Figs. 5, 6A and 6B which illustrate, respectively, a microcapsule (1) and a first (10) and a second (100) variant of â € œpailletteâ €, all the steps listed above are now described in detail.

Preparation of the microcapsules (â € œbeadsâ €)

The buffalo seminal material is diluted and added to a sodium alginate solution, preferably to a medium viscosity sodium alginate solution.

The dilution is preferably carried out with the BULLXcell <TM> diluent produced by IMV Technologies.

Preferably the diluted buffalo seminal material is preferably added in a 1: 1 ratio to a sodium alginate solution, preferably of medium viscosity, in a concentration approximately equal to 2% w / v and dissolved in BULLXcell <TM>.

Preferably the medium viscosity sodium alginate solution is manufactured by Sigma Aldrich.

For clarity, a medium viscosity sodium alginate solution has a viscosity of â ¥ 2,000 cP.

The suspension thus obtained is then extruded, preferably by means of hypodermic needles 32G, in a solution of BULLXcell <TM>, kept under preferably magnetic stirring and added with a saturated solution of barium chloride (BaC 2) such as to obtain bivalent ions Ba < ++> in a concentration between 10 and 75 mM, preferably between 25 and 60 mM, the optimum concentration being equal to 50 mM.

It is hereby intended to highlight that, according to the present invention, barium (Ba) is exclusively used since this element does not interfere with biological processes; in particular, unlike calcium (Ca), barium does not have a capacitating effect which is responsible for the early activation of spermatozoa and their equally early degeneration.

When each drop comes into contact with the BULLXcell <TM> and BaCl <2> solution, the divalent Ba <++> ion diffuses through the drop reacting with the alginate which thus gels.

The microcapsules thus formed are left under preferably magnetic stirring, preferably for 10 minutes, and subsequently collected and washed with BULLXcell <TM>.

Naturally, as will be evident to the expert in the field, the BULLXcell <TM> diluent used here preferably can be replaced with a diluent having equivalent chemical and physical properties; similarly, any other sodium alginate solution, preferably of medium viscosity, can be used instead of the one preferably indicated here and produced by Sigma Aldrich.

The spermatozoa are then trapped in a barium alginate gel matrix and the microcapsules (1) are thus prepared.

Preparation of doses (â € œpaillettesâ €)

After determining the average concentration of spermatozoa per microcapsule, carried out for each lot of microcapsules prepared, the number of microcapsules 1 to be loaded in each â € œpailletteâ € 10,100 is defined to obtain doses preferably from 20 million spermatozoa.

The suitable number of microcapsules (1) is then aspirated into the â € œpaillettesâ € (10,100), preferably by means of a suitable filling system, for example by means of a vacuum pump.

The concentration of spermatozoa in the ejaculate and in the microcapsules is preferably determined by an integrated system consisting of a fluorescence microscope, an image analysis system, an accurate calibrated sampler and a cell staining system intrinsic in the sampler. , for example ChemoMetec's NucleoCounter SP100 model. With this system, the spermatozoa, after a lysing treatment, are stained with propidium iodide, a dye capable of binding DNA and coloring all lysed cells red; the system then detects and counts the red spots in a fixed volume of suspension (spermatozoa and lysant).

Freezing of â € œpillettesâ €

The prepared â € œpaillettesâ € are subjected to a freezing protocol that guarantees the maintenance of the characteristics of quality, vitality and fertilization capacity.

After packaging, the sequins were placed on the ramps and placed in a showcase at 4 ° C for balancing, preferably lasting 3 hours. Freezing is carried out preferably using the Mini-Digitcool automatic freezer (IMV, France) preferably using a freezing curve that includes the following steps: from 4 ° C to -10 ° C with a speed of 10 ° C / min and from - 10 ° C to -110 ° C with a speed of 30 ° C / min. Once this temperature is reached, the sequins are transferred to a special container containing liquid nitrogen.

Dissolution of the microcapsules

The microcapsules contained in the â € œpaillettesâ €, once positioned inside the female reproductive system of the animal, specifically in the uterus, dissolve gradually, thus guaranteeing the release of spermatozoa for the entire duration the vitality of the ovulated oocyte and, consequently, a more effective fertilization of the animal.

Referring now to Figs. 1 to 4, the verification tests of the present invention will be illustrated; the characterization of the encapsulated seminal material, both before and after freezing, was carried out following the dissolution of the microcapsules carried out as will be illustrated below.

For greater clarity it is useful to specify that in said Figs. 1-4 shows, respectively, the mean percentage membrane integrity, the mean percentage total motility, the mean percentage progressive motility and the VAP (Average Path Velocity), and the relative standard deviations, of the following four treatment groups (n represents the sample size):

â € ¢ Control: semen diluted in BULLXcell <TM>,

â € ¢ Beads: semen diluted in BULLXcell <TM> and encapsulated,

â € ¢ Contr Scong: seminal material of the Control group subjected to a cryopreservation protocol and subsequently thawed,

â € ¢ Beads Scong: seminal material from the Beads group subjected to a cryopreservation protocol and subsequently thawed.

Determination of the membrane integrity

The spermatozoa were analyzed with the NucleoCounter SP100 system from the ChemoMetec company twice: the first after lysing treatment (in a similar way to the determination of the concentration), the second without lysing treatment. The difference between the two results gives the number of spermatozoa with intact membranes.

The results, shown in Fig. 1, demonstrate that the spermatozoa treated according to the present invention are not susceptible to deterioration and, consequently, guarantee an increase in fertility and prolificacy.

Evaluation of motility

The assessment of sperm motility was performed by means of the CASA system (Computer Assisted Sperm Analysis); the instrument used was the Hamilton-Thorne HTM-IVOS equipped with an integrated phase contrast microscope, with a negative phase contrast 10x objective and a stage thermostated at 37 ° C.

From the sample to be analyzed, 2 aliquots of 10µl of semen were prepared in two Makler <®> chambers, preheated to 37 ° C.

The analysis was performed on a minimum of four microscopic fields by analyzing at least 200 total spermatozoa.

Linear velocity (VSL) was measured, given by the distance between the first and last position taken by the spermatozoon (straight path) divided by time and the Average Path Velocity (VAP), given by the ratio between the length of the trajectory of the spermatozoon calculated by an algorithm using the moving average and time.

Total motility is the percentage of spermatozoa with a VAP greater than 25 µm / sec; progressive motility is instead the percentage of motile spermatozoa with a VSL / VAP ratio greater than 0.75.

The results, shown in Figs. 2, 3 and 4, show that the spermatozoa treated according to the present invention are vital and, consequently, guarantee the fertilization of the oocyte.

Dissolution of the microcapsules

As aforesaid, the characterization of the encapsulated seminal material, both before and after freezing, was carried out following the dissolution of the microcapsules.

The dissolution medium was prepared using an isotonic saline solution having a substantially neutral pH, preferably having a pH of 7.4, and comprising about 3% w / v of tribasic sodium citrate and about 5% w / v of EDTA.

The microcapsules were incubated with the above saline solution (preferably in a 2: 1 ratio) at 37 ° C for 40 minutes, under stirring.

The solution thus obtained was analyzed by means of the integrated microscopic fluorescence system / image analyzer / sampler, for example the NucleoCounter SP100 model by ChemoMetec, and by means of a CASA system, for example the HTM-IVOS model by the company. Hamilton-Thorne, in order to determine the concentration, membrane integrity and motility of the encapsulated semen.

Another aspect of the present invention relates to a process for the preparation of microcapsules, or â € œbeadsâ € incorporating seminal material comprising the steps of:

to. the seminal material is diluted and preferably added in a 1: 1 ratio to a sodium alginate solution, preferably to a medium viscosity sodium alginate solution;

b. the suspension thus obtained is extruded in a diluent solution, kept under preferably magnetic stirring and added with a saturated solution of barium chloride (BaCl <2>) such as to obtain bivalent ions Ba <++> in a concentration between 10 and 75 mM, preferably between 25 and 60 mM, the optimum concentration being equal to 50 mM;

c. when each drop comes into contact with the diluent and BaCl <2> solution, the divalent Ba <++> ion diffuses through the drop reacting with the alginate which thus gels;

d. the microcapsules (1) thus formed are left under preferably magnetic stirring, preferably for 10 minutes, and subsequently collected and washed with diluent, the spermatozoa thus remaining trapped in a matrix of barium alginate gel.

Preferably and precisely the seminal material used belongs to the buffalo species.

Another aspect of the present invention relates to a device for the instrumental insemination of bovids comprising a dose, or â € œpailletteâ €, comprising in turn one or more microcapsules, said dose preferably containing 20 million spermatozoa.

Preferably and precisely said device is used for the instrumental insemination of buffaloes, in which said at least one microcapsule incorporates buffalo seminal material.

On the basis of the foregoing, it is therefore understood that the process, and the relative device, object of the present invention, achieve the purposes and achieve the advantages mentioned above.

Finally, it is clear that numerous other variations can be made to the process, and to the relative device in question, without departing from the principles of innovation inherent in the inventive concept, just as it is clear that, in the practical implementation of the According to the invention, the materials, shapes and dimensions of the illustrated details may be any according to requirements, and may be replaced with other technically equivalent ones.

Where the construction features and techniques mentioned in the subsequent claims are followed by reference marks or numbers, these reference marks or numbers have been introduced with the sole aim of increasing the intelligibility of the claims themselves and, consequently, they do not present no limiting effect on the interpretation of each element identified, by way of example only, by such signs or reference numbers.

Claims (10)

CLAIMS 1. Process for the preparation of microcapsules, or â € œbeadsâ €, (1) incorporating seminal material of bovidae comprising the steps of: to. the seminal material is diluted and added to a sodium alginate solution; b. the suspension thus obtained is extruded into a diluent solution, kept under stirring and added with a saturated solution of barium chloride (BaCl <2>) such as to obtain bivalent ions Ba <++>; c. when each drop comes into contact with the diluent and BaCl <2> solution, the divalent Ba <++> ion diffuses through the drop reacting with the alginate which thus gels; d. the microcapsules (1) thus formed are left under stirring, preferably for 10 minutes, and subsequently collected and washed with diluent, the spermatozoa thus remaining trapped in a matrix of barium alginate gel. 2. Process according to claim 1, wherein the seminal material used belongs to the buffalo species. 3. Device for instrumental insemination of bovidae comprising a dose, or â € œpailletteâ €, (10,100) comprising in turn one or more microcapsules (1) prepared with the process according to claim 1, said dose containing 20 million spermatozoa. 4. Device according to claim 3 specifically for the instrumental insemination of buffaloes, in which said at least one microcapsule incorporates buffalo seminal material. 5. Use of the device according to claim 3 or 4 for the instrumental fertilization of buffaloes. 6. Process for instrumental insemination of bovidae including the phases of: the. preparing microcapsules, or â € œbeadsâ €, (1) incorporating seminal material, the formulation of said microcapsules (1) being such as to allow their dissolution in vivo; ii. prepare doses, or â € œpaillettesâ €, (10,100) including one or more of said microcapsules (1); iii. freezing at least one of said doses (10,100); iv. introducing at least one of said frozen doses (10,100) into a female bovid specimen; v. allow the in vivo dissolution of said microcapsules (1) to take place, consequently obtaining fertilization. 7. Process according to claim 6 specifically for the instrumental insemination of buffaloes, in which the seminal material used belongs to the buffalo species. 8. Process according to claim 7, wherein said buffalo seminal material is diluted and added to a sodium alginate solution; the suspension thus obtained is extruded in a diluent solution and in turn added with a saturated solution of barium chloride (BaCl <2>), thus obtaining the gelling of the alginate and thus producing said microcapsules (1) incorporating the spermatozoa. 9. Process according to claim 8, in which the average concentration of spermatozoa per microcapsule (1) is determined, the number of microcapsules (1) to be loaded in each â € œpailletteâ € (10,100) is defined to obtain doses of, for example, 20 millions of spermatozoa and aspirate the appropriate number of microcapsules (1) into the â € œpillettesâ € (10,100). Use of the process according to any one of claims 6 to 9 for the instrumental fertilization of buffaloes.