RU2736131C2 - Compositions and methods for improving milk productivity and reproductive health of mammals, based on using il-8 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to compositions and methods for improving mammalian reproductive health and increasing milk production by mammalian females. Methods include a step of administering an effective amount of IL-8 to a mammalian female, such that the reproductive health of the mammal is improved, or the mammal's milk productivity is increased, or the fat content in milk is increased. In another aspect, the present invention involves prevention and/or treatment of uterine conditions by administering IL-8 to a mammalian female.

EFFECT: disclosed are compositions and methods for improving milk productivity and reproductive health in mammals, based on using IL-8.

12 cl, 21 dwg, 1 tbl, 4 ex

Description

Cross-reference to related claims

This application claims priority from US patent application No. 62/099,643, filed January 5, 2015, the disclosure of which is incorporated herein by reference.

The technical field to which the invention relates

The present invention relates generally to the improvement of milk production and reproductive health in mammals through the administration of interleukin-8 (IL-8).

State of the art

As the world's population grows, and more importantly, as purchasing power parity per capita increases, the demand for animal protein (milk, meat and eggs) will grow steadily and inevitably; in order to avoid inflationary pressures, the supply of animal protein products must grow significantly and sustainably, with minimal expansion of agricultural land use. In addition, it has been reported that feed efficiency is the single largest contributor to changes in actual carbon dioxide emissions, and that improved feed conversion efficiency can reduce greenhouse gas emissions, both by reducing gut methane and manure yield. Postpartum uterine conditions such as metritis, endometritis and retained placenta are important for compliance reasons, contributing to cow discomfort and removal from the herd; coupled with deeply affected reproductive potential, reduced milk yield and treatment costs. Metritis and endometritis are commonly associated with mixed bacterial infection of the uterus, which includes E. coli, T. pyogenes, and F. necrophorum (Bicalho et al., 2012). A concomitant factor that increases susceptibility to uterine disease is the immunosuppression experienced by cows during the perinatal period (Drackley, 1999; Cai et al., 1994; Kimura et al., 1999; Hammon et al., 2006; Galvao et al. , 2010). There is an ongoing and unmet need for improved approaches to prevent and treat postpartum diseases, and to improve reproductive potential and milk production. The present invention addresses these and other needs.

The essence of the invention

In one aspect, the present invention includes the use of recombinant IL-8 to improve milk production in female mammals. Administration of IL-8 is also useful for the prevention and / or treatment of one or more diseases of the uterus and for the prevention or treatment of hyperketonemia in female mammals.

In one embodiment, the present invention provides a method for improving the health of a female mammal, and / or increasing milk production and / or fat content in milk produced by a mammal. The method includes the step of administering IL-8 to the female mammal so that at least the health of the female mammal is improved and / or the amount of milk in the mammal is increased and / or the fat content in the milk is increased.

In one embodiment, IL-8 is administered to a pregnant female mammal, or a female mammalian postpartum, eg, 12 months postpartum. In one embodiment, the administration of IL-8 is accompanied by the prevention or treatment of metritis or retained placenta, or inhibition of hyperketonemia in a mammal, or a combination thereof. In embodiments, the administration of IL-8 is oral, parenteral, subcutaneous, intramucosal, or intraperitoneal. Parenteral administrations include intramuscular, intravenous, intraarterial, intraperitoneal, intravaginal, intrauterine, and subcutaneous administration. In one embodiment, the administration of IL-8 is intrauterine administration.

In various aspects, the present invention provides an improvement in the health of a mammal by inhibiting the development of postpartum metritis, or by inhibiting retained placenta, or by increasing milk production in a mammal, including, but not limited to, increasing energy-corrected milk production, or by increasing the fat content of milk produced by a female mammal, or a combination of the above.

In embodiments, the female mammal is a bovine, such as a dairy cow. In embodiments, the female mammal is a member of a population of female mammals of the same species, and the administration of IL-8 is provided to other members of the population.

In another aspect, the present invention includes milk produced by a female mammal to which IL-8 has been administered, as well as milk products produced using such milk.

In another aspect, the present invention comprises a kit for i) improving the health of a female mammal, and / or ii) increasing milk production and / or fat content in milk produced by a mammal, the kit comprising IL-8 in one or more sealed containers, a device delivery and instructions for administering IL-8 to a female mammal in order to obtain i) or ii). The delivery device may be suitable for administering IL-8 to the uterus of a non-human mammal.

In another aspect, the present invention includes a finished product containing IL-8 in an airtight container, packaging and printed information, the printed information identifies IL-8 as the contents of the package and provides an indication that IL-8 should be used to improve female health. mammalian, and / or ii) increasing milk production and / or fat content in milk produced by mammals.

Brief Description of Drawings

Figure 1: Milk production (kg / day) per week of lactation for primiparous and multiparous cows. The error rates represent the standard error of the mean. Total milk production was higher for cows with L-IL8 and H-IL8 compared to control cows (P-value <0.01). The relationship between treatment and lactation week was not significant (P-value = 0.06). An asterisk (*) indicates that the weekly values differ (P-value <0.05).

Figure 2: Fat-corrected milk production (kg / day) during the first two months of lactation. The total fat-corrected milk production was higher for cows with L-IL8 and H-IL8 than for control cows (P-value = 0.02). The relationship between treatment and month of lactation was not significant (P-value = 0.89). The error rates represent the standard error of the mean.

Figure 3: Energetically corrected milk production (kg / day) during the first two months of lactation. The total energy-corrected milk production was higher for cows with L-IL8 and H-IL8 compared to the control counterparts (P-value = 0.02). The relationship between treatment and month of lactation was not significant (P-value = 0.56). The error rates represent the standard error of the mean.

Figure 4: Linear estimation of the number of somatic cells during the first two months of lactation. The overall linear estimate of the number of somatic cells did not depend on treatment (P-value = 0.02). The relationship between treatment and month of lactation was not significant (P-value = 0.09). The error rates represent the standard error of the mean.

Figure 5: Effect of treatment on farm diagnosed incidence of postpartum metritis in primiparous and multiparous cows.

Figure 6: Effect of treatment on the incidence of clinical endometritis in nulliparous and multiparous cows.

Figure 7: Concentration of beta-hydroxybutyrate (BHBA) in blood according to DIM. The total concentration of BHBA in the blood was 0.77 μmol / L (95% CI = 0.65 - 0.90), 0.62 μmol / L (95% CI = 0.50 - 0.74) and 70 μmol / L (95% CI = 0.58 - 0.82) for control cows, cows with L-IL8 and H-IL8, respectively (P-value = 0.22). The relationship between treatment and DIM was not significant (P-value = 0.66). The error rates represent the standard error of the mean.

Figure 8: DIM blood IL-8 concentration. The total concentration of IL-8 in the blood did not increase with intrauterine infusion of IL-8 (P-value = 0.17). The relationship between treatment and DIM was not significant (P-value = 0.16). The error rates represent the standard error of the mean.

Figure 9: Rectal temperature by DIM. Total rectal temperature did not differ between treatment groups (P-value = 0.47). The relationship between treatment and DIM was not significant (P-value = 0.13). The error rates represent the standard error of the mean.

Figure 10: DIM blood haptoglobin levels. The total blood haptoglobin level did not increase with intrauterine infusion of IL-8 (P-value = 0.96). The relationship between treatment and DIM was not significant (P-value = 0.48). The error rates represent the standard error of the mean.

Figure 11: The decline in body condition scores from day of birth to 35 DIM was independent of treatment (P-value = 0.99).

Figure 12: DIM Blood IGF-1 Levels. The total blood IGF-1 level did not increase with intrauterine infusion of IL-8 (P-value = 0.18). The relationship between treatment and DIM was not significant (P-value = 0.25). The error rates represent the standard error of the mean.

Figure 13: DIM Serum Glucose Concentration. Total serum glucose concentration did not increase with intrauterine infusion of IL-8 (P-value = 0.10). The relationship between treatment and DIM was not significant (P-value = 0.55). The error rates represent the standard error of the mean.

Figure 14: The effect of treatment on the incidence of subclinical ketosis.

Figure 15: Representative IL-8 amino acid sequence alignments from selected animal species. Sequences are shown from N- to C-terminus. The sequence for each species and the consensus sequence is contiguous across all the series. The Bos taurus sequence is SEQ ID NO: 1. The Bubalus bubalus sequence is SEQ ID NO: 4. The Cervus elephus sequence is SEQ ID NO: 5. The Ovis aries sequence is SEQ ID NO: 6. The Equus caballus sequence is SEQ ID NO: 7. The sequence of Homo sapiens is SEQ ID NO: 8. The sequence of Canis lupus familiaris is SEQ ID NO: 9. The sequence of Felus catus is SEQ ID NO: 10. The consensus of the specific mammalian sequences shown in the lower row, is SEQ ID NO: 11.

Figure 16: Effect of different intrauterine doses of IL-8 treatment on milk fat percentage in the first and second months of lactation. Bars are shown for the first month and second month from left to right as control, low IL-8, medium IL-8, and high IL-8.

Figure 17: Effect of different intrauterine doses of IL-8 treatment on daily milk production.

Figure 18: Effect of different intrauterine doses of IL-8 treatment on weekly milk production.

Figure 19: Effect of different intrauterine doses of IL-8 treatment on fat-corrected 3.5% milk. Bars are shown from left to right as controls, IL8-HIGH, IL8-MEDIUM, and IL8-LOW.

Figure 20: Effect of different intrauterine doses of IL-8 treatment on energy-corrected milk. Bars are shown from left to right as controls, IL8-HIGH, IL8-MEDIUM, and IL8-low.

Figure 21: Graphical representation of data showing that intravaginal administration of IL-8 significantly increases milk production. IL-8 was administered on day 0.

Detailed description of the invention

The present invention relates generally to the administration of an effective amount of IL-8 to female mammals for the purpose of improving the health of female mammals, which may include the prevention and / or treatment of one or more diseases of the uterus and / or hyperketonemia, and to increase milk production and / or fat content. in milk, and combinations thereof. The present invention thus encompasses the administration of an effective amount of IL-8 to a mammal such that milk production in the mammal is increased and / or the fat content of the milk produced by the mammal is increased and / or uterine disease is reduced in the mammal and / or hyperketonemia is reduced. ... In embodiments, the amount of milk produced by the mammal is increased and collected. In embodiments, reduced uterine disease includes, but is not necessarily limited to reduced endometritis and / or postpartum metritis and / or reduced retained placenta.

With regard to diseases of the uterus, as is known in the art, metritis generally includes inflammation of the wall of the uterus, while endometritis generally includes inflammation of the endometrium. In this regard, the discovery according to the present invention that exogenously administered IL-8 has beneficial effects on uterine health was unexpected, since among the known functions of IL-8 is its association with inflammation. In addition, the accidental discovery of the beneficial effects of IL-8 on milk fat content and milk production, as further described below, was unexpected. Considering these results, the methods of the present invention result in an increase in the health of the female mammal, as evidenced by, for example, an increase in milk production, an increase in fat-corrected milk production, an increase in energy-adjusted milk production, a decrease in the incidence of retained placenta , a decrease in the incidence or severity of metritis, or clinical endometritis, or postpartum metritis, or an improvement in body condition in a mammal at birth, or a decrease in ketosis, including, but not necessarily limited to, a decrease in hyperketonemia, or a decrease in rectal temperature, or a combination thereof. Thus, the present invention encompasses many ways in which the general health and reproductive function of female mammals can be improved.

One of ordinary skill in the art will understand that Energy Corrected Milk (ECM) is the amount of energy in milk based on milk, fat and protein and adjusted to 3.5% fat and 3.2% protein. A typical ECM formula is ECM = (0.327 X milk lb) + (12.95 X fat lb) + (7.65 X protein lb).

It is expected that the methods in accordance with the present invention will be applicable to any female mammal. In embodiments, the present invention is directed to veterinary approaches, and thus, in this aspect, relates to non-human mammals. In embodiments, the female non-human mammal administered with IL-8 is a ruminant, including, but not necessarily limited to, cattle, sheep, antelopes, deer, giraffes and their relatives, and may further include mock ruminants such as camels. In embodiments, the ruminant is a female cattle that is a member of the Bos genus, such as oxen, cows, and buffaloes. In one embodiment, the ruminant is a dairy cow. In embodiments, the dairy cow is a nulliparous or multiparous cow. In embodiments, the female mammal is an ungulate.

In one embodiment, the present invention includes the administration of IL-8 to a member of the genus Sus, and therefore encompasses the practice of the present invention to any pig, examples of which are not limited to the domestic pig (i.e., Sus domesticus), also commonly referred to as pig or domestic pig ...

The present invention also includes the administration of IL-8 to mammals other than cattle and ruminants, including, but not necessarily limited to equine, canine and feline. In embodiments, the present invention includes the administration of IL-8 to aquatic mammals such as cetacean mammals, examples of which are not necessarily limited to whales, dolphins, and porpoises. Thus, the present invention in certain aspects relates to companion animals as well as animals kept in conservation conditions, for example, in zoos or aquariums.

In addition, it is believed that the methods described in this application are acceptable for use in humans, for example, by administering IL-8 to a woman in order to increase milk production or increase the nutritional value of milk by increasing its fat content.

Certain implementations of the present invention may also preclude the introduction of IL-8 under certain circumstances. For example, in certain approaches, administration of IL-8 is not provided to a mammal that is not milked after administration of IL-8. In certain embodiments, the milk obtained after administration of IL-8 to, for example, a dairy cow is acceptable for human consumption. Thus, in certain embodiments, the administration of IL-8 occurs to a non-human mammal whose milk is expected to be and / or obtained, wherein the milk is intended for human consumption and / or is consumed by humans. In certain aspects, the present invention can exclude the administration of IL-8 to specific types of mammals. In one example, IL-8 is not administered to a rodent. The present invention may thus include the administration of IL-8 to all types of mammals, with the exception of rodents, specific examples of which include, but are not limited to, mice, rats and guinea pigs. In another example, primates, including either one or both anthropoid and non-human primates, may be excluded from the group of mammals to which IL-8 is administered. In one example, a mammal to which IL-8 is administered does not have thrombosis, including but not limited to deep vein thrombosis. In certain embodiments, the present invention may exclude the administration of IL-8 during certain periods of time, for example, in certain embodiments, the present invention may exclude the administration of IL-8 during pregnancy in order to facilitate fertilization, implantation, or in order to induce contractile activity uterus. In certain aspects, IL-8 is not administered to mammalian species in which acute inflammation after coitus is significant and / or promotes conception. In certain embodiments, IL-8 is not administered by direct infusion into breast tissue or nipple, and thus, in embodiments, administration of IL-8 does not induce or contribute to mastitis.

IL-8 is well known in the art as a chemokine produced by a number of different cell types, including macrophages. It is also referred to as CXCL8 and binds with specificity to the CXCR1 and CXCR2 receptors. It is produced as a precursor protein, which typically ranges from 99 amino acids (for human IL-8) up to 103 amino acids for other species, and is cleaved to produce the active isoforms. The cleaved version of human IL-8, which is most commonly secreted by human macrophages, is 72 amino acids in length. Therefore, while the present invention provides certain representative examples of the effect of administering recombinant bovine IL-8 to dairy cows, it is expected that any IL-8 expressed by any animal can be used in the methods of the present invention. In non-limiting embodiments, IL-8 is a recombinantly produced Bos taurus IL-8 that includes the following sequence or fragment:

MTSKLAVALL AAFLLSAALC EAAVLSRMST ELRCQCIKTH STPFHPKFIK ELRVIESGPH CENSEIIVKL TNGNEVCLNP KEKWVQKVVQ VFVKRAEKQD P (SEQ ID NO: 1).

In embodiments, IL-8 is a truncated form and is thus shorter than the IL-8 precursor sequence. In embodiments, IL-8 is at least 70 amino acids in length. In embodiments, IL-8 used in the methods of the present invention has at least 70 contiguous amino acids of IL-8, with at least 70 amino acids having at least 70.0% homology with the bovine sequence shown above and / or the consensus sequence shown in Figure 15 (bottom alignment row). In embodiments, IL-8 comprises or consists of a sequence that is 70-100% identical to a Bos Taurus sequence of 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 or more, its related amino acids. In embodiments, such sequence identity and length refers to an amino acid sequence starting at the N-terminus, or starting at any amino acid from the N-terminus through amino acid position 2-25, inclusive, and including each amino acid position in between. In embodiments, IL-8 comprises or consists of a sequence or fragment of any amino acid sequence shown in Figure 15. In one embodiment, IL-8 comprises an ELR change to AAR in the Bos Taura sequence shown in Figure 15 (SEQ ID NO: 1).

IL-8 used in the methods of the present invention can be obtained from any suitable source. In one embodiment, IL-8 is commercially obtained from a supplier. For example, human IL-8 expressed in E. coli and presented as a lyophilized powder can be obtained from Sigma Aldrich. Bovine IL-8 can be obtained from Kingfisher Biotech, Inc., Saint Paul, MN. Alternatively, IL-8 can be produced recombinantly using methods well known to those skilled in the art, for example using a protein expression system.

In a non-limiting and illustrative embodiment, IL-8 is produced recombinantly using the following approach or modifications thereof that will be obvious to one skilled in the art, given the advantages of the present invention. Plasmid construction. pET28-His-L-EK-IL8 was constructed by subcloning from Trc-His-L-EK-IL8 into pET28A (NOVAGEN, Darmstadt, Germany) using the NheI and XhoI restriction sites. The original Trc plasmid was constructed by PCR amplification of the codon optimized bovine IL-8 ΔSS cDNA using the following nucleotides; 5 '- C GGCGCC GTG CTG TCT CGT ATG TCC ACC GAA C (SEQ ID NO: 2) and 5' - G CTCGAG TCA CGG ATC TTG TTT TTC TGC ACG (SEQ ID NO: 3). The PCR product was TA cloned into the pGEM T vector (PROMEGA, Madison, WI) and was sequenced after screening for white / blue colonies. The correct clone was then digested with restriction enzymes SfoI and XhoI and ligated into the pTrcHis B vector (Invitrogen, Carlsbad, Calif.). In order to preserve the native version of IL-8 when digested with enterokinase, the Trc vector was prepared by digestion with BamHI followed by digestion with golden bean nuclease in order to remove the 5'-overhang and create a blunt end for ligation, then the vector was digested using XhoI. The final construct was confirmed by sequencing.

Expression of recombinant IL-8. In order to determine the preferred expression conditions of E. coli BL21 for the full version of IL-8 and the truncated form (no signal peptide), the coding sequences were cloned into a pET vector, a pilot time study was performed as follows. All growth stages were incubated at 37 ° C at 200 rpm. in LB broth or plates containing 300 μg / ml kanamycin, in 125 ml Erlenmeyer flasks or Petri dishes. Frozen stock cultures (-80 ° C) were reactivated overnight in 20 ml of medium. The next day, 0.4 ml of the growing culture was transferred to 40 ml of fresh medium and 1 mM IPTG was added when O.D. reached 600 nm, an aliquot of 1 ml was removed before induction of IPTG and at intervals of one hour for 4 hours. Each sample was centrifuged at 10,000 g for 5 min and the pellets were resuspended in lysis buffer (10 mM Tris-HCL; 1 mM EDTA; 0.1 N NaOH; 0.5% SDS). Then, insoluble proteins and cell debris were precipitated for 10 minutes at 13000 g at 4 ° C. The supernatant was boiled with Laemmli's buffer (63 mM Tris-HCL pH 6.8; 10% glycerol; 2% SDS (electrophoresis grade), 0.1% β-mercaptoethanol and 0.0005% bromophenol blue) for 5 minutes for loading in 12% SDS-polyacrylamide gel. Electrophoresis was performed at 80 V for 90 minutes. The gel was stained with a dye solution for 30 minutes and decolorized with a decolorizing solution (Bio-Rad) for 2 hours with vigorous shaking. We expressed (pET28-His-LEK-IL8) IL8 in E. coli. Expressed pET28-His-LEK-IL8 is partially soluble and suitable for purification.

Compositions containing IL-8 for use in the methods of the present invention can be presented in various forms and delivered in various ways. Compositions for use in humans or non-human mammals can be prepared by mixing IL-8 with any suitable pharmaceutically acceptable carriers, excipients and / or stabilizers. Some examples of compositions suitable for mixing with IL-8 can be found in: Remington: The Science and Practice of Pharmacy (2005) 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins. In certain aspects, IL-8 can be added to the diet of a mammal, and thus consumed as a dietary supplement to support reproductive health and / or milk production.

Compositions containing IL-8 can be administered to a mammal using any available method and route, including oral, parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intravaginal, intrauterine, and subcutaneous administration. The composition can be administered by an intramucosal approach. Administration of IL-8 can be performed before or after childbirth and can be performed during pregnancy.

In certain embodiments, subject to certain provisions, as further described in this application, compositions containing IL-8 are administered to a pregnant mammal and, thus, prenatal administration is used. In certain approaches, prenatal administration is performed during the period of mammogenesis, which varies from species to species, but is in the last third of the gestation period. As a non-limiting illustration, in one embodiment, the gestation period of a Holstein cow is 280 days. Thus, it is believed, without intending to be bound by theory, that administration of IL-8 after approximately 180 days of gestation aids breast development, resulting in increased milk production in the postpartum period.

In one embodiment, prenatal administration comprises intravaginal administration of a composition comprising IL-8. In one non-limiting example, intravaginal administration of a composition containing IL-8 is performed on a pregnant mammal, such as a dairy cow.

In certain embodiments, compositions comprising IL-8 are administered to a mammal that has recently given birth, and thus postpartum administration is used. In embodiments, postpartum intrauterine administration is used. In one non-limiting example, postpartum intrauterine administration of a composition containing IL-8 is administered to a mammal, such as a dairy cow, within 72 hours of delivery. Administration within a shorter or longer time postpartum is also encompassed by the present invention. In certain non-limiting examples, a composition containing IL-8 is administered immediately postpartum, and up to 20 weeks postpartum. In certain approaches, the present invention thus includes administration on the same day as labor, or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 days, inclusive, and including all ranges of integers in between.

In certain approaches, the present invention includes, as an alternative to the administration of exogenous IL-8, stimulating the production of endogenous IL-8, such that one or more of the effects described in this application are obtained. In non-limiting examples, stimulating the production of exogenous IL-8 includes administering to a mammal one or more IL-8 stimulating compounds and / or compositions, including, but not necessarily limited to, tumor necrosis factor alpha (TNF-α), lipopolysaccharide (LPS), interleukin 1 (IL-1), platelet activating factor (PAF), and / or other substances that can be used in embodiments of the present invention, taking into account the beneficial effect of the present invention.

In certain embodiments, the present invention comprises administering a composition comprising IL-8 to one or more mammals, a non-limiting example of which is dairy cow (s), such that any one or any combination of the following is achieved: i) an increase in milk production; ii) an increase in energy-adjusted milk production; iii) increasing the fat content of milk produced by mammals; iv) reducing the development of postpartum metritis and / or reducing the incidence of postpartum metritis when IL-8 is administered to multiple mammals; v) reducing the development of clinical endometritis and / or decreasing the incidence of clinical endometritis when IL-8 is administered to multiple mammals; vi) reducing hyperketonemia, such as reducing subclinical ketosis and / or the incidence of subclinical ketosis when IL-8 is administered to multiple mammals; and vii) inhibiting retained placenta. In embodiments, the above effects of administering IL-8 are achieved by using intrauterine administration of a composition containing IL-8, but it is believed that other routes of administration can also be used.

It will be recognized that any of the above results obtained from IL-8 administration can be compared with a reference value to assess the effect of IL-8 administration. Any suitable reference value can be used, and one skilled in the art will determine acceptable reference values given the beneficial effect of the present invention. In embodiments, the reference value can be a single value or a range of values. For example, the reference value can be a standardized curve or area on a graph. The reference value can include a positive or negative control. In embodiments, the reference value includes a measurement made from a sample obtained from a mammal that has not received IL-8, or has received a different amount of IL-8, or used a different dosing regimen for IL-8. In various aspects, the measurement of the result can be compared with a reference value to provide a qualitative or quantitative determination of the result, which can be positively or negatively correlated with the administration of IL-8. In certain embodiments, comparison with a reference value can be performed by a person skilled in the art of animal processing or research. For example, retained placenta and metritis can be diagnosed by trained farm personnel according to specific protocols known in the art, and specific measurements compared to a condition with no retained or no metritis can be made by those skilled in the art, regardless of whether whether or not a direct comparison is made with an acceptable reference value. For example, in certain embodiments, an altered uterine discharge, such as the appearance of a fetid, watery, red-brown uterine discharge accompanied by fever, can be used to diagnose postpartum metritis, whereas postpartum cows that do not produce uterine secretions with such characteristics, found to have no postpartum metritis.

The present invention includes the administration of IL-8 to any one or more mammals, for example, a plurality or population of mammals. In one embodiment, the plurality of mammals includes a group of dairy cows that may be present, for example, in a dairy farm of any size, which ranges from a few dairy cows to a commercial dairy farm that can house thousands of dairy cows.

As will be seen from the results presented in the examples and figures of the present invention, representative but non-limiting experiments demonstrate the above listed effects using intrauterine and intravaginal infusions, including a range of amounts of IL-8. Specific and non-limiting examples demonstrate aspects of the present invention using 9.5 mg, 1.125 mg, 0.095 mg, 0.0095 mg, and 11.25 μg of recombinant IL-8. Thus, the present invention demonstrates that a wide range of amounts of IL-8 can produce some or all of these effects, and given the beneficial effect of the present invention, one skilled in the art will understand how to modify the dose of IL-8 to obtain the desired result in any a specific mammal. In addition, the present invention includes the demonstration that doses of IL-8 that range from 9.5 mg to as little as 0.0095 mg result in a statistically significant increase in the percentage of milk fat. Accordingly, the present invention includes the administration of an effective amount of IL-8, wherein the effective amount of IL-8 is the amount that results in the desired result. In one embodiment, the amount of IL-8 is from 0.001 μg to 10 mg, including all integers and amounts in between up to 0.001 μg units, and all ranges of μg and mg in between. In embodiments, at least 11.25 μg of IL-8 is administered to the mammal. In this regard, the form and nature of a particular IL-8 dosing regimen will be determined by administration and other known variables taking into account factors such as size, health, age, type of mammalian species, number of previous births (if any), previous history of the uterus or other related conditions and risk factors associated with uterine health and milk production. In one embodiment, the mammal requires administration of IL-8 because of, for example, a risk or other predisposition to a uterine condition, or because of poor milk production. In embodiments, the administration of IL-8 is prophylactic or therapeutic, or both.

The IL-8 compositions of the present invention may be administered once or in a series of doses, and may be administered concurrently or sequentially with any other compound or composition intended to improve the overall health of a mammal, or for the specific purpose of promoting or enhancing IL-8 induced effects described in this application. In embodiments, the administration of IL-8 is used in combination with an antibiotic, hormone, or growth factor. In certain approaches, IL-8 is administered only once, but has a long-term effect on any one or a combination of health and / or milk production outcomes as described herein.

Administration of the IL-8 composition may result in an increase in milk production and / or milk with an increased fat content, for various periods of time after administration. The desired milk fat content can be determined using any suitable method, some of which are known in the art. For example, the milk fat content can be determined using the so-called Babcock test or the Gerber method. In embodiments, the fat content of the milk is increased. The present invention provides for the demonstration of an increase in milk fat in milk obtained from dairy cows after intrauterine and intravaginal infusions of recombinant IL-8. Thus, in certain approaches, the present invention includes methods for promoting higher fat milk production, and includes milk produced by such methods.

In certain aspects, the present invention includes increasing the milk fat in milk produced by a dairy cow relative to controlling, for example, the amount of milk fat in milk produced by a dairy cow that has not received IL-8 administration. In certain approaches, the increased milk fat includes an increase in milk fat (relative to control) of at least 0.01% to 0.5%, inclusive, and including all numbers up to the second after the decimal point in the decimal between them, and all ranges of such numbers. In certain approaches, the milk obtained in accordance with an embodiment of the present invention contains at least 3.4% milk fat, and may contain from 3.4% to 4.4% milk fat, including all numbers up to the second after comma in decimal between them, and all ranges of such numbers. In certain approaches, milk contains such amounts of milk fat when first obtained from a mammal. Thus, these amounts may be present in unprocessed milk.

The practice of the methods of the present invention has, in certain embodiments, one or more effects on a mammal that are long-term over a period of time. For example, we have demonstrated an increase in milk production in both fat-corrected and energy-corrected milk over 11 months after a single dose of IL-8. In certain embodiments, administration of IL-8 results in increased milk production or increased fat-corrected and / or energy-adjusted milk production and / or increased fat content in milk, over a period of at least 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, or 11 months after IL-8 administration. Longer periods of time are also covered. In certain approaches, IL-8 administration has a long-term effect on milk production that lasts for one lactation period, that is, the entire lactation period, immediately after or during which IL-8 is administered. In one example, lactation ends with the next pregnancy. The present invention includes, in one non-limiting approach, the administration of IL-8 in a single dose such that one or more effects on milk content and / or milk production, as described herein, persist for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 months, or the entire lactation period, after or during which a single dose of IL-8 is administered. In certain embodiments, one or more of the effects of IL-8 begins within a period of 1, 2, 3, 4, 5, 6, or 7 days after administration of IL-8 and may persist thereafter according to any of the time periods described in this application.

In certain aspects, the present invention includes increasing the amount of milk produced by mammals, such as a dairy cow. In certain aspects, the increase in milk production includes an increase of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 pounds of milk per day. The increase in milk production can be assessed in relation to a control such as a dairy cow that does not receive IL-8. One of ordinary skill in the art will understand that the value of any IL-8-induced change described herein can be taken as, for example, an average value determined from a group of mammals over a period of time.

In another embodiment, the present invention includes milk production, and includes milk itself, from a mammal treated with IL-8 as described above. This aspect includes administering IL-8 to a female mammal and collecting milk generated after administration. In one embodiment, the milk produced according to the method differs from other types of milk in that it has an increased fat content, such as milk fat content, as described above. In embodiments, containers are provided containing milk from a mammal treated with IL-8. The containers can be any container such as a consumer-oriented container such as a milk carton, or larger containers such as vats or containers suitable for sea transport or other transport of large quantities of milk. In embodiments, provided are products made using milk obtained according to the method described herein. Non-limiting examples of such products include cheese, yoghurt, milk-based creams and creams, ice cream, dairy-based fillings, and any other dairy product made with said milk. Thus, in embodiments, the dairy product may contain a milk derivative, such as one or more separated milk components, including, but not limited to, milk fat. Accordingly, milk can be processed to separate milk components for inclusion in various dairy products. The present invention includes the manufacture of such products using conventional approaches, but by replacing previously available milk with milk in accordance with the present invention.

In another aspect, the present invention provides a finished product such as a kit. The kit may include a pharmaceutical composition containing IL-8 in one or more sealed containers, that is, glass or plastic vials. The kit may include a syringe, catheter, or other delivery device. For example, in the case of a catheter, this could be an artificial insemination (A.I.) catheter, such as the Gilt A.I. or equivalents. The kit may also contain a pouch, such as a pouch, that is acceptable to contain the solution and is adapted for use with a catheter for administering the solution to a mammal, for example, by intravaginal or intrauterine delivery. The kit may optionally include instructions for the use of its contents, either written on paper or in a machine-readable format. The kit may also contain IL-8 to be mixed with a carrier, such as IL-8 in lyophilized form, in which case the kit may further include instructions for reconstituting the lyophilized IL-8 in the vehicle / solution for administration to a mammal. For example, the carrier can be sterile water, saline, or phosphate buffered saline. The carrier can be provided in one or more separate vials.

In another aspect, the present invention includes a finished product. The finished product contains IL-8 provided in the package. The package may include a container, or may itself be a container. Any suitable container can be used, such as a plastic or glass container, including, but not limited to, plastic or glass vials. In various embodiments, the finished product includes printed matter. The printed material may be part of the package, or may be provided on a label, or as a paper insert or other written material included in the package. The printed material provides information identifying IL-8 as the contents of the package and instructs the consumer how to use IL-8 to obtain any one or any combination of effects on mammals as described herein.

In view of the above, and without intending to be bound by any particular theory, the present invention relates in part to the observation that a factor contributing to increased susceptibility to diseases of the uterus is the immunosuppression experienced by cows during the perinatal period (Drackley, 1999 ; Cai et al., 1994; Kimura et al., 1999; Hammon et al., 2006; Galvao et al., 2010). Neutrophils are the main leukocyte type involved in placental separation (Kimura et al., 2002) and in bacterial clearance following infection of the uterus (Hussain, 1989) and breast (Paape et al., 2002). Blood neutrophil function begins to decline before delivery, reaches extreme decline shortly after delivery, and slowly returns to prenatal levels approximately 4 weeks after delivery (Kehrli and Goff, 1989; Goff and Horst, 1997). Several factors may explain the loss of neutrophil function, such as increased concentrations of estradiol and cortisol in the blood before calving, and deficiencies in nutrients and minerals such as vitamins A and E, calcium and selenium (Goff and Horst, 1997; Kimura et al., 2002 ; Hammon et al., 2006). In addition, neutrophils from cows with retained placental separation (RP) also had reduced migratory capacity and decreased myeloperoxidase activity (Kimura et al., 2002). Cows with the highest influx of neutrophils into the uterus have a reduced risk of bacterial infection and a reduced incidence of endometritis (Gilbert et al., 2007). It is also believed that migration of neutrophils into the mammary gland plays a role in the clearance of mastitis pathogens (Paape et al., 2002). IL-8 is a neutrophil chemoattractant; binding of IL-8 to its receptors (CXCR1 and CXCR2) in the neutrophil causes the activation of neutrophils, stimulates chemotaxis, and increases the ability to phagocytosis and kill (Mitchell et al., 2003). Since neutrophils play a role in maintaining endometrial health, it is believed that an appropriate stimulus is needed to selectively attract neutrophils to the uterus. However, continued inflammation as a result leads to the development of chronic uterine disease, which impairs the ability to reproduce and reduces the profitability of dairy products (Dubuc et al., 2011; Lima et al., 2013). Therefore, the present invention provides a paradoxical approach in one aspect of which it provides treatment and / or prevention of uterine conditions known to positively correlate with inflammation by administering IL-8, which is also known to promote inflammation. In this regard, and without intending to be limited by theory, we developed the present invention, investigating whether the administration of IL-8, despite its pro-inflammatory properties, can still attract and activate neutrophils in the uterus, resulting in an early influx of neutrophils into the uterine cavity. early detachment of the placenta, early clearance of bacterial contamination and, ultimately, the final positive result of healthier, more fertile dairy cows. As seen in the following examples, administration of IL-8 results in healthier and more fertile dairy cows and unexpectedly improves their milk production and milk fat content.

The following examples illustrate specific embodiments of the present invention and are not intended to limit it.

EXAMPLE 1

This example provides a description of the material and methods used to obtain the data described in example 2.

Farm and management

The study was conducted at a large commercial dairy farm located in Cayuga County near Ithaca, New York (NY). The farm milked 3,300 Holstein cows 3 times a day in a double parallel milking parlor with 52 stalls. The cows were housed in open-type stalls with concrete stalls covered with mattresses and bedding with dung solids. All cows were offered a TMR consisting of approximately 55% roughage (corn silage, haylage and wheat straw) and 45% concentrate (corn flour, soy flour, rapeseed, cottonseed and citrus pulp) based on DM. The diet was formulated to meet or exceed the NRC nutrient requirements for 650 kg lactating Holstein cows producing 45 kg 3.5% FCM (NRC, 2001). Farm reproductive management used a combination of Presynch, Ovsynch, Resynch, and estrus detection, with 25 to 30% of cows raised with a time-aligned AI and the remainder raised after estrus detection was purely activity monitoring (Alpro; DeLaval, Kansas City, MO).

Study design, treatment and sample collection

A total of 217 fresh cows were enrolled in the study. The cows were grouped by number of births and randomly assigned to one of three treatment groups: control, low dose IL-8 (L-IL8), and high dose IL-8 (H-IL8). Cows dispensed into H-IL8 and L-IL8 received an intrauterine infusion of 250 ml of saline containing 1.125 and 11.25 μg of recombinant IL-8, respectively. IL-8 was obtained using the above-described pET28-His-LEK-IL8 and consisted of the sequence SEQ ID NO: 1.

Control cows received an intrauterine infusion of 250 ml of saline solution as placebo. All fresh cows that were available during the registration period were included in the study. The random distribution was completed in Excel (Microsoft, Redmond, WA) using a random number function and imported into the Dairy Comp 305 farm program (Valley Agricultural Software, Tulare, CA).

Treatment was carried out no longer than 14 hours postpartum by the research team as follows: cows were restrained and the perineal area was cleaned and disinfected with 70% ethanol. Then the sterile catheter "Gilt" A.I. (Livestock concepts, Hawarden) attached to a 250 ml saline bag was inserted cranially into the vagina. The catheter was inserted into the uterus and the tip was applied to the uterine cavity and the treatment was pumped into the uterus. A sample and using a swab was collected from the tip of the catheter; she was cultured aerobically on CHROMagar-E. coli (CHROMagar, Paris, France) at 37 ° C.

Milk and blood samples were collected from 60 cows (20 cows per treatment group) during the first four days of lactation. To obtain serum samples, blood was collected from the coccygeal vein / artery using a Vacutainer tube without anticoagulant and a 20 gauge Vacutainer 62.54 cm needle (Becton, Dickinson and Company, Franklin Lakes, NJ). All blood samples were transferred to the laboratory on ice and centrifuged at 2000xg for 15 min at 4 ° C; serum was collected and frozen at -80 ° C.

Identification of the disease

Retention of placenta and metritis was diagnosed and treated by trained farm staff according to specific protocols developed by the Ambulatory and Production Medicine Clinic at Cornell University. After giving birth, cows were kept in one pen for up to 20 DIM. This pen was monitored by farm workers and the cows were sent for a full physical examination if they showed signs of lethargy and depression; cows with fetid, watery, reddish-brown uterine secretions accompanied by fever were diagnosed with postpartum metritis and were treated by farm workers. The farm staff performed blind treatment. Retained placental separation was defined as a condition in which cows were unable to detach their embryonic membranes within 24 hours after calving.

Clinical endometritis was assessed by the investigators at 35 ± 3 DIM and was defined as the presence of purulent or mucopurulent discharge by extracting vaginal mucus using a Metricheck device (Metricheck, SimcroTech, Hamilton, New Zealand). Vaginal discharge was assessed using a modified 0 to 5 scale (0 = no secretory material removed, 1 = clear mucus, 2 = pus spots in vaginal discharge, 3 = <50% pus in vaginal discharge, 4 => 50% pus in vaginal discharge, 5 = watery, fetal vaginal discharge). Cows that had a score ≥ 3 were considered clinical endometritis.

Rectal temperature was measured at registration, 3, 6 and 9 DIM using a digital thermometer (GLA M750, GLA Agriculture Electronics, Calif.) Equipped with an angled probe (11.5 cm, 42 °). Body condition scores were recorded at check-in and at 35 days of lactation (DIM) by one investigator using a 5-point quarter-point scale as previously described (Edmonson et al., 1989). Body condition score loss was defined as the difference between body condition score (BCS) at registration and 35 DIM.

Calf data (female, male, twins and stillbirth), labor assistance, day of pregnancy at birth, milk production and somatic cell counts were retrieved from the DairyComp 305 farm database (Valley Agricultural Software, Tulare, CA).

Blood and milk parameters

IL-8 concentrations in serum and milk samples were determined using a human IL-8 ELISA kit (R&D Systems Inc., Minneapolis, Minn.), Which was validated for use in cattle. Serum samples were also tested for beta-hydroxybutyrate (BHBA) concentration using a BHBA electronic measuring system (Precision Xtra, Abbott, Abingdon, UK) already approved for use in animals. Cows tested with more than 1.2 μmol / L BHBA on at least one of the first four days of lactation were considered to be in subclinical ketosis. Serum glucose levels were measured using a portable blood glucose meter (Accu-Check Active, Roche Diagnostics, Indianapolis, IN). Serum IGF-1 levels were determined using a human IGF-1 ELISA kit (R&D Systems Inc., Minneapolis, Minn.).

Serum haptoglobin concentration was determined using a colorimetric technique that measures the haptoglobin / hemoglobin complex by assessing differences in peroxidase activity. In short, 5 μl of plasma or distilled water (for blank determination) was added to 7.5 ml of O-dianisidine solution (0.6 g / L O-dianisidine, 0.5 g / L EDTA and 13.8 g / L sodium phosphate monobasic in distilled water; pH adjusted to 4.1) in a borosilicate tube. Twenty-five microliters of hemoglobin solution (0.3 g / L bovine hemoglobin in distilled water) was immediately added to each tube. All tubes were incubated in a water bath set at 37 ° C for 45 min. After incubation, 100 μl of freshly prepared hydrogen peroxide solution with a working concentration of 156 mM was added to each tube. All tubes were incubated for 1 hour at room temperature. After incubation, 200 μl from each tube was transferred to one well in a 96-well flat-bottomed polystyrene microplate, and the optical density (OD) was read immediately at 450 nm in a microplate reader (BioTek Instruments, Model EL 340, Winooski, VT). The OD of the blank sample was subtracted from the OD of all samples containing plasma. The results were presented as absorbance readings at 450 nm, considering that the method used does not contain a standard curve.

Statistical analysis

Analysis descriptive statistics was performed in JMP ^® PRO 10 using ANOVA and distribution function of the chi-square test for continuous and categorical variables, respectively. Ten mixed general linear models were established from the data using the MIXED SAS procedure (SAS Institute). The dependent variables that were evaluated in this study were: average daily milk production (kg / day), average fat-corrected milk production (kg / day), average daily energy-corrected milk (kg / day), linear SCC score, concentration Blood BHBA (μmol / L), blood IL-8 concentration (pg / ml), blood haptoglobin, blood IGF-1 concentration (ng / ml), serum glucose concentration (mg / dL) and rectal temperature ( ° C). The data included a series of repeated measurements for each dependent variable during the first eight weeks of lactation for average daily milk production; first two months of lactation for average fat-corrected milk production, average daily energy-adjusted milk production and linear SCC score; the first four days of lactation for blood BHBA concentration, blood IL-8 concentration, blood haptoglobin, blood IGF-1 concentration, and serum glucose concentration; and at 3, 6 and 9 days postpartum for rectal temperature. In order to properly assess the correlation within cows, the magnitude of the error was modeled by superimposing a first-order autoregressive covariance structure for all statistical models (which assumed a correlation between repeated measurements within cows). The independent variables proposed for the models were: treatment, number of births, placenta availability at check-in, E. coli intrauterine culture result, birth assistance, calf, body condition score at birth, day of pregnancy at birth, temperature at check-in. and the time of data collection. The models were offered biologically plausible two-way and three-way relationships. In addition, the variables and their corresponding association effects in all models were retained in the model at a P-value <0.10. The variable "treatment" was forced in all models.

In order to assess the effect of treatment on the likelihood of postpartum metritis, clinical endometritis, and subclinical ketosis, three mixed logistic regressions were established on the data using the GLIMMIX SAS procedure. The models included fixed effects of treatment, number of births, placenta at check-in, IUD culture of E. coli, labor and calf care, body condition score at birth, day of pregnancy at birth, and temperature at check-in. The models were offered biologically plausible two-way and three-way relationships. Moreover, the variables and their respective relationship effects in all models were retained in the model at a P-value <0.10. The variables "treatment", "number of births" and the effect of the relationship between number of births and treatment were constrained in all models. In order to obtain the parameters of the ratio of probability to specific groups for different concentrations of the influence of the relationship between the number of births and treatment, the root-mean-square mean of the GLIMMIX procedure (binary distribution) was used. P-values were adjusted for multiple comparisons using the HSD Tukey test. The chi-square distribution function of JMP®PRO 10 was used to estimate the proportion of cows that had detectable levels of IL-8 in milk samples and the incidence of postpartum metritis, clinical endometritis and subclinical ketosis.

EXAMPLE 2

This example represents the results obtained using the materials and methods described in example 1.

Table 1 presents descriptive statistics on the number of registered multiparous and primiparous animals, the number of registered animals that were positive for an intrauterine culture of E. coli, the number of registered animals with a placenta present at registration, the number of registered animals assisted in childbirth, the number registered animals that gave birth to a female, male, twins or stillborn calves, the average number of days of pregnancy at the time of delivery, the average fatness score at birth and the average rectal temperature at registration.

Table 1: Descriptive statistics of 213 cows studied enrolled in three treatment groups.

Control IL-8
(11.25 mcg) IL-8
(1125.00 mcg) P-value Registered primiparous cows (%) 32 (48) 41 (51) 31 (47) 0.86 Registered multiparous cows (%) 35 (52) 39 (49) 35 (53) Total 67 80 66 Reported cows testing positive for intrauterine E. coli (%) 42 (63) 39 (49) 30 (45) 0.10 Cows with placenta present at check-in (%) 21 (31) 23 (29) 27 (41) 0.28 Registered cows assisted in childbirth 2 (3) 3 (4) 12) 0.71 Registered cows that gave birth to heifers (%) 37 (55) 38 (48) 37 (56) 0.28 Registered cows that gave birth to bulls (%) 23 (34) 37 (46) 25 (38) Registered cows with twins at birth (%) 4 (5) 0 (0) 2 (3) Registered cows that had a stillbirth at birth (%) 3 (6) 5 (6) 2 (3) Average number of days of pregnancy at the time of delivery 274.9 277.4 275.2 0.06 Average BCS at birth 3.36 3.41 3.40 0.42 Average rectal temperature at registration 38.7 38.8 38.8 0.59 Total number of registered animals (%) 67 (31) 80 (38) 66 (31)

The effect of treatment on milk production per week of lactation for nulliparous and multiparous cows is shown in Figure 1. Overall milk production was greater in cows treated with IL-8; 33.1 kg / day (95% CI = 32.0 - 34.2), 35.6 kg / day (95% CI = 34.5 - 36.7) and 35.9 kg / day (95% CI = 34.9 - 37.0) for control cows, cows with L-IL8 and H-IL8, respectively (P-value <0.01). The variables “number of births”, “calf”, “average number of days of pregnancy at the time of delivery”, “rectal temperature at registration” and “week of lactation” were retained in the model. The relationship between treatment and lactation week was not significant (P-value = 0.06).

The effect of treatment on fat-corrected milk production during the first two months of lactation is shown in Figure 2. Total fat-corrected milk production was higher for cows treated with IL-8; 34.2 kg / day (95% CI = 32.6 - 35.7), 37.1 kg / day (95% CI = 35.7 - 38.5) and 36.6 kg / day (95% CI = 35.0 - 38.1) for control cows, cows with L-IL8 and H-IL8, respectively (P-value = 0.02). The variables “number of births,” “body condition score at delivery,” “mean number of days of pregnancy at the time of delivery,” “rectal temperature at registration,” and “month of lactation” were retained in the model. The relationship between treatment and month of lactation was not significant (P-value = 0.89).

The effect of treatment on energy-corrected milk production during the first two months of lactation is shown in Figure 3. Total energy-corrected milk production was higher for cows treated with IL-8; 32.8 kg / day (95% CI = 30.9 - 34.6), 35.7 kg / day (95% CI = 34.0 - 37.3) and 36.2 kg / day (95% CI = 34.3 - 38.1) for control cows, cows with L-IL8 and H-IL8, respectively (P-value = 0.02). The variables “number of births,” “body condition score at delivery,” “mean days of pregnancy at the time of delivery,” and “month of lactation” were retained in the model. The relationship between treatment and month of lactation was not significant (P-value = 0.56).

The effect of treatment on the linear SCC score during the first two months of lactation is shown in Figure 4. The overall linear score for somatic cell count did not differ between treatment groups; 2.46 (95% CI = 1.64 - 3.28), 2.44 (95% CI = 1.66 - 3.23) and 2.48 (95% CI = 1.65 - 3.31) for control cows, cows with L-IL8 and H-IL8, respectively (P-value = 0.99). The variables "help with childbirth" and "month of lactation" were retained in the model. The relationship between treatment and month of lactation was not significant (P-value = 0.09).

The effect of treatment on the farm diagnosed incidence of postpartum metritis depended on the number of births and is presented in Figure 5; the relationship between treatment and number of births was significant (P-value <0.01). For primiparous animals, cows with H-IL8 had a 7.43 higher probability of having postpartum metritis compared to control cows (P-value = 0.03), while the likelihood of having postpartum metritis for cows with L-IL8 and control cows was not. differed (P-value = 0.27). On the other hand, in multiparous animals, intrauterine treatment with IL-8 had a protective effect against postpartum metritis; control cows had a 7.14 (P-value = 0.02) and 5.88 (P-value = 0.02) increased likelihood of having postpartum metritis than cows with L-IL8 and H-IL8, respectively. In one non-limiting embodiment, the present invention relates to the prevention of postpartum metritis in multiparous animals. The effect of treatment on the incidence of clinical endometritis is shown in Figure 6. Intrauterine infusion of IL-8 did not confer protection against clinical endometritis. (P-value = 0.73).

The total concentration of BHBA in the blood was 0.77 μmol / L (95% CI = 0.65 - 0.90), 0.62 μmol / L (95% CI = 0.50 - 0.74) and 70 μmol / L (95% CI = 0.58 - 0.82) for control cows, cows with L-IL8 and H-IL8, respectively (P-value = 0.22). In addition, the relationship between treatment and DIM was not significant (P-value = 0.66, Figure 7).

The total concentration of IL-8 in the blood was 235.0 pg / ml (95% CI = 193.9 - 276.0), 275.5 pg / ml (95% CI = 233.7 - 317.4) and 287, 5 pg / ml (95% CI = 246.0 - 329.0) for control cows, cows with L-IL8 and H-IL8, respectively (P-value = 0.17, figure 8). The variables "rectal temperature at registration" and "DIM" were stored in the model. The relationship between treatment and DIM was not significant (P-value = 0.16).

Most of the milk samples tested for IL-8 concentration had levels below the detection limit of the assay used (1.5 pg / ml). Of all 182 samples tested, only 13.7% (25 samples) had an IL-8 concentration above the detection limit. Therefore, a very inconsequential conclusion can be drawn regarding the effect of intrauterine infusion of IL-8 on the concentration of this cytokine in milk. The proportion of pretreatment samples that had IL-8 concentrations above the detection limit was 40.0%, 43.0% and 23.5% for controls, L-IL8 and H-IL8, respectively (P-value = 0 ,ten). The proportion of samples collected after treatment with IL-8 levels above the detection limit of the assay was 2.2%, 20.0% and 0.0% for controls, L-IL8 and H-IL8, respectively (P-value <0, 01).

Total rectal temperature was 38.8 ° C (95% CI = 38.7 - 38.9), 38.8 ° C (95% CI = 38.8 - 38.9) and 38.8 ° C (95% CI = 38.8 - 38.9) for control cows, cows with L-IL8 and H-IL8, respectively (P-value = 0.47, figure 9). The variables “placenta at registration,” “body condition score at delivery,” “days of pregnancy at delivery,” and “DIM” were retained in the model. The relationship between treatment and DIM was not significant (P-value = 0.13). At 6 DIM, control cows had lower rectal temperatures compared to cows with H-IL8 (P-value = 0.02).

Haptoglobin levels during the first four days postpartum were unaffected by treatment (Figure 10); the total haptoglobin level was 0.13 (95% CI = 0.11 - 0.16), 0.13 (95% CI = 0.11 - 0.16) and 0.13 (95% CI = 0.11 - 0.15) for control cows, cows with L-IL8 and H-IL8, respectively (P-value = 0.96). The variables “number of births”, “calf” and “DIM” were retained in the model. The relationship between treatment and DIM was not significant (P-value = 0.48).

Treatment had no effect on body condition scores from day of delivery to 35 DIM (Figure 11). The average decrease in body condition in points was 0.23 (95% CI = 0.15 - 0.30), 0.23 (95% CI = 0.16 - 0.30) and 0.23 (95% CI = 0, 16 - 0.31) for control cows, cows with L-IL8 and H-IL8, respectively (P-value = 0.99). The variable "number of births" was stored in the model.

Total serum IGF-1 levels were 149.9 ng / ml (95% CI = 133.2 - 166.5), 172.0 ng / ml (95% CI = 153.6 - 190.4) and 153, 9 ng / ml (95% CI = 136.5 - 171.4) for control cows, cows with L-IL8 and H-IL8, respectively (P-value = 0.18, figure 12). The variable "DIM" was stored in the model. The relationship between treatment and DIM was not significant (P-value = 0.25). However, cows with L-IL8 had or are able to have higher blood IGF-1 levels on day 1 (P-value = 0.09), 2 (P-value = 0.01) and 3 (P-value = 0 , 08) after childbirth.

Total serum glucose was 218.5 mg / dL (95% CI = 195.2 - 241.8), 220.5 mg / dL (95% CI = 196.7 - 244.3), and 241.6 mg / dl (95% CI = 218.6 - 264.5) for control cows, cows with L-IL8 and H-IL8, respectively (P-value = 0.11, figure 13). The variables “number of births”, “calf” and “DIM” were retained in the model. The relationship between treatment and DIM was not significant (P-value = 0.53).

The effect of treatment on the incidence of subclinical ketosis is shown in Figure 14. Treatment with L-IL8 had a protective effect against subclinical ketosis; control cows had an 8.04 increased likelihood of having subclinical ketosis than cows with L-IL8 (P-value = 0.02). However, the incidence of subclinical ketosis did not differ between control cows and cows with H-IL8 (P-value = 0.30).

EXAMPLE 3

This example reproduces the approach described in examples 1 and 2 above, and further demonstrates that a wider dosage range of rIL-8 is effective for improving milk production and milk content. The study was also conducted on a commercial dairy farm located in Cayuga, NY. A total of 341 fresh cows were enrolled in the study in 116 days. Within 12 hours postpartum, cows were randomly assigned to receive intrauterine infusions with 9.5 mg rIL-8 (high IL-8, n = 86), 0.095 mg rIL-8 (mean IL-8, n = 82), 0.0095 mg rIL8 (low IL-8; n = 88), or no treatment (control; n = 85). Postpartum cows were treated within 12 hours of delivery. The cows were restrained in head restraint stations and their crotch area was disinfected with ethanol (70% v / v). A sterile Gilt artificial insemination catheter (Livestock Concepts Inc., Hawarden, IA), attached to a 250 ml saline bag containing the appropriate dose of rIL8, was inserted into the cranial vagina, manipulated through the cervix, and the treatment was infused into the uterine cavity ... Milk production was recorded daily, and monthly milk samples were submitted to a laboratory (DairyOne, Ithaca, NY) to evaluate milk components (protein and milk fat). Energy-corrected and fat-corrected 3.5% milk was calculated and reported in this application.

Treatment with rIL8 increased the percentage of milk fat regardless of the dose of treatment (Figure 16). Treatment with rIL8 also increased daily milk yield (Figure 17), weekly milk yield (Figure 18), fat-corrected 3.5% milk (Figure 19), and energy-corrected milk (Figure 20). The results are consistent with those obtained in the experiments described in Examples 1 and 2 and show that the rIL8-treated cows produced on average 10 pounds more milk per day compared to the controls.

EXAMPLE 4

This example demonstrates the intravaginal administration of IL-8. In order to obtain the data shown in Figure 21, a total of 60 cows were randomly assigned to receive placebo treatment (sterile saline; n = 30) or 1.125 mg rIL-8 (n = 30). Treatments and placebo were administered intravaginally. The registered cows were 30-80 days after delivery (late lactation). As can be seen from Figure 21, intravaginal treatment with IL-8 significantly increased milk production.

Although the invention has been described in specific embodiments, conventional modifications will be apparent to those skilled in the art, and such modifications are intended to be within the scope of the present invention.

Sources

Cai, T. Q., P. G. Weston, L. A. Lund, B. Brodie, D. J. McKenna and W. C. Wagner. 1994. Association between neutrophil functions and periparturient disorders in cows. Am. J. Vet. Res. 55: 934-943.

Drackley, J. K. 1999. ADSA foundation scholar award. biology of dairy cows during the transition period: The final frontier? J. Dairy Sci. 82: 2259-2273.

Dubuc, J., T. F. Duffield, K. E. Leslie, J. S. Walton and S. J. Leblanc. 2011. Randomized clinical trial of antibiotic and prostaglandin treatments for uterine health and reproductive performance in dairy cows. J. Dairy Sci. 94: 1325-1338.

Edmonson, A. J., I. J. Lean, L. D. Weaver, T. Farver, and G. Webster. 1989. A body condition scoring chart of Holstein dairy cows. J. Dairy Sci. 72: 68-78.

Galvao, K. N., M. J. Flaminio, S. B. Brittin, R. Sper, M. Fraga, L. Caixeta, A. Ricci, C. L. Guard, W. R. Butler and R. O. Gilbert. 2010. Association between uterine disease and indicators of neutrophil and systemic energy status in lactating holstein cows. J. Dairy Sci. 93: 2926-2937.

Gilbert, R. O., S. T. Shin, C. L. Guard, H. N. Erb and M. Frajblat. 2005. Prevalence of endometritis and its effects on reproductive performance of dairy cows. Theriogenology. 64: 1879-1888.

Goff, J. P. and R. L. Horst. 1997. Physiological changes at parturition and their relationship to metabolic disorders. J. Dairy Sci. 80: 1260-1268.

Hammon, D. S., I. M. Evjen, T. R. Dhiman, J. P. Goff and J. L. Walters. 2006. Neutrophil function and energy status in holstein cows with uterine health disorders. Vet. Immunol. Immunopathol. 113: 21-29.

Hussain, A. M. 1989. Bovine uterine defense mechanisms: A review. Zentralbl. Veterinarmed. B. 36: 641-651.

Kehrli, M. E., Jr and J. P. Goff. 1989. Periparturient hypocalcemia in cows: Effects on peripheral blood neutrophil and lymphocyte function. J. Dairy Sci. 72: 1188-1196.

Kimura, K., J. P. Goff and M. E. Kehrli Jr. 1999. Effects of the presence of the mammary gland on expression of neutrophil adhesion molecules and myeloperoxidase activity in periparturient dairy cows. J. Dairy Sci. 82: 2385-2392.

Kimura, K., J. P. Goff, M. E. Kehrli Jr and T. A. Reinhardt. 2002. Decreased neutrophil function as a cause of retained placenta in dairy cattle. J. Dairy Sci. 85: 544-550.

Ley, K., J. B. Baker, M. I. Cybulsky, M. A. Gimbrone Jr and F. W. Luscinskas. 1993. Intravenous interleukin-8 inhibits granulocyte emigration from rabbit mesenteric venules without altering L-selectin expression or leukocyte rolling. J. Immunol. 151: 6347-6357.

Lima, F. S., R. S. Bisinotto, E. S. Ribeiro, L. F. Greco, H. Ayres, M. G. Favoreto, M. R. Carvalho, K. N. Galvao and J. E. Santos. 2013. Effects of 1 or 2 treatments with prostaglandin F (2) alpha on subclinical endometritis and fertility in lactating dairy cows inseminated by timed artificial insemination. J. Dairy Sci. 96: 6480-6488.

Mitchell, G. B., B. N. Albright and J. L. Caswell. 2003. Effect of interleukin-8 and granulocyte colony-stimulating factor on priming and activation of bovine neutrophils. Infect. Immun. 71: 1643-1649.

NRC. 2001. Nutrient Requirements of Dairy Cattle. (7th rev. Ed.) Natl. Academy Press, Washington, D.C.

Paape, M., J. Mehrzad, X. Zhao, J. Detilleux and C. Burvenich. 2002. Defense of the bovine mammary gland by polymorphonuclear neutrophil leukocytes. J. Mammary Gland Biol. Neoplasia. 7: 109-121.

Watanabe, A., J. Hirota, S. Shimizu, S. Inumaru and K. Kimura. 2012. Single intramammary infusion of recombinant bovine interleukin-8 at dry-off induces the prolonged secretion of leukocyte elastase, inflammatory lactoferrin-derived peptides, and interleukin-8 in dairy cows. Vet. Med. Int. 2012: 172072.

--->

LIST OF SEQUENCES

<110> Cornell University

<120> COMPOSITIONS AND METHODS FOR IMPROVING DAIRY PRODUCTIVITY AND REPRODUCTIVE HEALTH OF MAMMALS BASED ON THE APPLICATION OF IL-8

<130> 018617.00665

<150> 62 / 099.643

<151> 2015-01-05

<160> 11

<170> PatentIn version 3.5

<210> 1

<211> 101

<212> PRT

<213> Bos taurus

<400> 1

Met Thr Ser Lys Leu Ala Val Ala Leu Leu Ala Ala Phe Leu Leu Ser

1 5 10 15

Ala Ala Leu Cys Glu Ala Ala Val Leu Ser Arg Met Ser Thr Glu Leu

20 25 30

Arg Cys Gln Cys Ile Lys Thr His Ser Thr Pro Phe His Pro Lys Phe

35 40 45

Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro His Cys Glu Asn Ser

50 55 60

Glu Ile Ile Val Lys Leu Thr Asn Gly Asn Glu Val Cys Leu Asn Pro

65 70 75 80

Lys Glu Lys Trp Val Gln Lys Val Val Gln Val Phe Val Lys Arg Ala

85 90 95

Glu Lys Gln Asp Pro

100

<210> 2

<211> 32

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 2

cggcgccgtg ctgtctcgta tgtccaccga ac 32

<210> 3

<211> 31

<212> DNA

<213> artificial sequence

<220>

<223> primer

<400> 3

gctcgagtca cggatcttgt ttttctgcac g 31

<210> 4

<211> 101

<212> PRT

<213> Bubalus bubalus

<400> 4

Met Thr Ser Lys Leu Ala Val Ala Leu Leu Ala Ala Phe Leu Leu Ser

1 5 10 15

Ala Ala Leu Cys Glu Ala Ala Val Leu Ser Arg Met Ser Thr Glu Leu

20 25 30

Arg Cys Gln Cys Ile Lys Thr His Ser Thr Pro Phe His Pro Lys Phe

35 40 45

Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro His Cys Glu Asn Ser

50 55 60

Glu Ile Ile Val Lys Leu Thr Asn Gly Lys Glu Val Cys Leu Asn Pro

65 70 75 80

Lys Glu Lys Trp Val Gln Lys Val Val Gln Val Phe Val Lys Arg Ala

85 90 95

Glu Lys Gln Asp Pro

100

<210> 5

<211> 101

<212> PRT

<213> CERVUS ELEPHUS

<400> 5

Met Thr Ser Lys Leu Ala Val Ala Leu Leu Ala Ala Phe Leu Leu Ser

1 5 10 15

Ala Ala Leu Cys Glu Ala Ala Val Leu Ser Arg Met Ser Thr Glu Leu

20 25 30

Arg Cys Gln Cys Ile Lys Thr His Ser Thr Pro Phe His Pro Lys Phe

35 40 45

Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro His Cys Glu Asn Ser

50 55 60

Glu Ile Ile Val Lys Leu Thr Asn Gly Lys Glu Val Cys Leu Asn Pro

65 70 75 80

Lys Glu Lys Trp Val Gln Lys Val Val Glu Val Phe Val Lys Arg Ala

85 90 95

Glu Lys Gln Asp Pro

100

<210> 6

<211> 101

<212> PRT

<213> Ovis aries

<400> 6

Met Thr Ser Lys Leu Ala Val Ala Leu Leu Ala Ala Phe Leu Leu Ser

1 5 10 15

Ala Ala Leu Cys Glu Ala Ala Val Leu Ser Arg Met Ser Thr Glu Leu

20 25 30

Arg Cys Gln Cys Ile Lys Thr His Ser Thr Pro Phe His Pro Lys Phe

35 40 45

Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro His Cys Glu Asn Ser

50 55 60

Glu Ile Ile Val Lys Leu Thr Asn Gly Lys Glu Val Cys Leu Asp Pro

65 70 75 80

Lys Glu Lys Trp Val Gln Lys Val Val Gln Ala Phe Leu Lys Arg Ala

85 90 95

Glu Lys Gln Asp Pro

100

<210> 7

<211> 97

<212> PRT

<213> Equus caballus

<400> 7

Met Thr Ser Lys Leu Ala Val Ala Leu Leu Ala Val Phe Leu Leu Ser

1 5 10 15

Ala Ala Leu Cys Glu Ala Ala Val Val Ser Arg Ile Thr Ala Glu Leu

20 25 30

Arg Cys Gln Cys Ile Lys Thr His Ser Lys Pro Phe Asn Pro Lys Leu

35 40 45

Ile Lys Glu Met Arg Val Ile Glu Ser Gly Pro His Cys Glu Asn Ser

50 55 60

Glu Ile Ile Val Lys Leu Val Asn Gly Ala Glu Val Cys Leu Asn Pro

65 70 75 80

His Thr Lys Trp Val Gln Ile Ile Val Gln Ala Phe Leu Lys Arg Thr

85 90 95

Glu

<210> 8

<211> 100

<212> PRT

<213> Homo sapiens

<400> 8

Met Thr Ser Lys Leu Ala Val Ala Leu Leu Ala Ala Phe Leu Ile Ser

1 5 10 15

Ala Ala Leu Cys Glu Gly Ala Val Leu Pro Arg Ser Ala Lys Glu Leu

20 25 30

Arg Cys Gln Cys Ile Lys Thr Tyr Ser Lys Pro Phe His Pro Lys Phe

35 40 45

Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro His Cys Ala Asn Thr

50 55 60

Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu Leu Cys Leu Asp Pro

65 70 75 80

Lys Glu Asn Trp Val Gln Arg Val Val Glu Lys Phe Leu Lys Arg Ala

85 90 95

Glu Asn Ser Leu

100

<210> 9

<211> 101

<212> PRT

<213> Canis lupus familiaris

<400> 9

Met Thr Ser Lys Leu Ala Val Ala Leu Leu Ala Ala Phe Val Leu Ser

1 5 10 15

Ala Ala Leu Cys Glu Ala Ala Val Leu Ser Arg Val Ser Ser Glu Leu

20 25 30

Arg Cys Gln Cys Ile Lys Thr His Ser Thr Pro Phe His Pro Lys Tyr

35 40 45

Ile Lys Glu Leu Arg Val Ile Asp Ser Gly Pro His Cys Glu Asn Ser

50 55 60

Glu Ile Ile Val Lys Leu Phe Asn Gly Asn Glu Val Cys Leu Asp Pro

65 70 75 80

Lys Glu Lys Trp Val Gln Lys Val Val Gln Ile Phe Leu Lys Lys Ala

85 90 95

Glu Lys Gln Asp Pro

100

<210> 10

<211> 101

<212> PRT

<213> Felus catus

<400> 10

Met Thr Ser Lys Leu Val Val Ala Leu Leu Ala Ala Phe Met Leu Ser

1 5 10 15

Ala Ala Leu Cys Glu Ala Ala Val Leu Ser Arg Ile Ser Ser Glu Leu

20 25 30

Arg Cys Gln Cys Ile Lys Thr His Ser Thr Pro Phe Asn Pro Lys Leu

35 40 45

Ile Lys Glu Leu Thr Val Ile Asp Ser Gly Pro His Cys Glu Asn Ser

50 55 60

Glu Ile Ile Val Lys Leu Val Asn Gly Lys Glu Val Cys Leu Asp Pro

65 70 75 80

Lys Gln Lys Trp Val Gln Lys Val Val Glu Ile Phe Leu Lys Lys Ala

85 90 95

Glu Lys Gln Asn Ala

100

<210> 11

<211> 101

<212> PRT

<213> artificial sequence

<220>

<223> consensus IL-8 based on the sequences shown in Figure 15

<220>

<221> misc_feature

<222> (28) .. (28)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (30) .. (30)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (71) .. (71)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (75) .. (75)

<223> Xaa can be any naturally occurring amino acid

<220>

<221> misc_feature

<222> (79) .. (79)

<223> Xaa can be any naturally occurring amino acid

<400> 11

Met Thr Ser Lys Leu Ala Val Ala Leu Leu Ala Ala Phe Leu Leu Ser

1 5 10 15

Ala Ala Leu Cys Glu Ala Ala Val Leu Ser Arg Xaa Ser Xaa Glu Leu

20 25 30

Arg Cys Gln Cys Ile Lys Thr His Ser Thr Pro Phe His Pro Lys Phe

35 40 45

Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro His Cys Glu Asn Ser

50 55 60

Glu Ile Ile Val Lys Leu Xaa Asn Gly Lys Xaa Val Cys Leu Xaa Pro

65 70 75 80

Lys Glu Lys Trp Val Gln Lys Val Val Gln Val Phe Leu Lys Arg Ala

85 90 95

Glu Lys Gln Asp Pro

100

<---

Claims (13)

1. A method for the prevention or treatment of postpartum uterine diseases selected from metritis and retained placenta in a female mammal, where the mammal is a cattle, the method comprising the step of administering to the female mammal an effective amount of interleukin-8 (IL-8 ), so that after administration there is an improvement in the health of the female mammal. 2. A method according to claim 1, wherein the female mammal is pregnant or is within twenty weeks of delivery. 3. A method according to claim 1, wherein improving the health of the mammal comprises reducing retained placenta separation. 4. The method according to claim 1, wherein the prevention of metritis comprises inhibiting the development of postpartum metritis. 5. The method of claim. 1, characterized in that the administration of IL-8 is intrauterine administration or intravaginal administration. 6. The method according to claim 1, wherein the female mammal is a member of a population of female mammals of the same species, the method further comprising the step of administering IL-8 to other members of the population such that other members after introduction improves the health of the female mammal. 7. The method according to claim 1, characterized in that the cattle is a dairy cow. 8. A method according to claim 1, wherein the female mammals are dairy cows, and the health of the female mammal is improved. 9. A kit for the prevention or treatment of postpartum diseases of the uterus, selected from metritis and retained placenta, in a female mammal, where the mammal is a cattle, and the kit contains IL-8 in one or more sealed containers, a delivery device and instructions for administering IL-8 to a female mammal. 10. The kit of claim 9, wherein the instructions for administering IL-8 comprise instructions for administering IL-8 to a dairy cow. 11. The kit of claim 9, wherein the delivery device is a catheter suitable for intrauterine or intravaginal administration of IL-8 to a dairy cow. 12. A finished product for the prevention or treatment of postpartum uterine diseases, selected from metritis and retained placenta, in a female mammal, where the mammal is cattle, and the product contains IL-8 in a sealed container, packaging and printed information, while printed the information identifies IL-8 as the contents of the pack and provides an indication that IL-8 should be used for the prevention or treatment of postpartum uterine disease selected from metritis and retained placenta.

Images