Use of casein peptides for fertility

Abstract

The current disclosure relates to a method of increasing reproductive efficiency in mammals comprising administering a composition comprising at least one milk derived peptide.

Description

P-635757-PCUSE OF CASEIN PEPTIDES FOR FERTILITY SEQUENCE LISTING STATEMENT

[0001] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 22, 2026, is named P-635757-PC-ST26 and is 66,603 bytes in size.BACKGROUND OF THE INVENTION

[0001] Dairy cow farms have implemented highly appropriate management for the last few decades, aiming at optimizing production, including reproductive efficiency. Perturbations to the dam’s health, nutrition and environment during pregnancy may program permanent structural and physiological modifications during the calving and post calving period. Dairy farms depend largely on the reproductive efficiency of dairy cows. It is established that early reproduction cyclicity results in increased pregnancy per artificial insemination (Al) and decreased time to pregnancy, which is expected to increase profitability for the dairy enterprise. Heat stress, long lactation period, uterine diseases, are highly prevalent in high-producing dairy cows and are associated with decreased pregnancy per Al services, extended interval to pregnancy, increased culling, and economic losses.

[0002] The casein protein comprises three fractions, a, P and K, according to their electrophoretic mobility. Casein hydrolysate is the hydrolyzed form of casein which includes, among others, the active beta-casein-derived peptide. It has been established that casein hydrolysate plays a role in immune responses against microbial infections.

[0003] There is still a great need for methods of increasing reproductive efficiency.P-635757-PCSUMMARY OF THE INVENTION

[0004] In some aspects, disclosed herein is a method of increasing reproductive efficiency in mammals comprising administering a composition comprising at least one milk derived peptide.

[0005] In some related aspects, increasing reproductive efficiency comprises reducing the number of days from calving to conception, increasing insemination success rates, reducing total number of inseminations for successful conception, reducing mean number of inseminations for successful conception, or any combination thereof.

[0006] In some further related aspects, the mammal experience conception after 1-10 inseminations.

[0007] In some further related aspects, the administration is at the beginning of the dry period. In some aspects, the dry off period comprises 28 to 120 days.

[0008] In some aspects, the milk derived peptide comprises a casein derived peptide.

[0009] In some aspects, the casein derived peptide comprises natural peptide, synthetic peptide, semi-synthetic peptide, or any combination thereof.

[0010] In some further related aspects, the casein derived peptide comprises one or more fragments of P-casein, aS 1 -casein, aS2-casein, K-casein, optimally further comprises amino acids with different lengths or any combination thereof.

[0011] In some related aspects, the casein derived peptide comprises a casein hydrolysate. In some further related aspects, the casein derived peptide comprises a phosphopeptide. In some further related aspects, the phosphopeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1 - SEQ ID NO 26.

[0012] In some related aspects, the composition is free of antimicrobials and comprises an acceptable carrier.P-635757-PC

[0013] In some related aspects, the administration comprises between 1 Opg to 10,000 mg of the milk derived peptide, per administration.

[0014] In some related aspects, the administration comprises intramammary infusion to a single teat or a plurality of teats. In some further related aspects, the administration comprises between one to eight administrations per teat. In some further related aspects, the administration comprises intervals of from about 1 hour to about 72 hours.

[0015] In some aspects, disclosed herein is a composition comprising at least one milk derived peptide for increasing reproductive efficiency in mammals.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The subject matter regarded as the invention is particularly pointed out and distinctly claimed in the concluding portion of the specification. The invention, however, both as to organization and method of operation, together with objects, features, and advantages thereof, may best be understood by reference to the following detailed description when read with the accompanying drawings in which:

[0017] Fig. 1 is graphic illustration of time from the beginning of insemination process until successful conception, in treatment groups A (bCNH 1200 mg), B (bCNH 2400 mg) vs Positive Control. Kaplan-Meier curve showed higher conceptions rate in group B.

[0018] It will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements.P-635757-PCDETAILED DESCRIPTION OF THE PRESENT INVENTION

[0019] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the present invention.A method of increasing reproductive efficacy

[0020] In some embodiments, disclosed herein is a method for increasing reproductive efficiency in mammals comprising administering a composition comprising at least one milk derived peptide.

[0021] In some embodiments, the increase in reproductive efficiency comprises reducing the number of days from calving to conception, increasing insemination success rates, reducing total number of inseminations for successful conception, reducing mean number of inseminations for successful conception, or any combination thereof. In one embodiment, the increase in reproductive efficiency comprises reducing the number of days from calving to conception. In another embodiment, the increase in reproductive efficiency comprises increasing insemination success rates. In another embodiment, the increase in reproductive efficiency comprises reducing the total number of inseminations for successful conception. In another embodiment, the increase in reproductive efficiency comprises reducing the mean number of inseminations for successful conception.

[0022] A skilled in the art would understand that artificial insemination in cows is a reproductive technology that involves introducing sperm into a cow's reproductive tract to achieve pregnancy, rather than relying on natural mating. This technique allows for the use of genetically superior bull sperm, enabling better herd genetics and improving milkP-635757-PCproduction and overall herd health. Artificial insemination is commonly used in dairy and beef cattle breeding programs to increase efficiency and control over reproduction.

[0023] In another embodiment, the increase in reproductive efficiency comprises reducing the number of days from calving to conception and increasing insemination success rates. In another embodiment, the increase in reproductive efficiency comprises reducing the number of days from calving to conception and reducing total number of inseminations for successful conception. In another embodiment, the increase in reproductive efficiency comprises reducing the number of days from calving to conception and reducing mean number of inseminations for successful conception. In another embodiment, the increase in reproductive efficiency comprises reducing the number of days from calving to conception and reducing mean number of inseminations for successful conception. In another embodiment, the increase in reproductive efficiency comprises increasing insemination success rates and reducing total number of inseminations for successful conception. In another embodiment, the increase in reproductive efficiency comprises increasing insemination success rates and reducing mean number of inseminations for successful conception. In another embodiment, the increase in reproductive efficiency comprises reducing the total number of inseminations for successful conception and reducing mean number of inseminations for successful conception.

[0024] In another embodiment, the increase in reproductive efficiency comprises a combination of reducing the number of days from calving to conception, increasing insemination success rates, reducing total number of inseminations for successful conception and reducing mean number of inseminations for successful conception.

[0025] In one embodiment, the mammal experience conception after 1-10 inseminations. In another embodiment, the mammal experience conception after 1 insemination. In anotherP-635757-PCembodiment, the mammal experience conception after 2 inseminations. In another embodiment, the mammal experience conception after 3 inseminations. In another embodiment, the mammal experience conception after 4 inseminations. In another embodiment, the mammal experience conception after 5 inseminations. In another embodiment, the mammal experience conception after 6 inseminations. In another embodiment, the mammal experience conception after 7 inseminations. In another embodiment, the mammal experience conception after 8 inseminations. In another embodiment, the mammal experience conception after 9 inseminations. In another embodiment, the mammal experience conception after 10 inseminations.

[0026] In one embodiment, the administration is at the beginning of the dry period.

[0027] In some embodiments, the dry period comprises 7 to 150 days. In some embodiments, the dry period comprises 28 to 120 days. In some embodiments, the dry period comprises 28 to 60 days. In some embodiments, the dry period comprises 40 to 49 days. In some embodiments, the dry period comprises 50 to 59 days.

[0028] In one embodiment, the dry period comprises 28 days. In another embodiment, the dry period comprises 30 days. In another embodiment, the dry period comprises 35 days. In another embodiment, the dry period comprises 30 days.

[0029] In another embodiment, the dry period comprises 40 days. In another embodiment, the dry period comprises 49 days. In another embodiment, the dry period comprises 50 days. In another embodiment, the dry period comprises 59 days. In another embodiment, the dry period comprises 60 days.Casein peptides

[0030] In some embodiments, the method as described comprises administering a composition comprises at least one milk derived peptide.P-635757-PC

[0031] A skilled artisan would understand that milk peptides are derived from milk proteins by enzymatic breakdown by digestive enzymes or by the proteinase enzymes produced by lactobacilli during the fermentation of milk, or by plant derived sources, for example papain and bromelain.

[0032] In some embodiments, the milk derived peptide comprises a casein derived peptide.

[0033] Casein is a protein in non-human mammal’s milk, also found in human mammal’s milk known to include the subgroups aSl, aS2, p and K. Casein is defined according to the amino acid sequences of each of the subgroups aSl, aS2, p and K. In the context of the present disclosure, when referring to casein, it is to be understood as also including acid casein, salts of casein, phosphorous containing casein and rennet casein.

[0034] The term "protein" as used herein refers to amino acid residues, connected by peptide bonds. A protein sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group. Amino acids, as used herein, refer to naturally occurring and synthetic amino acids, as well as amino acid analogs, and amino acid mimetics, that function in a manner similar to the naturally occurring amino acids. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0035] The casein-derived peptide may be a single peptide or a mixture of different peptides which may be independently selected from a naturally occurring peptide, a semisynthetic peptide, a synthetic peptide or a recombinant peptide. It should be further noted that the peptides according to the present disclosure may be produced synthetically, or byP-635757-PCrecombinant DNA technology, or by any other technology. Methods for producing peptides are well known in art.

[0036] In some embodiments, the casein-derived peptide may comprise a casein protein breakdown product which occurs when casein protein is cleaved by enzymes or acids to peptide fragments (also known in the art by the term "casein hydrolysate"). A casein hydrolysate is to be understood as the hydrolyzed form of casein (protein). Casein hydrolysate includes, for example, the active beta-, alpha S1-, alpha S2-, kappa-caseinderived peptide known to those versed in the art. In some embodiments, the casein-derived peptide is or comprises a casein hydrolysate.

[0037] In some embodiments, the casein derived peptide comprises natural peptide, synthetic peptide, semi-synthetic peptide, or any combination thereof. In another embodiment, the casein derived peptide comprises a natural peptide. In another embodiment, the casein derived peptide comprises a synthetic peptide. In another embodiment, the casein derived peptide comprises a semi-synthetic peptide. In another embodiment, the casein derived peptide comprises a combination of natural peptide and synthetic peptide. In another embodiment, the casein derived peptide comprises a combination of natural peptide and semi-synthetic peptide. In another embodiment, the casein derived peptide comprises a combination of synthetic peptide and semi-synthetic peptide. In another embodiment, the casein derived peptide comprises a combination of natural peptide, synthetic peptide and semi-synthetic peptide.

[0038] Natural casein-derived peptides are typically obtained following enzymatic hydrolysis, the enzyme may be any mammal peptidase, such as, without being limited thereto, plasmin, pancreatin, trypsin, chymotrypsin, neutrase, alcalase, pepsin, carboxypeptidase, cathepsin, as well as plant peptidase such as, without being limitedP-635757-PCthereto, papain, bromelain, as well as enzymes from microorganism source. For example, a naturally occurring casein-derived peptide may be the result of an enzyme activity such as plasmin on casein subunits P-casein, asl- and as2-casein or K-casein. In some embodiments, a casein hydrolysate is obtained by cleavage of the casein protein with trypsin.

[0039] Synthetic peptide may be obtained by any methods known in the art of peptide synthesis including chemical synthesis and recombinant DNA technology. For example, the peptides may be synthesized by using standard solid phase techniques.

[0040] In one embodiment, the synthetic peptide is a recombinant peptide.

[0041] A semi-synthetic casein-derived peptide may be obtained by chemical hydrolysis of casein, e.g. by prolonged boiling in a strong acid (acid-HVP) or strong base or using a chemical agent such as Cyanogen bromide (CNBr). The casein-derived peptide may also be obtained by molecular engineering, e.g. using recombinant DNA, in molecular techniques known in the art. In such an embodiment, the casein-derived peptide is a recombinant peptide.

[0042] In one embodiment, the recombinant peptide is produced by fermentation, tissue culture or combination thereof. In another embodiment, the recombinant peptide is produced by fermentation. In another embodiment, the recombinant peptide is produced by tissue culture. In another embodiment, the recombinant peptide is produced by a combination of fermentation and tissue culture.

[0043] In one embodiment, the tissue culture comprises mammary gland tissue .

[0044] In some embodiments, the casein derived peptide comprises one or more fragments of P-casein, aS 1 -casein, aS2-casein, K-casein or any combination thereof. In one embodiment, the casein derived peptide comprises one or more fragments of P-casein. In another embodiment, the casein derived peptide comprises one or more fragments of aSl-P-635757-PCcasein. In another embodiment, the casein derived peptide comprises one or more fragments of aS2-casein. In another embodiment, the casein derived peptide comprises one or more fragments of K-casein. In another embodiment, the casein derived peptide comprises one or more fragments of combination of P-casein, aS 1 -casein, aS2-casein and K-casein.

[0045] In one embodiment, the casein derived peptide further comprises amino acids with different lengths.

[0046] In one embodiment, the casein derived peptide comprises a casein hydrolysate.

[0047] In one embodiment, the casein derived peptide comprises a phosphopeptide.

[0048] As used herein, the term "phosphopeptide" designates a phosphorylated peptide in form of a conjugated peptide in which the non-peptide portion is a residue of phosphoric acid. The expression "phosphopeptide" or "phosphoserine" designates conjugated serine in which the non-peptide portion is a residue of phosphoric acid.

[0049] In some embodiments, the casein-derived peptide is a single peptide or mixture of a phosphopeptide, namely, which contains a single phosphorous group or is a phosphorus-enriched peptide. In some embodiments, the casein-derived peptide is any phosphoserine, phosphotyrosine, phosphothreonine, and / or phosphohystidine-enriched casein-derived peptides (casein phosphopeptide, CPP) and monovalent cation phosphocaseinates, such as sodium, potassium, calcium or ammonium phosphocaseinates.

[0050] In some embodiments, the casein-derived peptide is a phosphor-peptide.

[0051] The phosphor-peptide may be genetically engineered casein-derived peptides as well as peptidomimetics of casein-derived peptides. For example, phosphorylation of amino acids such as at least one serine residue may be performed by any method as is known in the art. The term "casein-derived peptide" also encompasses peptide fragments or peptidomimetic products obtained from or corresponding to one or more sections of caseinP-635757-PCprotein. The peptidomimetic peptide may be for example a peptoid or a semipeptoid, which are peptide analogs, having, for example, modifications such as, but are not limited to, cyclization, N-terminus modification, C-terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S-O, O-C-NH, CH2-O, CH2-CH2, S-C-NH, CH-CH or CF-CH, backbone modification and residue modification.

[0052] As used herein, the term “casein-derived peptide” further encompasses any derivatives, analogues, variants or homologues of any of the peptides. The term "derivative" is used to define amino acid sequences (peptide), with any insertions, deletions, substitutions, and modifications to the amino acid sequences (peptide) that do not alter the activity of the original peptides. By the term “derivative” it is also referred to homologues, variants and analogues thereof, as well as covalent modifications of polypeptides made according to the present invention.

[0053] In some embodiments, the modified, synthetic, semi-synthetic or other types of analogs of the naturally occurring casein-derived peptides are in some embodiments at least 75%, at times 85%, 90%, 95% and even 99% identical (in sequence) to a naturally occurring casein-derived peptide when the two sequences are optimally aligned. Further, any non-naturally occurring casein-derived peptide to be used in accordance with the present disclosure may retain at least part of the biological activity of the naturally occurring casein protein.

[0054] The present disclosure also encompasses homologues of the casein-derived peptide. The term "homologues” is used to define amino acid sequences (peptide) which maintain a minimal homology to the amino acid sequences defined by the invention, e.g. have at least about 65%, at least about 75%, at least about 85%, or at least about 95% overallP-635757-PCsequence homology with the amino acid sequence of any of the peptide as structurally defined above, e.g. of a specified sequence.

[0055] The term "about" as used herein indicates values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. As used herein the term "about" refers to ± 10 %.

[0056] In some embodiments, the casein-derived peptide may also include a chemical modification of a naturally occurring peptide, e.g. where one or more amino acids are deleted, substituted or modified, e.g. by removal of a side group, substitution of a side group or the introduction of a chemical group. Without being limited thereto, the chemical modification may include acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPI anchor formation, covalent attachment of a lipid or lipid derivative, methylation, myristoylation, pegylation, prenylation, phosphorylation, ubiquitination, or any similar process. When referring to replacement of an amino acid sequence by another, it is likely that the replacement is a conservative substitution. For example, one or more amino acid residues within a casein sequence is substituted by another amino acid of a similar polarity or charge. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Nonetheless, non-conservative substitutions may also take place as long as it does notP-635757-PCsignificantly change the desired (casein like) biological activity of the resulting caseinderived peptide analog.

[0057] A casein-derived peptide in accordance with the present disclosure is characterized by a molecular weight of between about an average 100 to an average 10,000 Dalton (e.g. between 2 to 100 amino acids) at times between about an average 100 to an average 7,000 Dalton and at times between an average 1,000 to an average 5,000 Daltons.

[0058] A casein-derived peptide in accordance with the disclosure is characterized by a length of from 2 to 100 amino acids, at times between 4 amino acids to 40 amino acids, at times from 4 amino acids to 30 amino acids, at times 4 amino acids to 10 amino acids, at times between 10 amino acids to 50 amino acids.

[0059] In some embodiments, the casein-derived peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1 - SEQ ID NO. 26.

[0060] In one embodiment, the phosphopeptide comprises an amino acid sequence denoted as Ser-Ser-Ser-Glu (SEQ ID NO:1), wherein at least one Ser residue, at least two Ser residues or three Ser residues are phosphorylated (phosphorylated serine is denoted herein as Ser(p) or S(p)).

[0061] In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Ser-Ser-Ser-Glu-Glu (SEQ ID NO:2), wherein at least one Ser residue, at least two Ser residues or three Ser residues are phosphorylated.

[0062] In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Ser(p)-Ser(p)-Ser(p)-Glu-Glu (SEQ ID NO:3).

[0063] In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as RELEELNVPGEIVES(p)LS(p)S(p)S(p)EESITR (SEQ ID NO:4). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted asP-635757-PCQMEAESIS(p)S(p)S(p)EEIVPDSVEQK (SEQ ID NO: 5). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as KNTMEHVS(p)S(p)S(p)EESIISNETYK (SEQ ID NO:6). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as KVNELSKNIGS(p)ES(p)TEDQ (SEQ ID NO:7). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as PTLNREQLS(p)TS(p)EENSKKTVD (SEQ ID NO: 8). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as ELEELNVPGEIVES(p)LS(p)S(p)S(p)EESITR (SEQ ID NO:9). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as RELEELNVPGEIES(p)LS(p)S(p)S(p)EESITR (SEQ ID NO: 10). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as QMEAES(p)IS(p)S(p)S(p)EEIVPNS(p)VEQK (SEQ ID NO: 11). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as KNTMEHVS(p)S(p)S(p)EESIIS(p)QETYK (SEQ ID NO: 12). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as KVNELSKDIGS(p)ES(p)TEDQ (SEQ ID NO: 13). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as ESnS(p)QETYKQEKNMAINPSKENLCSTFCKEWRNANEEETSIGS(p)S(p)S(p)EES( p)AEVATEEVKITVDDKHYQKALNEINQFYQKFPGYLQYLYQGPIVLNPWNQVLR NAVPITPTLNREQLS(p)TS(p)EENSKKTVN (SEQ ID NO: 14). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as ELEELNVPGEIES(p)LS(p)S(p)S(p)EESITR(SEQ ID NO: 15).P-635757-PC

[0064] In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as XI (n)- Ser(P)-Ser(P)-Ser(P)-X2(m)-Lys (SEQ ID NO: 16), wherein at least one of XI and X2 is independently selected from a positively charged amino acid and wherein each one of n and m is independently selected from 0, 1 and 2.

[0065] In some embodiments, the positively charged amino acid is selected from the group consisting of lysine, arginine and histidine. In some embodiments, the positively charged amino acid is lysine. In some other embodiments, the positively charged amino acid is arginine. In some other embodiments, the positively charged amino acid is histidine.

[0066]

[0106] According to some embodiments, the formula of SEQ ID NO:16 further comprises a blocking group (also denoted herein as a protecting group) at the C-terminus. In some embodiments, the carboxyl group at the C terminus of the peptide is protected with a protecting group. The protecting group is selected from, but not limited to an amide (i.e., the hydroxyl group at the C terminus is replaced with a primary amine (NH2), secondary amine, or tertiary amine) or ester (i.e. the hydroxyl group at the C terminus is replaced with an ester).

[0067] According to some embodiments, the blocking group is selected from the group consisting of amide and ester. According to some embodiments, the blocking group is amide.

[0068] In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys-Lys-Ser(P)-Ser(P)-Ser(P) (SEQ ID NO: 17). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys-Lys-Ser(P)-Ser(P)-Ser(P)-Lys (SEQ ID NO: 18). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys-Lys-Ser(P)-Ser(P)-Ser(P)-Lys-Lys (SEQ ID NO: 19). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys-Ser(P)- Ser(P)-Ser(P)-Lys-Lys (SEQ ID NO:20). In another embodiment, theP-635757-PCphosphopeptide comprises an amino acid sequence denoted as Lys- Ser(P)-Ser(P)-Ser(P)-Lys (SEQ ID NO:21). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys-Ser(P)-Ser(P)-Ser(P) (SEQ ID NO:22). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Ser(P)-Ser(P)-Ser(P)-Lys-Lys (SEQ ID NO:23). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Ser(P)-Ser(P)-Ser(P)-Lys (SEQ ID NO:24). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as Lys-Lys-Ser(p)-Ser(p)-Ser(p)-NH2 (SEQ ID NO:25). In another embodiment, the phosphopeptide comprises an amino acid sequence denoted as RELEELNVPGEIVES(p)LS(p)S(p)S(p)EESITRINK (SEQ ID NO:26).

[0069] The casein-derived peptide according to the invention may comprise “L” as well as “D” form residues. While the amino acid residues of the peptide sequences set forth in SEQ ID NOs:l-26 are all in the "L" isomeric form, residues in the "D" isomeric form can substitute any L-amino acid residue so long as the resulting peptide analog retains at least part of the biological activity of the corresponding “L” isomer. One reason for designing casein-derived peptides comprising at least one D-amino acid is to increase stability of the peptide to proteolytic degradation.

[0070] In one embodiment, the composition is free of antimicrobials and comprises an acceptable carrier. In another embodiment, the composition is further free from hormones, genetic modified organism, or combination thereof. A skilled artisan would understand that a genetically modified organism (GMO) is an animal, plant, or microbe whose DNA has been altered using genetic engineering techniques.

[0071] In one embodiment, the milk derived protein is measured by UV at a range from 204 to 220 nm.P-635757-PCDosage and administration

[0072] In some embodiments, the methods of the present disclosure comprise administering between lOpg to 10,000mg of the milk derived peptides, per administration.

[0073] In some embodiments, the methods of the present disclosure comprise administering between lOpg to 9,000mg. In some embodiments, the methods of the present disclosure comprise administering between 100 pg to 10,000mg. In some embodiments, the methods of the present disclosure comprise administering between Img to 8000mg. In some embodiments, the methods of the present disclosure comprise administering between 500mg to 7000mg. In some embodiments, the methods of the present disclosure comprise administering between lOOOmg to 6000mg. In some embodiments, the methods of the present disclosure comprise administering between 1500mg to 6500mg. In some embodiments, the methods of the present disclosure comprise administering between 2000mg to 6000mg. In some embodiments, the methods of the present disclosure comprise administering between 2500mg to 5500mg. In some embodiments, the methods of the present disclosure comprise administering between 3000mg to 5000mg. In some embodiments, the methods of the present disclosure comprise administering between 3500mg to 4500mg.

[0074] In one embodiment, the methods of the present disclosure comprise administering lOpg. In another embodiment, the methods of the present disclosure comprise administering 100 pg. In another embodiment, the methods of the present disclosure comprise administering Img. In another embodiment, the methods of the present disclosure comprise administering 500mg. In another embodiment, the methods of the present disclosure comprise administering lOOOmg. In another embodiment, the methods of the present disclosure comprise administering 1500mg. In another embodiment, the methods of theP-635757-PCpresent disclosure comprise administering 2000mg. In another embodiment, the methods of the present disclosure comprise administering 2500mg. In another embodiment, the methods of the present disclosure comprise administering 3000mg. In another embodiment, the methods of the present disclosure comprise administering 3500mg. In another embodiment, the methods of the present disclosure comprise administering 4000mg. In another embodiment, the methods of the present disclosure comprise administering 4500mg. In another embodiment, the methods of the present disclosure comprise administering 5000mg. In another embodiment, the methods of the present disclosure comprise administering 5500mg. In another embodiment, the methods of the present disclosure comprise administering 6000mg. In another embodiment, the methods of the present disclosure comprise administering 6500mg. In another embodiment, the methods of the present disclosure comprise administering 7000mg. In another embodiment, the methods of the present disclosure comprise administering 7200mg. In another embodiment, the methods of the present disclosure comprise administering 8000mg. In another embodiment, the methods of the present disclosure comprise administering 10,000mg.

[0075] In another embodiment, the administration comprises intramammary infusion to a single teat or a plurality of teats. In another embodiment, the administration comprises intramammary infusion to a single teat. In another embodiment, the administration comprises intramammary infusion to a plurality of teats.

[0076] In some embodiments, the methods of the present disclosure comprise between one to eight administrations to a single teat. In one embodiment, the methods of the present disclosure comprise one administration. In another embodiment, the methods of the present disclosure comprise two administrations. In another embodiment, the methods of the present disclosure comprise three administrations. In another embodiment, the methods of theP-635757-PCpresent disclosure comprise four administrations. In another embodiment, the methods of the present disclosure comprise five administrations. In another embodiment, the methods of the present disclosure comprise six administrations. In another embodiment, the methods of the present disclosure comprise seven administrations. In another embodiment, the methods of the present disclosure comprise eight administrations.

[0077] In some embodiments, the administrations of the present disclosure comprise subsequent administrations. In one embodiment, the administrations are immediately one after the other.

[0078] In some embodiments, the administrations of the present disclosure comprise intervals of from about 1 hour to about 72 hours . In one embodiment, the administrations comprise intervals of about 1 hour. In another embodiment, the administrations comprise intervals of about 4 hours. In another embodiment, the administrations comprise intervals of about 5 hours. In another embodiment, the administrations comprise intervals of about 8 hours. In another embodiment, the administrations comprise intervals of about 10 hours. In another embodiment, the administrations comprise intervals of about 12 hours. In another embodiment, the administrations comprise intervals of about 15 hours. In another embodiment, the administrations comprise intervals of about 16 hours. In another embodiment, the administrations comprise intervals of about 20 hours. In another embodiment, the administrations comprise intervals of about 24 hours. In another embodiment, the administrations comprise intervals of about 25 hours. In another embodiment, the administrations comprise intervals of about 28 hours. In another embodiment, the administrations comprise intervals of about 30 hours.another embodiment, the administrations comprise intervals of about 35 hours. In another embodiment, the administrations comprise intervals of about 36 hours.anotherP-635757-PCembodiment, the administrations comprise intervals of about 40 hours. In another embodiment, the administrations comprise intervals of about 45 hours. In another embodiment, the administrations comprise intervals of about 50 hours. In another embodiment, the administrations comprise intervals of about 55 hours. In another embodiment, the administrations comprise intervals of about 60 hours. In another embodiment, the administrations comprise intervals of about 65 hours. In another embodiment, the administrations comprise intervals of about 70 hours. In another embodiment, the administrations comprise intervals of about 72 hours.

[0079] In some embodiments discloses herein a composition comprising at least one milk derived peptide for increasing reproductive efficiency in mammals.EXAMPLES EXAMPLEI— evaluating treatment effect on the reproductive performance.

[0080] The objective of this example was to treat cows at dry-off (with bovine casein hydrolysate (bCNH) vs. routinely used antibiotic) and evaluate the effect of the treatment on the reproductive performance.Materials and methods

[0081] Two bCNH dose levels were used. The experimental groups were as follows: (1) A, bCNH-1 at a dose of 1200 mg; (2) B, bCNH-1 at a dose of 2400 mg (2x1200 mg simultaneous); (3) antibiotic-treated (positive) control (Nafpenzal DC®), according to routine dry off treatment for which farmers decided to perform artificial insemination (Al) service. The number of cows included in the analysis in the treatment arms were as follows: cows which underwent artificial insemination (Al) - total 268 cows (A-98, B-80, positive control - 90 cows), from which 229 cows were successfully inseminated (A-83, B - 68, positive control - 78 cows).P-635757-PC

[0082] Calving date, first artificial insemination (Al) date, total number of Al attempts, conception date, were obtained. Pregnancy diagnoses were performed starting from 42 days post insemination and again before drying off to confirm pregnancy.

[0083] The examined fertility parameters were:(1) Proportion of overall conception success in each treatment group(2) Proportion of cases of successful insemination (insemination that led to conception) at the first insemination attempt (service)(3) Number of open days - days from calving to conception(4) Number of inseminations (services) performed until successful conception(5) Mean / Median time (days) from 1st insemination (service) until successful conception Statistical methods

[0084] Continuous variables were described using mean, standard deviation, median, minimum and maximum. Categorical variables were described using proportions. Chi Square Test (Fisher’s Exact test) was used to compare proportions of overall or first conception successes in each bCNH-treated group as compared with the control group. Mann-Whitney non parametrical test for independent samples was applied for analysis of non-normally distributed data, such as number of insemination trials until success and number of days from the beginning of insemination process until the end of follow up. Kaplan-Meier survival analysis performed for assessing time-in-insemination until of successful insemination, log-rank test performed to estimate the difference among time-in-insemination in treatment groups.ResultsProportion of overall conception success in each treatment groupP-635757-PC

[0085] A total of 268 cows (all parities) underwent insemination, of which 229 cows (85%) conceived. When compared among the treatment groups, no significant difference in overall conception proportions was found, as shown in Table 1, for all cows:Table 1. Conception proportions per treatment group.Proportion of cases of successful insemination at the first insemination attempt (service)

[0086] In the current study, overall, 34% of 1startificial inseminations led to conception, as seen in Table 2 below. The 1stinsemination conception proportion was calculated for each treatment group for all cows and for cowsTable 2. Conception proportions after 1st insemination (number of cases, % of total).P-635757-PCFor all cows, the proportion of insemination success at 1 st insemination in group B (2400mg bCNH) was significantly higher than in the control group, 47% v).30% (p = 0.04).Number of inseminations until conception

[0087] Number of inseminations to conception is related to various factors: health of reproductive organs (endometritis / metritis occurrence and its severity), metabolic morbidity after calving (such as milk fever, ketosis, non-esterified fatty acids (NEFA, or free fatty acids), beta-hydroxybutyrate (BHB) concentration, body condition score (BCS), and weather.An overall average of 34% of cows in this study conceived after the first insemination, as described above. The maximal number of inseminations attempted for cows participating in this study was 10. Details are shown in Table 3.

[0088] Table 3. Distribution of cows according to number of inseminations before conception success, per treatment group.The average number of inseminations needed was calculated for each treatment group.P-635757-PCTime until successful inseminationDescriptive characteristics of rest days (days from calving to the first insemination), days open (days from calving to conception) and days in insemination (days from the beginning of insemination to conception) per treatment group, are shown below, for all cows (Table4).Table 4: Rest days, days open, days in insemination per treatment group.

[0089] Additionally, the mean number of inseminations that led to a successful conception was calculated for all cows and per treatment group, as shown in Table 5.

[0090] Table 5. Number of inseminations performed until a successful conception, per treatment group.

[0091] On average, number of inseminations attempts per treatment group was either similar between bCNH-treated cows and control (p-value > 0.5) or lower in bCNH-treated cows (for group B, 2400mg bCNH). Cows in group B required on average 2.3 inseminationP-635757-PCattempts, while cows in the control group had on average 3 insemination attempts to conceive (p=0.037). Both groups had the same median number of insemination attempts, 2.Analysis of insemination interval

[0092] Insemination interval (days in insemination) was non-normally distributed; thus, a statistical non-parametrical approach was used in the analysis. Table 6. Descriptive characteristics of insemination interval (days), all parities.

[0093] The median insemination interval for all cows of parties in group B (2400mg bCNH) was 0 days, which corresponds to the results of the analysis of proportion of conceptions after 1 insemination. Median insemination interval in the control group was 39 days, significantly higher than in group B (p = 0.031) (Table 6).

[0094] Additionally, the time from the beginning of insemination process (first insemination service) until successful conception to all cows, per treatment group, is shown in Fig. 1. In a Kaplan-Meier curve, "censored" refers to the data points where the exact event time (such as death, failure, relapse, etc.) is unknown because the subject was lost to followup, withdrew from the study, or the event had not yet occurred by the end of the study period. The censored observations are typically marked with a symbol, like a "+" or a vertical tickP-635757-PCmark, on the curve at the time they were censored. This indicates that the survival time for these participants is incomplete. Despite this, they still contributed to the analysis up to the time they were censored.Conclusions

[0095] Generally, it is considered for lactating cows, the herd breeding goal should be a conception rate of 40% or more, or fewer than 2.5 services per conception. The dry-off treatment with bCNH demonstrated no negative effect on reproduction parameters that were analyzed. The total proportion of conceived cows did not differ significative among the treatment groups and was 85% in bCNH groups A and B, while in positive control - 87%. Cows treated with bCNH required either similar (3.0-3.1 services on average, p-value > 0.5) or lesser (2.3 services on average, p-value = 0.037) number of inseminations attempts to conceive as compared to antibiotic-treated cows (positive control) (3.0 services on average). Similarly, the number of bCNH-treated cows that conceived at the first insemination service was either similar to (28%-33%, p-value > 0.7) or higher (47%, p-value = 0.04) than that of control cows (30%).

[0096] Specifically, treatment with 2400mg bCNH (in group B) had an advantage of earlier conception. A higher proportion of cows in group B conceived after a single insemination attempt as compared to antibiotic-treated (control) cows (p = 0.04), which led to less artificial insemination attempts and less time from first service to conception compared with antibiotic-treated cows (p = 0.037 and p = 0.031, respectively).

[0097] Since the value of the whole herd or the herd value is the combination of the individual values of the cows on the herd according to the herd structure, knowing the specific value of a cow, its pregnancy value, its cost of pregnancy gain - loss, provide to farmers with a better guide in the quest for improved economic efficiency related to herdP-635757-PCreproductive management. Intramammary administration of bCNH showed, from this clinical study, a safe fertility performance and following the concept of herd value, an economic advantage of reproductive performance.

[0098] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

P-635757-PCCLAIMSWhat is claimed is:

1. A method of increasing reproductive efficiency in mammals comprising administering a composition comprising at least one milk derived peptide.

2. A method according to claim 1, wherein said increasing reproductive efficiency comprises reducing the number of days from calving to conception, increasing insemination success rates, reducing total number of inseminations for successful conception, reducing mean number of inseminations for successful conception, or any combination thereof.

3. A method according to claims 1 and 2, wherein said increasing reproductive efficiency comprises reducing the number of days from calving to conception.

4. A method according to claims 1 and 2, wherein said increasing reproductive efficiency comprises increasing insemination success rates.

5. A method according to claims 1 and 2, wherein said increasing reproductive efficiency comprises reducing total number of inseminations for successful conception.

6. A method according to claims 1 and 2, wherein said increasing reproductive efficiency comprises reducing mean number of inseminations for successful conception.

7. A method according to claims 1-6, wherein said mammal experience conception after 1-10 inseminations.

8. The method according to claims 1-7, wherein said administration is at the beginning of the dry period.P-635757-PC9. The method according to claim 8, wherein said dry off period comprises 28 to 120 days.

10. The method according to claim 8, wherein said dry off period comprises 28 to 60 days.

11. The method according to claim 8, wherein said dry off period comprises 40 to 49 days.

12. The method according to claim 8, wherein said dry off period comprises 50 to 59 days.

13. The method according to any of the proceeding claims, wherein said milk derived peptide comprises a casein derived peptide.

14. The method according to claim 13, wherein said casein derived peptides comprises natural peptide, synthetic peptide, semi -synthetic peptide, or any combination thereof.

15. The method according to claim 14, wherein said synthetic peptide is a recombinant peptide.

16. The method according to claim 15, wherein said recombinant peptide is produced by fermentation, tissue culture or combination thereof.

17. The method according to claim 16, wherein said tissue culture comprises mammary gland tissue.

18. The method according to claims 13-17, wherein said casein derived peptide comprises one or more fragments of P-casein, aS 1 -casein, aS2-casein, K-casein, optimally further comprises amino acids with different lengths or any combination thereof.P-635757-PC19. The method according to claims 13-18, wherein said casein derived peptide comprises a casein hydrolysate.

20. The method according to claims 13-19, wherein said casein derived peptide comprises a phosphopeptide.

21. The method according to claim 19, wherein said phosphopeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 1 - SEQ ID NO 26.

22. The method according to any of the proceeding claims, wherein said composition is free of antimicrobials and comprises an acceptable carrier.

23. The method according to any of the proceeding claims comprising administering between 10 pg to 10,000mg of said milk derived peptides, per administration.

24. The method according to any of the proceeding claims, wherein said administration comprises intramammary infusion to a single teat or a plurality of teats.

25. The method according to any of the proceeding claims, wherein said administration comprises between one to eight administrations per teat.

26. The method according to any of the proceeding claims, wherein said administration comprises intervals of from about 1 hour to about 72 hours.

27. A composition comprising at least one milk derived peptide for increasing reproductive efficiency in mammals.

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