WO2008016958A2

WO2008016958A2 - Reproductive cell media compositions and methods for improving fertility

Info

Publication number: WO2008016958A2
Application number: PCT/US2007/074935
Authority: WO
Inventors: Ludwig Simmet
Original assignee: Minitube of America Inc
Current assignee: Minitube of America Inc
Priority date: 2006-08-01
Filing date: 2007-08-01
Publication date: 2008-02-07
Anticipated expiration: 2009-02-01
Also published as: WO2008016958B1; WO2008016958A3

Abstract

Disclosed are compositions for reproductive cells which comprise a suitable reproductive cell media and a milk- or colostrum-derived product. The milk- or colostrum-derived product may comprise a growth factor, such as a transforming growth factor or insulin-like growth factor, or α-lactalbumin. The compositions may be optionally substantially free of bovine serum derived products. Methods for improving fertility in animals using compositions of the invention are also disclosed.

Description

REPRODUCTIVE CELL MEDIA COMPOSITIONS AND METHODS FOR IMPROVING FERTILITY

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional

Patent Application No. 60/821 ,044, filed August 1 , 2006.

INTRODUCTION

[0002] Bovine Spongiform Encephalopathy (BSE) is a transmissible, neurodegenerative disorder in cattle that was first recognized in the United Kingdom in 1986. Subsequently, the discovery of an indigenous, North American case of BSE in Canada in 2003 provoked international concern about safe sourcing of bovine materials. Non-affected countries prohibited the importation and use of materials derived from cattle in food and cosmetic products, resulting in a disruption of international trade in bovine products. Some of these bans have since been partially lifted. However, many countries continue to prohibit the importation of bovine meat and blood products from BSE-affected countries.

[0003] Bovine serum has been used to prepare a number of biological products, including cell culture products. In particular, bovine serum albumin (BSA) is a commonly used ingredient in cell culture compositions. However, at present there is a justified trend to remove all material of bovine serum origin from manufacturing processes for vaccines, pharmaceuticals, cosmetics and cell culture components. In 1993, the Food and Drug Administration (FDA) recommended against the use of bovine derived materials from cattle which have resided in, or originated from, countries where BSE outbreaks have occurred. Similarly, the current European Union (EU) guidelines on minimizing the risk of BSE transmission address geographical origin, the age of the animals, the breeding and slaughtering conditions, the tissue to be used and processing conditions to diminish the risk of cross-contamination during slaughtering or collection of the starting tissue. Other countries have also implemented regulations to control the risk of BSE transmission by bovine serum products.

[0004] On the other hand, there is no evidence that either bovine milk or bovine colostrum pose any risk of BSE transmission. Accordingly, importation and use of biological products derived from these sources are not restricted by any country. SUMMARY OF THE INVENTION

[0005] In one aspect, the invention provides a composition comprising a reproductive cell medium and a milk- or colostrum-derived growth factor.

[0006] In another aspect, the invention provides a composition comprising a reproductive cell medium, α-lactalbumin and a growth factor.

[0007] In another aspect, the invention provides a method of increasing the number of offspring of an artificially inseminated mammal, comprising inseminating the mammal with semen and a composition comprising a reproductive cell medium comprising a milk- or colostrum-derived growth factor.

[0008] In another aspect, the invention provides a method of increasing the number of offspring of an artificially inseminated mammal, comprising inseminating the mammal with semen and a composition comprising a reproductive cell medium, α- lactalbumin and a growth factor.

DESCRIPTION OF THE INVENTION

[0009] The invention is related to colostrum- and milk-derived biosubstitutes for blood and serum products in general, and for use in cell media compositions in particular. Not only does the present invention circumvent restrictions on importation and use of bovine serum products, cost advantages may also be realized by use of the present invention. The inventors have discovered that replacement of certain serum products with colostrum- and milk-derived equivalents in sperm culture medium surprisingly results in significantly improved fertilization rates in some in vitro fertilization protocols, although no particular degree of improvement in fertilization rates are required for the benefits of the present compositions to be realized.

[0010] The invention provides colostrum- and milk-derived products for use in cell media compositions. Suitably, the cell media compositions are reproductive cell media compositions, wherein the reproductive cells to be cultured are mammalian reproductive cells. More suitably, the cell media compositions are sperm cell media compositions wherein the sperm cells to be cultured are mammalian sperm cells. [0011] As used herein, the term "reproductive cells" encompasses sperm cells, oocytes, and embryos of any mammal, bird, or fish, including livestock (e.g., pigs, cows, horses, sheep and the like) and humans. As used herein a "reproductive cell medium" is a medium used for the collection, holding, processing, in vitro fertilization, sexing, culturing, or storing (including long-term cryopreservation) of mammalian, avian, or piscian reproductive cells, and includes both solid and liquid compositions, as well as solid compositions that are reconstituted or mixed with a liquid carrier, such as water, for use. Suitably the reproductive cell medium is formulated to maintain the viability of the reproductive cells.

[0012] The term "sperm cell medium," as used herein, refers to any medium used for the collection, holding, processing, in vitro fertilization, sexing, culturing, or storing (including long-term cryopreservation) of mammalian, avian or piscian sperm cells and/or semen. Suitably, the sperm cell medium may serve to maintain the viability of the sperm cells and to increase the volume of semen.

[0013] Milk- and colostrum-derived products suitable for use in the present invention are commercially available, or may be purified from milk or colostrum using standard methods. An example of a commercially available milk-derived product is α- lactalbumin from BIOPURE Corp. As used herein, a "milk-derived" or "colostrum-derived" product is any biological product purified from milk or colostrum, respectively, using standard purification methods. As used herein, the term "purified" refers to material that is at least partially separated from components which normally accompany it in its native state. Purity of polypeptides is typically determined using analytical techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A polypeptide that is the predominant species present in a preparation is "substantially purified." The term "purified" denotes that a preparation containing the polypeptide may give rise to essentially one band in an electrophoretic gel. The term "polypeptide" refers to two or more amino acid moieties linked by amide bonds, and includes peptides and proteins. The term "substantial homology" refers to a polypeptide sequence that performs substantially the same function as the parent sequence and has at least 60%, or more preferably, 75%, and most preferably, 90% or 95% or greater, sequence identity.

[0014] In one embodiment, suitable colostrum-derived products are growth factors. In particular embodiments, the growth factors are IGF-1 and/or TGF-β2. Suitably, colostrum-derived IGF-1 is included in sperm cell media compositions at a concentration ranging from about 50 ng/L to about 500 ng/L. More suitably, IGF-1 is included in sperm cell media compositions at a concentration ranging from about 100 ng/L to about 200 ng/L. Most suitably, colostrum-derived IGF-1 is included at a concentration of about 160 ng/L Suitably, colostrum-derived TGF-β2 is included in sperm cell media compositions at a concentration ranging from about 10 ng/L to about 200 ng/L. More suitably, colostrum-derived TGF-β2 is included in sperm cell media compositions at a concentration ranging from about 40 ng/L to about 100 ng/L. Most suitably, colostrum-derived TGF-β2 is included at a concentration of about 55 ng/L. Cell media compositions suitably comprise both IGF-1 and TGF-β2. A suitable ratio of IGF-1 to TGFβ2 is about 5:1 to about 2:1. Most suitably, the ratio of IGF-1 to TGFβ2 is about 3:1.

[0015] In another embodiment, a suitable milk-derived product is α-lactalbumin.

Suitably, α-lactalbumin is included in reproductive cell media compositions at concentrations ranging from about 1.0 to about 5.0 g/L. Most suitably, α-lactalbumin is included in reproductive cell media compositions at a concentration of about 2.5 g/L.

[0016] In a particularly suitable embodiment of the invention, the composition comprises cell media, colostrum-derived IGF-1 , colostrum-derived TGF-β2 and milk- derived α-lactalbumin. The composition also suitably includes a reproductive cell, e.g., a sperm cell or oocyte. Most suitably, the composition includes a porcine or bovine sperm cell or oocyte.

[0017] Suitably, compositions of the invention are substantially free of bovine serum-derived products. As used herein, a "serum-derived product" is any biological product purified from serum using standard purification methods.

[0018] Another embodiment of the invention provides a method of increasing the number of offspring of an artificially inseminated mammal, comprising inseminating the mammal with a composition comprising sperm and a milk- and/or colostrum-derived product. The number of offspring is increased relative to a suitable control. In the context of the invention, a suitable control is a mammal artificially inseminated with a composition comprising sperm, but not comprising a milk- or colostrum-derived product. An additional suitable control is a mammal artificially inseminated with a composition comprising sperm and a bovine serum-derived product, but not comprising a milk- or colostrum-derived product. In preferred embodiments, the artificially inseminated mammal is a pig or a cow. [0019] The following examples are provided to assist in a further understanding of the invention. The particular materials and conditions employed are intended to be further illustrative of the invention and are not limiting upon the reasonable scope of the appended claims.

Example 1. Sperm cell media formulation

[0020] A sperm cell culture media was formulated to include the following ingredients:

[0021] The above components were dry-mixed using standard blending techniques.

Example 2. Fetal recovery using media formulated with serum-derived versus colostrum- derived additives. [0022] Gilts were inseminated with one of three compositions or a control composition, as defined below, and were killed after the first trimester. The artificial insemination (Al) compositions were as follows:

[0023] X1 = ENDURAGUARD (Minitube, Verona, Wl) (with 2.5 g/L BSA and serum-derived TGF-β2 (9.3 ng/L) and serum-derived IGF-1 (450 ng/L)) + sperm cells (500 x 10⁶).

[0024] X2 = ENDURAGUARD (Minitube, Verona, Wl) base formulation (with 2.5 g/L BSA and colostrum-derived IGF-1 (160 ng/L) and colostrum-derived TGFβ2 (55 ng/L)) + sperm cells (500 x 10⁶).

[0025] X3 = ENDURAGUARD (Minitube, Verona, Wl) base formulation (with 2.5 g/L α-lactalbumin and colostrum-derived IGF-1 (160 ng/L) and TGFβ2 (55 ng/L)) + sperm cells (500 x 10⁶).

[0026] The total number of both fetuses and corpora lutea (CL) were counted.

The results are presented below:

[0027] As shown above, the X3 composition containing α-lactalbumin, colostrum- derived IGF-1 and colostrum-derived TGFβ2 resulted in a trend suggesting improvement in the average number of fetuses per gilt in comparison to the X1 and X2 compositions.

Example 3. Fetal recovery using media formulated with serum-derived versus colostrum- derived additives.

[0028] Gilts are inseminated with compositions described below, and are killed after the first trimester. The artificial insemination (Al) compositions are as follows: [0029] X1 = ENDURAGUARD (Minitube, Verona, Wl) base formulation (with 2.5 g/L BSA and serum-derived IGF-1 (160 ng/L) and TGFβ2 (55 ng/L)) + sperm cells (500 x 106).

[0030] X2 = ENDURAGUARD (Minitube, Verona, Wl) (with 2.5 g/ L α-lactalbumin and serum-derived TGF-β2 (9.3 ng/L) and IGF-1 (450 ng/L)) + sperm cells (500 x 106).

[0031] The total number of both fetuses and corpora lutea (CL) are counted. The

X2 composition is expected to result in an improvement in the average number of fetuses per gilt in comparison to the X1 composition.

[0032] As used in this specification and the appended claims, the singular forms

"a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing "a polypeptide" includes a mixture of two or more polypeptides. It should also be noted that the term "or" is generally employed in its sense including "and/or" unless the content clearly dictates otherwise. All publications, patents and patent applications referenced in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications, patents and patent applications are herein expressly incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference. In case of conflict between the present disclosure and the incorporated patents, publications and references, the present disclosure should control.

[0033] It also is specifically understood that any numerical value recited herein includes all values from the lower value to the upper value, i.e., all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended.

[0034] The invention has been described with reference to various specific embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

CLAIMSWe claim:

1. A composition comprising a reproductive cell medium and a milk- or colostrum- derived growth factor.

2. The composition of claim 1 , wherein the growth factor comprises IGF-1.

3. The composition of claim 1 , wherein the growth factor comprises TGF-β2.

4. The composition of claim 1 , comprising milk- or colostrum-derived IGF-1 and milk- or colostrum-derived TGF-β2.

5. The composition of claim 4, wherein the ratio of IGF-1 to TGF-β2 is from about 5:1 to about 2:1.

6. The composition of any of claims 1 to 5, further comprising α-lactalbumin.

7. The composition of any of claims 1 to 6, wherein the reproductive cell medium is a sperm cell medium.

8. The composition of any of claims 1 to 7, wherein the reproductive cell medium is reproductive mammalian cell medium.

9. The composition of any of claims 1 to 8, wherein the composition is substantially free of bovine serum-derived products.

10. The composition of any of claims 1 to 9, further comprising a reproductive cell.

11. The composition of claim 10, wherein the reproductive cell is a sperm cell or oocyte.

12. The composition of any of claims 10 to 11 , wherein the reproductive cell is a mammalian cell.

13. The composition of any of claims 10 to 11 , wherein the reproductive cell is a porcine or bovine cell.

14. A method of increasing the number of offspring of an artificially inseminated mammal, comprising inseminating the mammal with the composition of claim 10.

15. The method of claim 14, wherein the mammal is a pig or cow.