RU2438309C2 - Spermatozoid conservation and controlled delivery/release - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: this invention refers to animal breeding. Biopolymer particles for spermatozoid conservation include spermatozoids encapsulated in biopolymer gel mesh. Biopolymer includes alginate saturated with goluronic acids. To obtain particles, alginate and spermatozoid mix is added to gel-forming solution. Invention is used in animal breeding.

EFFECT: extended storage term and vitality of spermatozoids.

33 cl, 4 dwg, 5 tbl, 5 ex

Description

Field of Invention

The present invention relates to biopolymer particles for preserving sperm. The present invention also relates to a method for preserving, storing and controlled delivery / release of sperm and to the use of biopolymer particles according to the present invention in breeding animals.

State of the art

Artificial insemination (AI) is a method in which spermatozoa are placed in the uterus or cervix of an animal by artificial means more likely than by natural mating. It is widely used as a breeding method and in breeding animals to enhance the desired characteristics, in particular in the case of farm animals such as cattle, pigs, sheep, poultry and horses, but also in the case of pets, such as thoroughbred dogs, aquatic animals or endangered species.

Typically, sperm are harvested, diluted, and then preserved by, for example, cryopreservation. The use of cryopreservation methods suggests that spermatozoa from a particular animal species can withstand such treatment that does not result in too much damage to the quality of the sperm, their viability and ability to fertilize. Then the sperm are transported to the location of the females either in cryopreserved or fresh form, depending on what is suitable in this case. It is very important that the spermatozoa remain viable until insemination and for a sufficient period of time inside the female animal after insemination until the egg (s) reaches the point of fertilization.

Artificial insemination of farm animals has been used since the 1940s and is currently widely used in agricultural production, in particular for breeding dairy breeds of cows, as well as pigs. A review of the development of modern AI and the problems of breeders regarding the use of artificial insemination and sperm preservation is disclosed in RHFoote (2002), American Society of Animal Science (http://www.asas.org/symposia/esupp2/Footehist.pdf) and book "Reproduction in farm animals", edited by B. Hafez, ESEHafez. - 7th ed., Philadelphia, Lippincott Williams & Wilkins, 2000. - XIII, ISBN 0-683-30577-8 (ib.). Artificial insemination has become an important economic tool for breeding animals in agricultural production, both in relation to breeding animals with specific preferred genetic characteristics, and for livestock in general.

However, there are some restrictions on obtaining pregnancies in female animals as a result of artificial insemination. For example, the shelf life and viability of harvested spermatozoa, both during storage after thawing in the case of cryopreserved sperm and after insemination, are essential for a successful breeding result. The availability of suitable canning techniques for a particular animal species varies. For cattle, cryopreservation methods are widely used. On the other hand, spermatozoa from other species, such as pigs, are less tolerant to cryopreservation methods, resulting in less flexibility regarding the processing and storage of sperm.

In addition, in order to obtain spermatozoa with suitable fertilizing ability, it is desirable that the conservation method used also ensures that the fertilizing ability is maintained after insemination. A great deal of effort and research has been focused on canning methods aimed at providing storage methods and means that ensure that spermatozoa retain their fertilizing ability for a longer time after collection and until insemination.

If the shelf life regarding the preservation of the fertilizing ability after insemination is short, it is more difficult to find the right moment for insemination in relation to ovulation. In the case of a short shelf life property, good sperm preservation methods are needed that provide a longer shelf life and therefore a longer fertilization capacity. There is still no affordable preservation method that provides a sufficient shelf life and viability of spermatozoa for a sufficient period after insemination to meet the needs of breeders for flexibility of application, in particular when there is a long distance between the location of the males and, therefore, the place where the sperm is collected , and female recipients, and it takes time for transportation.

In addition, a preservation method that provides a more controlled and long-term availability of sperm would reduce the need for artificially stimulated ovulation by hormonal treatment. This would be preferable both economically, in accordance with the needs of the consumer, and in relation to animal health.

Therefore, there is a need for methods to ensure that sperm retain fertility for a longer time after insemination, and retain fertility for a longer time when they are placed inside a female recipient.

Currently, artificial insemination (AI) in cattle is carried out on a large scale using cryopreserved sperm. Cryopreserved sperm can be stored in liquid nitrogen for decades prior to use. However, when the sperm thaw, AI must be performed within a few hours. After insemination, cryopreserved spermatozoa have a fertilizing potential for approximately 12-24 hours, and AI must be performed approximately 12-24 hours before ovulation. Therefore, there is a need for preservation methods for breeding cattle, providing sperm that have the characteristics of a sufficient shelf life and which preferably retain the fertilizing ability for several days.

Some countries use bovine sperm stored in a liquid to reduce the number of sperm cells per dose of AI. Sperm have a fertilization capacity of approximately 24-36 hours before insemination. AI should be performed within approximately 24 hours after sperm collection. However, bovine liquid preserving sperm cells are burdened by some disadvantages, such as shortened / reduced shelf life and reduced distribution volume.

In pigs, cryopreserved spermatozoa are used only for special purposes, such as export, long-distance transportation, as well as for the control of infectious diseases. AI in pigs is usually done with liquid preservation sperm. The storage time of semen (sperm) liquid preservation should depend on which diluent is used. Spermatozoa diluted with short-acting diluents retain fertility for approximately 2–3 days, while sperm diluted with long-acting diluents can retain fertility for up to 5–6 days. After insemination, the fertilizing ability of the sperm lasts for about 12-24 hours. Most sows inseminate twice during heating at intervals of approximately 24 hours. With a more flexible system providing a longer storage time before insemination (e.g., one week) and / or prolonged storage and release of sperm after insemination (e.g., more than 24 hours), livestock production would have more efficient productivity and distribution, and livestock breeders would have less the need for precision insemination periodization relative to ovulation.

In horse breeding, the breeder mainly depends on the availability of freshly collected sperm due to the lack of acceptable conservation methods for equine sperm. This is a big problem in horse breeding and in the horse racing industry, since the preferred stallion producer for a particular breeding mare is most often located in another country, which requires a long transportation time. In addition, due to the lack of suitable conservation methods for equine sperm, AI of fresh equine sperm must be carried out within 24 hours after sperm collection. Horse breeding is thus accompanied by an undesirable time deficit, which often leads to reduced sperm quality or reduced fertilization due to incorrect insemination in relation to ovulation.

Therefore, there is a need for canning methods that guarantee both a longer shelf life in relation to the necessary time for transport delivery and a longer shelf life after insemination. The canning method applicable to horse sperm would be of great economic and practical value and would possibly lead to a revolution in horse breeding.

A sperm preservation system that provides sufficient viability both during storage and before and after insemination would be beneficial for livestock breeding in general. A more flexible canning system would make the work of breeding all kinds of animals easier and, as a result, would lead to an increase in successful fertilization. A system where the breeder is less dependent on matching the most preferred time of insemination with respect to ovulation provides greater flexibility.

In order to increase the fertilizing ability of the erupted sperm, several conservation methods have been investigated, including cryopreservation and liquid canning.

Several studies of sperm storage inside capsules have been published. Encapsulation methods have been used in these studies, which result in a particle where the sperm are located inside the fluid core surrounded by a semipermeable membrane.

Nebel et al. (1985), Microencapsulation of bovine spermatozoa. J. Anim. Sci. 1985 60 (6): 1631-39, described a method for encapsulating bovine sperm in capsules made from alginate in combination with polylysine. This method is based on a previously published method from Lim and Sun (1980), Microencapsulated islets as bioartificial endocrine pancreas. Science 1980 210 (4472): 908-10, which developed the production of encapsulated insulin based on the encapsulation of Langerhans islet cells. Nebel et al. (1985) reported the results of both storage at 37 ° C and 335 inseminations, and the results for encapsulated spermatozoa were compared with non-encapsulated control samples. In this study, large differences between the encapsulated and control samples were not reported. Other published studies have also demonstrated that sperm can be encapsulated inside capsules by similar methods and can retain their functionality in vivo (Munkittrick et al. (1992) Accessory sperm numbers for cattle inseminated with protamine sulfate microcapsules. J. Dairy Sci., 75 (3): 725-31 (Bovine), Vishwanath et al. (1997) Selected times of insemination with microencapsulated bovine spermatozoa affect pregnancy rates of synchronized heifers. Theriogenology, 48: 369-76 (Bovine), and Maxwell et al. (1996). Survival and fertility of micro-encapsulated ram spermatozoa stored at 5 ° C. Reprod. Dom. Anim., 31: 665-73 (Sheep). Munkittrick et al. (1992), and Vishwanath et al. (1997 ) reported, however, that the encapsulated sperm are not me lyayutsya as effective as untreated spermatozoa inseminated at one and the same time. This was attributed to the fact that sperm encapsulated may require some time for release from the capsules before fertilization can occur.

Conte et al. (1998) in EP 0922451 BI published by Universita di Pavia and Universita Degli Studi Di Milano, and Torre et al. (2002), Boar semen controlled delivery system: storage and in vitro spermatozoa release. J. Contol. Release, 85: 83-89, have developed an alternative method for encapsulating boar sperm. In this method, sperm are added to a solution containing calcium or barium, and this suspension is transferred dropwise into a solution containing alginate. A capsule of calcium or barium alginate spontaneously forms around the drop as soon as it enters the alginate solution. This method is claimed to be softer in relation to cells compared to the method described by Nebel et al. (1985). Another advantage is that a very small dilution of the sperm solution, which is claimed to be favorable for cell viability, is involved in this method.

Faustini et al. (2004), Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 ° C, Theriogenology, 61 (1): 173-184, reported a significantly larger fraction of spermatozoa with intact acrosome and less leakage of enzymes from sperm stored in an encapsulated state, where this method was compared with untreated sperm stored in the same conditions.

In a recent article by Weber et al. (2006), Design of high-throughput-compatible protocols for microencapsulation, cryopreservation and release of bovine spermatozoa. Journ. Biotechnol., 123: 155-163, also described a new system for encapsulating bovine sperm. This system is designed for the production of encapsulated spermatozoa with high throughput using capsules of Ca-alginate or cellulose sulfate and polydiallyldimethylammonium chloride (pDADMAC).

Finally, Chou et al., US 6596310 BI (2003), discloses a method for artificial insemination by time-controlled release of sperm from capsules or hard granules, where the sperm is stored in an uncapitated state by using an energy source that does not support capacitation.

Summary of the invention

The present invention is based on the surprising discovery that sperm entering into biopolymer matrices, where this biopolymer matrix is composed of alginate enriched with guluronic acid, results in spermatozoa with better conservation characteristics in terms of shelf life, viability and fertilization. Biopolymer particles and their sperm can, therefore, be used in artificial insemination of animals.

One of the non-limiting advantages of this biopolymer sperm preservation system is that it provides benefits by giving sperm fertility for a longer period after insemination and thus makes the insemination time less ovulating than critical.

The biopolymer particles according to the present invention can be used in animal breeding and in livestock production in agricultural production. Biopolymer particles are thus applicable when the goal is to obtain animals with particularly desirable characteristics. Biopolymer particles are also applicable when the goal is to obtain animals in general, for example, for the production of cattle in the meat direction.

According to the present invention, the biopolymer particles can be used directly and seeded as such by the recipient animal if the biopolymer particles, such as alginate particles, are dissolved under physiological conditions inside the recipient animal (i.e., inside the uterus or cervix). The biopolymer particles of the present invention thus provide controlled release of sperm. Sperm can also be dissolved from a biopolymer particle before insemination of the sperm.

As discussed above, prior art generally proposes encapsulation of sperm in biopolymer capsules, where the sperm are contained in the liquid core of the biopolymer capsule. Only one reference (Chou et al., US 6,596,310 BI) mentions the possibility of enclosing spermatozoa in a solid polymer matrix, although not a single example of the hard granules described therein is provided.

Instead, the authors of the present invention have discovered another approach to sperm immobilization. According to the present invention, sperm cells are enclosed within a solid gel network formed by alginate gels, where the alginate used is enriched in guluronic acid. The authors of the present invention made the discovery that immobilization inside a solid gel network has several advantages compared to encapsulation inside capsules and granules disclosed in the prior art.

Without being bound by a specific theory, it is believed that by using biopolymer particles and the method according to the present invention, this conclusion results in immobilization, for example, in limiting the natural mobility of sperm due to restrictive bonds of the gel network. Physical restriction of motility due to high cell concentrations and the presence of high viscosity polymers may be one of the factors that affects the survival and maintenance of sperm function in the tail of the epididymis (Watson 1993). Sperm cells are stored for extended periods of time (more than 1 week) in the tail of the epididymis (Watson 1993). The use of alginate enriched with guluronic acid makes it possible to obtain biopolymer particles containing spermatozoa contained in them, which are useful in practice as a preservation system, and which can be used in artificial insemination, for example, in breeding animals.

Thus, according to one aspect, the invention provides a biopolymer particle for preserving spermatozoa, where the sperm are enclosed in a biopolymer gel network, and where the biopolymer particle in which the sperm is enclosed contains alginate enriched in guluronic acid.

According to another aspect of the present invention, a sperm-containing biopolymer comprises calcium alginate.

According to another aspect, said biopolymer comprises a low viscosity alginate.

According to yet another aspect of the invention, the concentration of alginate in the biopolymer particles of the invention is at least 0.1%.

According to yet another aspect of the invention, the concentration of alginate in the biopolymer particles of the invention is between at least 0.1% and 6% of alginate.

According to yet another aspect of the invention, the concentration of alginate in the biopolymer particles of the invention is at least 1%.

In addition, according to one aspect of the invention, the concentration of sperm in biopolymer particles is at least 0.1 × 10 ⁶ sperm / ml, such as at least 100 × 10 ⁶ sperm / ml, such as up to at least 2.5 × 10 ⁹ sperm / ml.

According to yet another aspect, the particles of the present invention are stored in a solution, for example, where the ratio of storage solution: biopolymer particles is at least between 1: 1 and 1: 100.

In yet another aspect, the sperm can be enclosed in conjunction with compounds or agents that are useful in the sense of fertilizing ability and / or animal health, such as, for example, one or more of one of the compounds or agents selected from a non-limiting group of diluents , cryoprotectants, antibiotics, antibodies, antioxidants, proteins and hormones.

According to another aspect of the invention, spermatozoa are enclosed together with one or more antioxidants selected from the group consisting of pyruvate, 2,2,6,6-tetramethylpiperidin-1-oxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidine -1-oxyl, superoxide dismutase, catalase, glutathione peroxidase, butylated hydroxytoluene, butylated hydroxyanisole (guaiacol).

In one aspect, the biopolymer particles are coated. This shell may be selected from a non-limiting group consisting of polylysine, chitosan, cellulose sulfate, hydroxypropyl methyl cellulose and polydiallyldimethylammonium chloride.

The biopolymer particles of the present invention comprise sperm collected from an animal selected from a non-limiting group consisting of pigs, cattle, horses, sheep, goats, rabbits, poultry, pets, such as thoroughbred dogs, aquatic animals, and endangered species animals, preferably pigs, cattle, fur animals and horses. The biopolymer particles of the present invention can thus be used in pregnancies in female animals of the same species using conventional methods of artificial insemination, such as AI, IVF (in vitro fertilization), and VTsIS (intracytoplasmic sperm injection).

According to one aspect of the invention, the sperm used to form said particle are contained in seminal fluid.

Perhaps the biopolymer particle can be further processed by dehydration, cryopreservation or lyophilization.

The invention also provides a method for producing particles according to the invention, in which a mixture of guluronic acid enriched alginate and sperm is added dropwise to a gelling solution, the gelling solution according to one aspect of the invention contains one or more of the following ions selected from a non-limiting group, consisting of calcium, sodium, barium and magnesium, preferably calcium and sodium ions.

In addition, biopolymer particles can optionally be formed directly in the seed container.

According to one aspect of the method of the invention, the particles are further processed by dehydration, cryopreservation or lyophilization.

The present invention also provides the use of biopolymer particles according to the present invention in breeding animals such as pigs, cattle, horses, sheep, goats, rabbits, poultry, pets, such as thoroughbred dogs, aquatic animals and endangered species. According to one aspect of the invention, biopolymer particles are used in breeding pigs, cattle or horses. In one aspect, the particles are seeded directly. According to another aspect, the particles are dissolved before insemination. In yet another aspect, the particles of the invention are used together with free, immobilized sperm.

Finally, the present invention provides a method for fertilizing an animal, in which spermatozoa preserved in the particles of the present invention are administered to a female recipient of the animal.

A brief description of the graphic materials

Figure 1 shows the in vitro storage of bovine sperm (panel A) and boar (panel B) at ambient temperatures (bull 20 ° C, boar 18 ° C). Mobility values are given as functions of storage time in comparative samples and in samples with immobilized sperm.

Figure 2 shows the in vitro storage of bovine sperm (panel A) and boar (panel B) at 37 ° C. Mobility values are given as functions of storage time in comparative samples and in samples with immobilized sperm.

Figure 3 shows in vitro storage of sperm at ambient temperatures (18 ° C). Mobility values are given as functions of storage time in comparative samples and in samples with immobilized spermatozoa. In storage experiments, a different amount of storage medium was used relative to the number of granules, as indicated by the batch number. Sperm cells are immobilized on granules with a diameter of 1 mm at a concentration of 1 × 10 ⁹ sperm cells / ml.

Figure 4 - storage in vitro of boar sperm at ambient temperature (18 ° C). Mobility values are given as functions of storage time in comparative samples and in samples with immobilized sperm. In storage experiments, granules with different concentrations of immobilized spermatozoa were used, as indicated by the batch number. Sperm cells are immobilized on granules with a diameter of 3 mm.

DETAILED DESCRIPTION OF THE INVENTION

As described previously, research has previously focused on sperm encapsulation in order to facilitate in vitro fertilization and artificial insemination. The inventors have taken a fundamentally different approach to sperm immobilization, where spermatozoa are enclosed inside a gel or biopolymer network inside a solid gel particle, and where the biopolymer network consists of alginate enriched in guluronic acid. This approach is fundamentally different from the encapsulation of spermatozoa previously described in the prior art, where the sperm are contained inside a liquid core capsule, where the sperm can make circular movements, as in their natural environment. The sperm-containing biopolymer particles of the present invention are independent of the use of solutions, which ensures that the sperm are maintained in a non-capacitated state.

The term “enclose” or “enclosing,” as used herein, should be understood as immobilization of spermatozoa, resulting in preventing the natural possibility of sperm movement. The degree of immobilization should vary depending on the characteristics of the biopolymer particles, such as mechanical strength, and on the type of polymer used. However, it should be understood that the natural possibility of movement of the sperm contained in the biopolymer particles of the present invention, which the sperm would otherwise have possessed if stored in a liquid, such as a liquid capsule core, was prevented.

The term "biopolymer particle", as used here, means a particle, which, when it contains sperm, provides reduced ability to move. The biopolymer particles of the present invention are comprised of a network forming material consisting of alginate gel enriched with guluronic acid. The function of the biopolymer particle with respect to sperm preservation is independent of the three-dimensional shape of the biopolymer particle according to the present invention. Thus, the biopolymer particles according to the present invention can have various shapes, such as, for example, a spherical or cylindrical shape.

Alginate is a polymer that consists of guluronic acid (G) and mannuronic acid (M). Alginate is a common component of the cell wall in all types of brown algae (Phaeophyceae), and is usually extracted with an alkaline solution. The ratios of mannuronic acid to guluronic acid (M / G) in alginate vary widely depending on specific alginophytes (brown algae producing alginate) and over different seasons.

Alginate is also synthesized by the bacteria Pseudomonas and Azotobacter (Svanem et al. (2001), The Journal of Biological Chemistry, Vol 276: 34, 31542-31550, Jain et al. (2003), Molecular Microbiology, 47 (4), 1123-1133 , Scott and Quatrano (1982), Applied and Environmental Microbiology, 44: 3, 754-756).

Alginates are widely used, for example, in the food industry, as, for example, as stabilizers and for regulating viscosity, in the pharmaceutical and cosmetic industries as, for example, a baking powder. For various purposes, both alginates enriched in guluronic acid and mannuronic acid are available, respectively (Mancini et al., (1999), Journal of Food Engineering 39, 369-378), and various methods for producing alginates enriched in guluronic acid are known (see WO 8603781, US 4990601, US 5639467).

The term "alginate enriched in guluronic acid" or "G-enriched alginate", as used here, means alginate containing higher amounts of guluronic acid than mannuronic acid in the polymer chains that make up the polysaccharide. Examples of the understanding of the term “G-enriched” as commonly used by those skilled in the art are apparent from the prior art set forth in the two preceding paragraphs.

Alginate gels are formed due to interactions between divalent ions, such as Ca ²⁺ , and block the structure of guluronic acid in the polymer chain of alginate. Thus, the formation of alginate gels can be carried out under very mild conditions, and it is suitable for immobilizing cells. Alginate gels are thus widely used to immobilize various types of cells. However, immobilization in alginate is most often used as a starting point for the subsequent formation of various types of liquid core capsules.

When referring hereinafter to alginate, alginate gel, alginate network or biopolymer particle according to the present invention, it should be understood that said alginate, alginate gel, alginate particle or biopolymer network or particle according to the present invention is enriched with guluronic acid.

Non-limiting examples of suitable types of alginate are FMC LF 10/40, FMC LF 10/60 and FMC LF 20/60, available from FMC Biopolymer AS, Drammen, Norway, or A2033 from Sigma, Oslo, Norway.

The term "shelf life", as used here, means the time during which the sperm retain fertility, both in terms of storage in vitro before insemination, and in vivo after insemination in the recipient animal. The specific shelf life of spermatozoa of different origin may vary. However, the present preservation system, where the spermatozoa are encapsulated in biopolymer particles according to the present invention, provides a longer shelf life after insemination compared to the inseminated sperm after conventional storage methods after sperm collection. In accordance with one example of the invention, the porcine sperm retain excellent fertility for more than 12 hours after insemination, more preferably more than 24 hours after insemination. According to yet another example of the invention, bovine spermatozoa retain excellent fertilization ability for more than 12 hours after insemination, more preferably more than 24 hours after insemination.

The term "breeding", as used here, means any method used to achieve pregnancy in a female animal. A non-limiting list of such methods includes IVF, artificial insemination and VTsIS. In addition, breeding encompasses both breeding in relation to obtaining animals with particularly desirable characteristics, and in relation to commercial products.

The term "sperm", as used here, includes sperm as such, as well as sperm contained in seminal fluid, that is, sperm can be used directly in the formation of biopolymers according to the present invention. However, sperm isolated from seminal fluid, possibly contained in other suitable storage solutions, can also be used to form biopolymer particles according to the present invention.

The term "seed container", as used here, includes all types of packages suitable for containing and storing sperm.

The prior encapsulation methods disclosed in the prior art and such as discussed above are labor intensive, and immobilization methods typically comprise several stages. However, the preparation of biopolymer particles according to the present invention can be prepared in a one-step method. More precisely, calcium alginate particles containing spermatozoa are easily formed with minimal physiological and chemical stress on immobilized cells. Alginate particles of various sizes and shapes and with varying types of alginate, alginate concentrations and immobilized sperm concentrations can be obtained.

For the purpose of concluding spermatozoa according to the present invention, the use of alginate as a biopolymer material is particularly suitable due to the fact that i) gels can be formed under mild conditions, ii) alginate is non-toxic to both spermatozoa and the recipient animal, and iii) alginate dissolves under physiological conditions and, therefore, is able to release sperm inside the uterus or cervix.

According to one aspect of the present invention, alginate particles are prepared by adding a solution containing alginate and spermatozoa dropwise to a gel-forming solution, resulting in the formation of alginate granules with spermatozoa enclosed therein.

The preparation of alginate granules is known to those skilled in the art, for example, as disclosed in Smidsrad, O. and Skjak-Brak, G. (1990) Alginate as immobilization matrix for cells. Trends in Biotechnology 8, 71-78, and in US 6,497,902.

Various gel solutions can be used to form biopolymer particles, such as alginate granules. Various solutions that are suitable for the formation of various biopolymer particles are within the knowledge of those skilled in the art. According to the present invention, divalent ions (also referred to as gelling ions), such as calcium and barium, can be used to form alginate particles, for example, to achieve gelling of alginate. Alginates form gels in the presence of many divalent ions and multivalent ions. The use of calcium ions results in rapid gelation and relatively strong particles. On the other hand, the use of barium ions results in even stronger particles that are more stable under physiological conditions. It should be understood that the type and amount of gelling ion to be used to form biopolymer particles according to the present invention can vary, inter alia, with the desired gel strength, type and concentration of the alginate used, guluronic acid content and G-block length alginate, the type and source of sperm used, the type of additional agents that need to be included in the particles (such as antibiotics, diluents, antioxidants, etc.), and so e modifications are within the scope of the present invention. Based on the prior art and on the guidance of the present description, one skilled in the art should identify and determine, without undue stress, various compounds that can be co-contained with sperm in biopolymer particles according to the present invention.

According to one aspect of the present invention, calcium ions and sodium ions are used to form biopolymer particles. Thus, by varying the concentration of alginate and the degree of crosslinking, it is possible to obtain particles with different characteristics of sperm release and those that, for example, are adapted to a particular animal species or to specific ovulation conditions in various animals.

In addition, the concentration of alginate affects the mechanical characteristics of biopolymer particles and, therefore, also the characteristics of the dissolution of particles. The concentration of alginate may thus vary depending on the dissolution characteristics required in each case depending on, for example, the recipient animal. A person skilled in the art, based on general knowledge and based on the guidance provided here and without undue burden, will determine the various applicable concentrations of G-rich alginate for use in the preparation of the biopolymer particles of the present invention. According to one aspect of the invention, the concentration of alginate in the biopolymer particles according to the present invention is between at least 0.1 and 6%.

The concentration of sperm contained in the biopolymer particles according to the present invention may vary, for example, depending on the type / source of sperm, breed, recipient animal, insemination techniques or system, fertilization techniques or system, the presence of other agents included in the particles (antibiotics, antioxidants, diluents, proteins, etc.). In principle, there is no lower limit for the concentration of sperm, and this concentration may vary depending, for example, on the method of fertilization, the origin of the sperm, the recipient animal, the dissolution characteristics of biopolymer particles, etc. With the guidance of the present description and general knowledge, a person skilled in the art should, without undue burden of experimentation, determine a suitable sperm concentration, which should be used in each case based on the desired method of fertilization, the origin of the sperm, the recipient animal, etc. It should be borne in mind that when using VTsIS, lower sperm counts can be used as the desired method of fertilization compared to, for example, artificial insemination.

According to one aspect of the invention, the sperm concentration is thus at least 0.1 x 10 ⁶ sperm / ml.

The discoveries of the present invention show that for some animals, sperm survival is highly enhanced with increasing sperm concentration. Therefore, in accordance with one example of the present invention, at least 2.5 x 10 ⁹ porcine sperm / ml enclose in a biopolymer particle (see figure 4). However, 100 × 10 ⁶ pork sperm / ml also result in pregnancies in female animals. For bovine spermatozoa, the same concentration range is applicable as for porcine sperm.

The sperm concentration in biopolymer particles can also be modified by changing the sperm count relative to the amount of alginate before gelation, or by concentrating or diluting the sperm solution before mixing the alginate solution and the sperm containing solution. However, since the modification of the sperm count ultimately leads to the modification of the concentration of alginate in the obtained particles, the concentration of alginate in the initial solution of alginate must be adjusted if the concentration of alginate in the biopolymer particles should be maintained. Due to the high viscosity of alginate in solution, in practice it may be difficult to use an alginate solution with more than 4-6% alginate. Thus, in one aspect of the invention, the biopolymer particles are composed of low viscosity alginate.

In addition, the sperm contained in the biopolymer particles of the present invention are somewhat isolated from the environment and are not released until the calcium alginate gel network dissolves. In addition, the calcium alginate gel network dissolves slowly under physiological conditions, which causes the slow release of sperm from the immobilization matrix. Due to this effect, sperm with a fertilizing ability may be present for longer periods of time. Dissolution rates can be controlled using various types of coatings on the immobilization matrix. Various useful coatings are known, including, but not limited to, polylysine, chitosan, cellulose sulfate, hydroxypropyl methyl cellulose or polydiallyldimethylammonium chloride.

To further improve sperm quality, sperm can be combined with various solutions that are important for sperm survival and viability. According to one embodiment of the invention, a diluent is included in the biopolymer particle of the present invention.

Sperm thinners are commonly used for several purposes:

- to increase the volume

- to standardize the number of sperm in a dose of AI

- to obtain a buffer capacity

- to provide a physiological environment in relation to osmolarity

- to maintain sperm cell nutrition

- to control microbiological infection (antibiotics), or in the case of cryopreservation

- to protect sperm cells from damage during freezing and thawing.

It is within the knowledge of those skilled in the art to select an appropriate diluent that can be used in biopolymer particles according to the present invention, based, for example, on the type (origin) of spermatozoa, type, method of preservation, storage temperature, method of insemination, zoosanitary requirements and international law . Dairy diluents and Tris are just a few non-limiting examples of diluents among the wide variety of applicable commercially available sperm diluents. Other useful diluents are those described by Aalbers, JG, Rademaker, JHM, Grooten, HJG and Johnson, LA, 1983: Fecundity of boar semen stored in BTS, Kiev, Zorlesco, and Modena extenders under field conditions. J. Anim. Sci. 75 (Suppl. 1), 314-315, and Aalbers, JG, Johnson, LA, Rademaker, JHM and Grooten, HJG, 1984: Use of boar spermatozoa for Al: fertility and morphology of semen diluted in BTS and used for insemination within 24 hrs or 25 to 48 hrs after collection. Proc. 10th Int Congr. Anim. Reprod and Artif. Insemin. Urbana IL, Vol II, No. 180, pp 1-3, commonly known as BTS. BTS is commercially available from Minitub i Tyskland (Minitub Abfull und Labortechnik GmbH & Co.KG, Hauptstrasse 41, DE-84184 Tiefenbach, Germany).

Another useful diluent is the Tri X-Cell ™ diluent commercially available from IMV (Instrument de Medecine Veterinaire, 10, rue Georges Clemenceau, B.P. 81, FR-61302 L'AIGLE Cedex, France).

Different diluents vary in composition, pH, buffer capacity, osmolarity and antibiotics. Many are published, while others are trade secrets. The simplest diluents consist only of various solutions of sugars, such as, for example, lactose and glucose. Based on the general knowledge of those skilled in the art in the field of sperm preservation, it should be understood that various diluents can be used according to the present invention. The types of diluents used according to the present invention may vary depending on the origin of the sperm, the type of polymer used, the preservation method, storage temperature, etc. It is within the knowledge of those skilled in the art to determine and select the type and suitable amount of diluent used without deviating from the scope of the invention disclosed in the description and the attached claims.

According to the present invention, biopolymer particles of various sizes can be formed in accordance with methods known in the prior art. Which size is suitable depends on various factors, such as the source of sperm, the type and size of the fertilization device used, the size and shape of the seed container used, etc. The biopolymer particles according to the present invention can be formed by a diameter suitable for all prior art insemination methods to ensure ease of handling, handling, transportation and preservation.

According to yet another aspect of the invention, biopolymer particles are formed inside a container, such as, for example, an insemination tube, resulting in particles perfectly adapted to the size of the container. Thus, in the case of the formation of biopolymer particles inside the container, there may not be a need for any storage solution.

In one aspect of the invention, the biopolymer particles comprise sperm collected from cattle. According to yet another aspect of the invention, the present biopolymer particles comprise sperm collected from pigs.

Although the present invention is illustrated with alginate particles containing spermatozoa collected from cattle and pigs, respectively, sperm from other animal species can also be enclosed in biopolymer particles. Without limitation, spermatozoa collected, for example, from horses, sheep, goats, rabbits, poultry, pets such as thoroughbred dogs, aquatic animals and various endangered species can also be enclosed in biopolymer particles.

In addition, there are no indications that true biopolymer particles cannot be useful in preserving human sperm, and therefore are useful in the treatment of infertility. Thus, it should be understood that the term “animal,” as used herein, also encompasses humans.

In addition, sperm preservation methods are also widely required in fish farming. Thus, spermatozoa derived from aquatic animals can also be enclosed in biopolymer particles according to the present invention, and therefore they are also encompassed by the term “animal” as used herein.

Sperm immobilization in a biopolymer gel network can also be used to create a microenvironment that is favorable for storing sperm even inside the female genital organs. This can be done with additional solutions for joint conclusion, which are favorable in relation to sperm, the degree of fertilization, animal health, etc. For example, non-limiting examples of substances that increase viability during storage are antioxidants or proteins, hormones, etc. Sperm can also be co-concluded with other agents or compounds that are favorable in light of the fertilizing ability or health of the animal. Such agents or compounds can be, for example, antibiotics, such as, for example, streptomycin, neomycin, gentamicin, penicillin, lincomycin, spectinomycin, amoxicillin, tylosin, etc., which can be used to control or prevent sexually transmitted diseases, cryoprotectants, antibodies, hormones or compounds that increase reproductive efficiency, for example, such as compounds known from US 5972592.

Thus, according to yet another aspect of the invention, biopolymer particles contain, in addition to spermatozoa, compounds that are favorable to the fertility or health of the animal, such as, for example, antioxidants, antibiotics, antibodies, hormones or agents that increase reproductive efficiency, or combinations. According to one preferred embodiment of the present invention, the biopolymer particles comprise antioxidants, for example, such as pyruvate.

The biopolymer particles of the present invention may possibly be stored in well-known sperm storage solutions, such as, for example, milk diluents, PBS (phosphate buffered saline), BTS, Tri X-Cell ™, EDTA diluents, Kiev, Modena diluents , MR-A, Androhep, Acromax, Triladyl®, Biladyl®, Bioxcell, Biociphos plus, etc., containing or not containing antioxidants, such as, for example, pyruvate, 2,2,6,6-tetramethylpiperidin-1- oxyl (usually abbreviated as Tempo), 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (usually abbreviated as Tempol), superoxide dismutase, catalase, glutathione peroxidase, butylated oxytoluene (usually abbreviated as BHT), butylated hydroxyanisole (usually abbreviated as BHA). The choice of storage solution is not critical as far as this storage solution does not contain compounds that can adversely affect the characteristics of biopolymer particles. For example, in the case of alginate particles, the storage solution should not contain too large amounts of chelating agents, resulting in the removal of divalent ions and, therefore, the dissolution of the gel.

During storage, the proportion of biopolymer particles relative to the storage solution may vary depending on the type and concentration of G-rich alginate, sperm source, the method of fertilization used, etc. In accordance with the examples of the present invention, the ratio of the storage solution to the biopolymer particles is at least 1: 1, such as at least 1: 2, at least 1: 3, at least 1: 4, at least 1 : 5, at least 1: 6, at least 1: 7, at least 1: 8, at least 1: 9, or at least 1:10. According to the present invention, a ratio of storage solution to biopolymer particles of up to at least 1: 100 can be used.

In addition, the biopolymer particles according to the present invention can possibly be stored both at ambient temperature, for example, at 18 ° C or 20 ° C at physiological temperature (37 ° C), and under cooling conditions, for example, at 5 ° C . In addition, biopolymer particles can be further processed by dehydration, cryopreservation or lyophilization. Then, in the case of cryopreservation, the biopolymer particles are thawed before insemination.

Sperm, which are usually present in a liquid, can be stored for a long period of time when enclosed in a biopolymer particle according to the present invention. According to another embodiment of the invention, the biopolymer particles are used directly to inseminate the recipient animal. Perhaps biopolymer particles can be dissolved to release sperm before insemination. However, the biopolymer particles can also, according to yet another embodiment, be used together and in combination with free sperm.

Although the invention has been described in connection with its specific embodiments, it should be understood that it is capable of further modifications. It is intended that this application cover any variations, uses, or adaptations of the invention, generally following the principles of the invention and including those deviations from the present description that come from well-known and accepted practice in the field of technology to which the invention belongs, and which can be applied to the essential features disclosed above, and in accordance with the scope of the attached claims.

The above description of various aspects of the present invention reveals the general nature of the invention, and one skilled in the art, by applying general knowledge in the field of artificial insemination and breeding technology (including the contents of the references cited here), easily modifies and / or adapts the present biopolymer particles and their preparation and use for various applications without unnecessary experimentation, without deviating from the general concept of the present invention and the scope of the attached claims. Therefore, it is understood that such adaptations or modifications are within the meaning of a number of equivalents of the disclosed embodiments, based on the provisions and guidelines provided herein. It should be understood that the terminology used here is for description purposes and not limitation. Thus, the terminology of the present application should be interpreted by a person skilled in the art in light of the provisions and guidelines presented herein, in combination with the knowledge of those skilled in the art.

The following non-limiting examples further illustrate the present invention.

Examples

Example 1 Immobilization of sperm in alginate

MATERIALS AND METHODS

Materials

The following chemicals were used: CaCl ₂ .H ₂ O, K ₂ HPO ₄ , NaH ₂ PO ₄ , NaCl and sodium citrate from Riedel de Naën (Seelze, Germany); glucose monohydrate from Norsk Medisinaldepot (Oslo, Norway); sodium alginate (PROTANAL LF 10/60), supplied by FMC Biopolymer A / S (Drammen, Norway).

Sperm source

Bull sperm were collected in the Geno livestock complex at Hallsteingard in Trondheim, Norway. Boar sperm were harvested at the Norsvin livestock complex at Hamar, Norway. Bull sperm were diluted 1 + 2 in a milk diluent shortly after ejaculation. Boar sperm were bred 1 + 4 in TRI X-CELL ™ (IMV Technologies, L'Aigle Cedex, France). Neither this dilution, nor the choice of dilution buffers is critical for the immobilization technique or for the long-term survival of the sperm after immobilization, but is exclusively done to facilitate storage until the sperm can be processed further.

Buffer solutions

The following culture media were used. Modified IVT: 3 g / L glucose, 20 g / L sodium citrate, 2.1 g / L NaHCO ₃ , 1.16 g / L NaCl, 3 g / L EDTA, pH 7.35. BTS (with 3 mM Ca): 37 g / L glucose, 0.44 g / L CaCl ₂ , 4.5 g / L sodium citrate, 1.25 g / L NaHCO ₃ , 1.25 g / L EDTA, 0 , 74 g / l KCl, 1 g / l neomycin, pH 7.2. Gelling solution for granules: 7.3 g / l CaCl ₂ , 5.96 g / l NaCl. Milk diluent: 110 g / l skim milk Molico, 120 ml / l egg yolk, 6.25 g / l streptomycin, 1.5 million IE / l penicillin.

Cell immobilization

Sperm cells were harvested in Geno or Norsvin livestock complexes and bred as described above. Before immobilization, sperm cells were concentrated by centrifugation (800 g, 20 min, 20 ° C). The amount of supernatant that needs to be removed in each batch depends on the desired sperm concentration in the finished granules. After removing the excess supernatant, the cell pellet is carefully resuspended by gentle agitation. Then the cell suspension is carefully mixed with a sterile 6% (w / v) sodium alginate solution. The alginate solution is added in a volume ratio of 2 parts of the cell suspension to 1 part of the alginate solution (A large number of different combinations of the concentration of alginate, volume ratios of alginate solution and cell suspension have been studied. Typical values and methods are given here).

A mixture of alginate and cells is added dropwise to the gelling solution for the granules (see above). In order to obtain granules with a diameter of 3 mm, the solution is added through the tips of the syringe with an inner diameter of 0.5 mm. In order to obtain granules with smaller diameters (up to less than 1 mm in diameter) use a system based on the use of air flow in order to limit the size of the droplets that form on the needles. Sodium chloride is used in the gel-forming solution to ensure a uniform polysaccharide concentration throughout the granule (Smidsrad, O. and Skjak-Braek, G. (1990) Alginate as immobilization matrix for cells. Trends in Biotechnology 8, 71-78). In order to limit the amount of Ca ²⁺ in the granules, the granules are mixed for no more than 8 minutes in a gelling solution for the granules. The entire immobilization procedure is carried out at ambient temperature.

In vitro storage of immobilized sperm at ambient temperature

One of the goals of the immobilization technique is to extend the period of time during which sperm can be stored until insemination. Therefore, experiments were carried out in which sperm cells were stored in vitro at ambient temperature in buffer solutions. Comparative samples with non-immobilized sperm from the same ejaculate are included in all experiments. Sperm quality is assessed by measuring motility at various points in time during the storage period. At each point in time, the samples are analyzed and recorded the mobility of the immobilized sperm and the mobility of the control samples. A number of combinations of storage solutions and cell densities were studied in order to optimize the experimental system (data not shown). The experimental conditions described below are typical and have been used in the experiments described below.

In experiments with bovine sperm, approximately 2 ml of granules with immobilized sperm were transferred to 13 ml centrifuge tubes and a milk diluent solution was added at a volume ratio of 1 + 2. Control samples with freely suspended sperm are prepared by obtaining a total dilution of 1 + 15 in 13 ml centrifuge tubes (approximately 6 ml volume is used in all tubes). In previous experiments, it was shown that this dilution is almost optimal for limiting mobility during storage in comparative samples (data not shown).

In experiments with boar sperm, sperm cells were stored at 18 ° C in BTS (with 3 mM Ca). In the experiments described below, control samples were diluted 1 + 10 in all experiments to obtain a total volume of 20 in 50 ml centrifuge tubes (It was previously shown that this dilution is almost optimal for limiting mobility during storage). Granules containing immobilized sperm were transferred to 50 ml centrifuge tubes and BTS (with 3 mM CaCl ₂ ) was added in a 1 + 3 ratio. Granules containing immobilized sperm were washed with a storage solution before being transferred to a storage solution.

Storage of immobilized sperm at 37 ° C

In order to assess sperm survival at physiological temperatures, sperm motility was recorded during sperm storage at 37 ° C. Bull sperm were transferred to a buffer solution before storage experiments at 37 ° C. As described above, comparative samples from the same ejaculate were included in each experiment. Tests were performed in 13 ml centrifuge tubes with a total volume of 8 ml in each tube. Comparative samples were diluted 1 + 4 in the storage solution. For immobilized sperm, 3 ml of granules containing sperm were added to 6 ml of buffer solution and stored in a 13 ml centrifuge tube. Boar sperm were treated in a similar manner except that normal BTS (with 3 mM Ca) was used.

Mobility assessment

Sperm motility was evaluated using microscopic evaluation. Samples (typically 1 ml) were taken from storage containers and transferred to 1.5 ml Eppendorf tubes. The tubes were allowed to warm for a minimum of 15 minutes in a fuser at 37 ° C before evaluation. During the measurement, 2.5 μl of the sample was added onto a heated glass slide and immediately examined using a light microscope. The number of motile spermatozoa in each sample was established up to almost 5% of the interval. If practicable, the experimenter is not aware of the identification of the samples during the evaluation.

Immobilized sperm must be released from the granules before assessing motility. In order to dissolve the alginate granules, 1 part of the granules was added to 3 parts of an IVT solution in a 13 ml centrifuge tube. Then these tubes were placed in a rotating tube drum and allowed to mix for about 30 minutes before evaluation.

Results and discussion

Immobilized Sperm Survival

The data of experiments on the storage of sperm in comparative samples (with non-immobilized sperm) and in samples with immobilized sperm are presented in figure 1 for sperm of both the bull and the boar. For boar sperm, data are presented for 2 different granule sizes. The reason for this is that the granule size must be below 1 mm in order to use these granules for intrauterine insemination with existing equipment.

As can be seen from the data presented in figure 1, it turns out that immobilized sperm cells have slightly lower mobility than sperm cells in comparative samples, a short time after immobilization. This difference in motility can be caused by both the immobilization procedure and the dissolution procedure, since granules containing immobilized sperm cells must be dissolved before the mobility assessment. However, there is no need to dissolve the granules before insemination, since the granules under physiological conditions should dissolve slowly (data not shown).

The difference in motility between comparative samples and immobilized sperm decreases during the storage period, since it turns out that mobility in comparative samples decreases faster than in immobilized samples, especially for boar sperm. For immobilized boar sperm, motility of approximately 50% was recorded after 5 days of storage in granules with a diameter of 3 mm in this particular experiment. At this time, 10% mobility was recorded in comparative samples. These results show that immobilization affects the cells or the local environment of the cells favorably for their ability to withstand in vitro storage at 18 ° C.

For bovine sperm there is no significant difference in motility between comparative samples and immobilized sperm in the last period of the presented experiment.

Further optimization of storage conditions and immobilization procedures may, however, improve the storage periods of immobilized bovine sperm. Measurements of glucose consumption and lactate production of immobilized bovine sperm and non-immobilized sperm in comparative samples show that sperm stored inside the calcium alginate gel network have a reduced metabolic rate (see Example 2).

In order to assess whether immobilized sperm cells can withstand physiological conditions, that is, conditions where sperm activity should be maximum, storage experiments were carried out at a temperature of 37 ° C. data from in vitro storage experiments of bull and boar sperm during storage at 37 ° C are shown in FIG. 2.

As can be seen in experiments on storage in vitro at ambient temperatures, there is a difference in mobility between comparative samples and samples with sperm immobilized with alginate after sample preparation. However, for sperm of both the bull and the boar, motility decreases quite quickly in comparative samples with non-immobilized sperm during storage at 37 ° C. In samples with immobilized sperm, motility decreases much more slowly. After 15 hours of in vitro storage at 37 ° C, boar sperm motility at both pellet diameters is still above 50%. Indeed, mobility is still about 30% after 60 hours of storage at 37 ° C in granules with a diameter of 3 mm.

For bovine spermatozoa, it turns out that motility decreases faster than for boar sperm in this experimental system. However, as for boar sperm, there is a significant difference between the rates of incidence of mobility during storage in this experiment for bovine sperm. After 24 storage, immobilized bovine spermatozoa still have 45% motility, while mobility was not recorded in comparative samples.

It must be remembered that the data presented in FIG. 2 were obtained using an artificial experimental system that is not representative of the in vivo situation in a female animal. The data presented in figure 2, however, indicate that the sperm of both the bull and the boar survive and can remain motile for quite long periods when immobilized in the gel network of calcium alginate even at physiological temperature. These data may also indicate that immobilization in calcium alginate gel networks can be used to limit sperm motility during storage at physiological temperatures. This can be especially important since the insemination time is critical in order to obtain good fertilization results. Insemination time is critical because sperm have a limited survival time after insemination. These results may therefore indicate that sperm immobilization combined with controlled release after insemination can be used to increase the time period from insemination to fertilization.

Insemination Tests

Insemination tests were performed in total in 9 sows in one herd. These sows were intended for culling immediately after weaning the previous brood of piglets, but culling was delayed until about 4 weeks after insemination.

In a slaughterhouse, the genitals were collected immediately after removal from the cavity and examined for 15 minutes. The number of corpus luteum in the ovaries and the number of embryos (if any) in the horns of the uterus were counted and recorded. As a result of abortion at this early stage, the possibility of observing each present embryo (even degenerated) is relatively high. As a result of observing the number of full-term born piglets, there is a high risk of losing a high percentage of embryos, since any fertilized egg and embryo that died before ossification is absorbed by the maternal system, and no residues will be visible at this time. As many as 30% of fertilized pig eggs can die and disappear from fertilization to ossification. In the trials of the inventors, a good indicator of the probability of fertilization can be calculated for each sow by counting the number of embryos and the number of yellow bodies at 4 weeks.

The periodization of insemination varied from normal to one day earlier than usual, and both single and double inseminations were carried out. The results of these insemination tests are presented in table 1.

Table 1: The number of inseminated sows, pregnancy rate and fertilization rate (in pregnant animals) in insemination trials Way No sows Pregnant (%) Fertilization rate (%) Imm. sperm, day 1 ¹ +2 5 4 (80%) 57% Re-dissolved imm. sperm, day 1 + 2 four 2 (50%) 66%

Insemination tests have also been performed with bovine sperm. These tests are less easy to quantify, as large experimental herds are not available. These tests were carried out in a single herd with 20 available heifers. Heifers were synchronized by estrus before insemination. Good fertilization rates have been achieved using immobilized bovine sperm. In this study, 6 out of 12 heifers inseminated with immobilized sperm stored for 24 hours were fertilized, as confirmed by the pregnancy control.

Spermatozoa were immobilized in alginate, they were stored in an immobilized state at ambient temperature for various periods, inseminated with dissolved or undissolved. The periodization of insemination ranged from normal to one day earlier than usual, and only single inseminations were carried out. Pregnant animals were checked by rectal examination 5-7 weeks after insemination. The results of these tests are shown in table 2.

Table 2: Cattle insemination tests: Method used, number of inseminated heifers and pregnancy rate Insemination test Way No heifers Pregnant (%) one Sperm, imm., Storage for 24 hours at 18 ° C, dissolved immediately before insemination 12 6 (50%) 2 Sperm, imm., Storage for 24 hours at 18 ° C, insemination insoluble one day earlier than usual with respect to estrus 3 1 (33.3%) 3 Sperm, imm., Storage for 48 hours at 18 ° C, dissolved immediately before insemination 5 4 (80%) ¹ Day 1 = sperm collection day

These data show that sperm immobilized in granules of calcium alginate not only retain motility, but also retain all the functional properties necessary for fertilization. Thus, Geno and Norsvin have demonstrated that sperm immobilized in a calcium alginate gel can be used for insemination, and they can cause fertilization in both cattle and pigs.

Example 2. The energy consumption of immobilized sperm

Not limited to any particular theory, it is believed that sperm immobilization results in reduced energy consumption by sperm, and that this is useful in terms of shelf life and fertility. Thus, to study this theory, bovine spermatozoa were immobilized in alginate granules at a concentration of about 150 × 10 ⁶ sperm / ml and added to a 2-fold granule volume with milk dilution buffer. Comparative samples with freely suspended spermatozoa were prepared from the same ejaculate, and they contained the same total sperm concentration per total volume in milk dilution buffer. Samples were prepared under anaerobic conditions by blowing N ₂ before storage at 20 ° C. Samples of milk dilution buffer were taken over the storage period and lactate production was measured by sperm in the samples by HPLC. The results are presented in table 3:

Table 3: The production of lactate by immobilized and freely suspended sperm during storage at ambient temperatures. Values are in nanomoles of lactate produced by 350 million sperm. Storage time (day) Immobilized sperm Comparative samples one 4.21 16.1 2 6.00 20.6 3 6.90 22.1

Example 3: Immobilization of semen from a black fox (vulpes fulva) Good quality mixed sperm from three adult male black fox (Vulpes fulva) was diluted in an EDTA diluent of 228 × 10 ⁶ sperm cells per ml and transferred to the laboratory. Three hours after collection and dilution, an aliquot of sperm was centrifuged at 2000 rpm for 10 minutes. The precipitate was mixed with alginate (LF10 / 60) and granules were formed as described in this patent application. The granules were stored in modified Biladyl at 18 ° C. The remaining semen was stored in EDTA at 18 ° C as a control.

The granules were dissolved after 48 hours. Both dissolved semen and control semen were heated to 35 ° C for 20 minutes, after which mobility was assessed as a percentage of motile spermatozoa using phase contrast microscopy at 10 × and 25 × magnification on a heating table at 38 ° C.

Table 4: Sperm motility of a black-brown fox during storage in an immobilized and non-immobilized (control) state Mobility upon arrival Mobility after storage for 48 hours Control sperm 75% 45-50% Immobilized semen (after dissolution) 55-60%

Example 4: Piglets born full-term after a single intrauterine insemination with boar sperm immobilized in alginate

Mixed semen from 3 adult boars was treated according to the present invention, and the granules were used for intrauterine insemination of a sow, which was subsequently farrowed after full gestation. The sow was weaned from its previous litter on February 12, 2007 and was seeded with sperm immobilized in alginate granules once on February 12, 2007. The sow gave birth to 16 live pigs on June 10, 2007. There were no stillborn piglets in this litter, and all pigs looked normal in a clinical study.

Example 5: Immobilization of ram sperm in alginate

Sperm from two rams for AI was collected using an artificial vagina and transferred to the laboratory for ten minutes. Sperm was diluted 1 + 3 in a skim milk diluent (Curtis PG, Forteath AD, Polge C. Survival of bull sperm frozen in milk diluents containing varying concentrations of glycerol and fructose. Proc IVth Int Congr Anim Reprod 1961; 3: 952-956 ), after which alginate was added and granules were formed as described in patent application 0613288.0, Example 1. The granules were stored in a skim milk diluent either at 5 ° C or at ambient temperature. Immobilized sperm were released from the granules before microscopic assessment of motility as a percentage of motile sperm 24 and 48 hours after immobilization. For practical reasons, controls were not included in this study. However, viability parameters, including motility, should deteriorate during storage of ram sperm at 5 ° C or 20 ° C for 30 hours (Paulenz H, Soderquist L, Perez-Pe R, Berg KA. Effect of different extenders and storage temperatures on sperm viability of liquid ram semen. Theriogenology 2002: 57 (2): 823-36).

Table 5. The mobility of immobilized ram sperm stored at different temperatures for 24 hours and 48 hours Storage temperature Mobility after 24 hours of storage Mobility after 48 hours of storage Ram 1 5 ° C 75-85% 70-80% 20 ° C 70-80% 65-75% Ram 2 5 ° C 75-85% 70-80% 20 ° C 70-80% 65-75%

Claims (33)

1. Biopolymer particles for preserving sperm, where the sperm are enclosed in a biopolymer gel network, and where the biopolymer in which the sperm are enclosed contains alginate enriched in guluronic acid. 2. Particles according to claim 1, where the biopolymer in which the sperm are enclosed includes calcium alginate. 3. Particles according to claim 1, where the concentration of alginate is at least 0.1%. 4. Particles according to claim 3, where the concentration of alginate is between at least 0.1% and 6% alginate. 5. Particles according to claim 4, where the concentration of alginate is at least 1%. 6. Particles according to claim 4, where the concentration of alginate is at least 2%. 7. Particles according to claim 4, where the concentration of alginate is 6%. 8. Particles according to claim 1, having a sperm concentration of at least 0.1 × 10 ⁶ sperm / ml. 9. Particles of claim 8, having a sperm concentration of at least 100 × 10 ⁶ sperm / ml. 10. Particles of claim 8, having a sperm concentration of at least 2.5 × 10 ⁹ sperm / ml. 11. Particles according to claim 1, where the particles are stored in solution. 12. The particles according to claim 11, where the ratio of the solution for storage: biopolymer particles is at least between 1: 1 and 1: 100. 13. Particles according to claim 1, where the sperm are enclosed together with one or more than one antioxidant. 14. The particles of claim 13, wherein the antioxidant is selected from the group consisting of pyruvate, 2,2,6,6-tetramethylpiperidin-1-oxyl, 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, superoxide dismutase, catalase, glutathione peroxidase, butylated hydroxytoluene, butylated hydroxyanisole. 15. Particles according to claim 1, where the specified particle is coated. 16. The particles of claim 15, wherein the coating is selected from the group consisting of polylysine, chitosan, cellulose sulfate, hydroxypropyl methyl cellulose or polydiallyldimethylammonium chloride. 17. The particles according to claim 1, where the sperm cells are additionally enclosed in conjunction with compounds or agents that are useful for the fertilizing ability and / or health of the animal. 18. The particles of claim 17, wherein the spermatozoa are enclosed in conjunction with one or more of one of the compounds or agents selected from the group consisting of diluents, cryoprotectants, antibiotics, antibodies, antioxidants, proteins and hormones. 19. Particles according to claim 1, where the sperm are derived from an animal selected from the group consisting of pigs, cattle, horses, sheep, goats, rabbits, poultry, pets, such as thoroughbred dogs, fur animals, aquatic animals and endangered species of animals. 20. Particles according to claim 19, where the sperm are collected from an animal selected from the group consisting of pigs, cattle, fur animals and horses. 21. The particles according to claim 1, where the sperm are contained in seminal fluid. 22. Particles according to claim 1, where the particles are further processed by dehydration, cryopreservation or lyophilization. 23. Particles according to claim 1, where the particles form directly in the seed container. 24. The method of producing particles according to claim 1, where a mixture of alginate and sperm is added to the gelling solution. 25. The method according to paragraph 24, where the gelling solution contains one or more than one of the following ions selected from the group consisting of calcium, sodium, barium, magnesium. 26. The method according A.25, where the gel-forming solution contains calcium and sodium ions. 27. The method according to any one of paragraphs.24-26, where the particles are further processed by dehydration, cryopreservation or lyophilization. 28. The use of biopolymer particles according to claim 1 in animal breeding. 29. The application of claim 28, wherein the particles are used in breeding animals selected from the group consisting of pigs, cattle, horses, sheep, goats, rabbits, poultry, pets, such as thoroughbred dogs, fur animals, aquatic animals and endangered species of animals. 30. The application of claim 28, wherein the particles are seeded directly. 31. The use of claim 28, wherein the particles are dissolved before insemination. 32. The application of claim 28, wherein the particles are used together with free sperm. 33. The method of fertilizing an animal using sperm preserved in particles according to claim 1, comprising the steps of (a) obtaining particles according to claim 1, in which the sperm are preserved, and (b) these particles are introduced to the recipient female animal.

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