WO2020149859A1

WO2020149859A1 - Use of zinc butyrate and chromium propionate for the treatment and prevention of woody breast disease in broilers

Info

Publication number: WO2020149859A1
Application number: PCT/US2019/014302
Authority: WO
Inventors: Farrah Christine PHILLIPS; Mitchell James POSS; Karen Pollock; Jon RUBACH
Original assignee: Kemin Industries, Inc.
Priority date: 2019-01-18
Filing date: 2019-01-18
Publication date: 2020-07-23

Abstract

The present invention relates to compositions and methods to reduce the severity and incidence of woody breast disease in poultry. One aspect of the present invention relates to the administration of zinc butyrate and chromium propionate, preferably in the animal's feed. The invention is surprisingly effective in promoting healthy muscle tissue development in poultry without affecting broiler growth, feed conversion efficiency or breast meat yield.

Description

USE OF ZINC BUTYRATE AND CHROMIUM PROPIONATE FOR THE

TREATMENT AND PREVENTION OF WOODY BREAST DISEASE IN BROILERS

BACKGROUND OF THE INUENΉON

Large, fast-growing broilers are susceptible to woody breast disease, which affects the pectoralis major muscle group of chickens, and is characterized by muscle hardness caused by large deposits of collagen. Muscle tissue affected by severe woody breast was labeled by the

USDA as“unfit for human food” in 2017 and must be trimmed and discarded. Because the whole breast is the most profitable cut for producers and processors, any breast meat losses can significantly impact their margins on the entire bird.

Woody breast is a muscle myopathy that results in the necrosis of muscle fibers with macrophage infiltration. In response to the necrosis, fibrosis takes place, leading to connective tissue synthesis and the replacement of muscle-specific proteins by highly cross-linked collagen. The highly cross-linked collagen gives the muscle its woody or very stiff phenotype.

While the exact cause of woody breast is not known, breast muscles with the woody breast phenotype appear to have reduced capillary blood supply to the muscle. Without sufficient capillaries, muscle damage may occur as lactic acid, a by-product of anaerobic respiration (which is the metabolic pathway used in the breast muscle), cannot be cleared through the capillaries and remains in the muscle.

Woody breast continues to confound the poultry' industry' a decade after its discovery. The condition does not harm the birds or cause them to act differently, and it does not harm people if eaten. It does, however, cause the breast meat on chicken to become unusually tough, with a coarse texture, prompting complaints from consumers and leading to large amounts of affected poultry products going into another supply chain, to be processed as ground chicken. The condemned portion of the meat is taken at a loss from the producer and represents a very large-scale production problem in die US that needs to be solved. The heavier the bird and the larger the quantity of meat on it, the more likely it is to develop woody breast. Poultry' processing plants typically wait until each carcass has been de-feathered before allowing breasts to be touched by human hands, which increases the expense of the process and slows production.

The muscle fibers of woody breasts exhibit increased degeneration and active repair, deposits of connective tissue and fat and infiltration of immune cells. These characteristics suggest that the tissue is actively undergoing a wound-healing response that requires the removal of degraded muscle cells and stimulating muscle repair. These changes also suggest that the muscle is experiencing oxidative stress due to reduced oxygen levels.

Recent approaches for preventing and treating woody breast syndrome have focused on factors that increase oxygen and antioxidant levels in the muscle and which reduce environmental stressors like heat and infection. Other treatments have focused on varying the birds’ diet and nutrition. Regarding the latter, while it has been found that keeping birds on the recommended growth curve and within standard weight ranges can help reduce the occurrence of woody breast, no dietary supplement has been found to reduce incidence of the condition in the field.

Another tactic for reducing woody breast syndrome is by growing the large breeds more slowly by restricting feed intake and/or nutrition. However, this approach is expensive because it takes longer for the birds to reach the desired body weight and does not guarantee a woody breast- free flock. Further, using heritage strains that grow more slowly than commercial hybrids and have lower breast meat yields is even more expensive than simply restricting feed intake. With either approach, the increase in cost for poultry is inevitably passed on to customers.

As the demand for inexpensive boneless, skinless breast meat has risen, the industry has pushed the performance of the modem broiler to its genetic limits, thus creating a need for nutritional supplements that can alter gene expression and tissue composition to improve meat quality without sacrificing breast meat yield. For these and other reasons, there is a need for the present invention.

SUMMARY OF THE INVENTION

The present invention relates to compositions and methods for reducing the incidence and severity of woody breast using the combination of the trace minerals, zinc and chromium, with butyric acid. This unique combination therapy optimizes muscle cell development and function while simultaneously reducing the secretion and/or signaling of pro-fibrotic molecules. The combination of a salt of zinc and butyric acid with chromium propionate is preferably used as a feed additive for broilers to reduce both woody breast severity and incidence. The invention works to reduce woody breast disease incidence and severity in vivo by inhibiting collagen production.

In contrast to prior treatments, the methods and processes disclosed herein are effective without negatively impacting broiler performance or breast meat yield.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 A shows toxicity of zinc butyrate on CEDMs at increasing concentrations. FIG. IB is a quantification of collagen production in CEDMs treated with or without

TGFb and zinc butyrate at increasing concentrations.

FIG. 1C is a quantification of collagen production in CEDMs treated with or without

TGFb and zinc butyrate and its individual active components for collagen inhibition.

FIG. ID shows representative images of cells from one experiment after staining for collagen.

FIG. 2 A shows the average pen weight of breast fillets for each of the treatment groups

FIG. 2B is the frequency of breast fillets scored as normal or with moderate to severe woody breast.

FIG. 3 A shows the weight of breast fillets for each of the treatment groups

FIG. 3B is the frequency of breast fillets scored as normal or with moderate to severe woody breast.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Woody breast disease incidence and severity is linked to the growth rate of the birds, which means large, fast-growing broiler chickens fed ad libitum develop the disease more often than small, slow-growing breeds or birds with limited access to food. The size of the breast correlates with woody breast disease, thus suboptimal growth conditions will reduce woody breast disease incidence and severity. Reducing the growth rate, however, comes with the associated cost of lengthening the growth period and/or reduced breast meat weight and yield.

The present inventors have discovered that the combination of zinc and butyric acid salt and chromium effectively prevents woody breast disease in the muscle tissue without reducing breast size or yield. In vitro zinc butyrate, but not zinc sulfate or butyric acid alone, reduces collagen production by chicken myofibroblasts. Collagen is the cause of these undesirable features of woody breast, thus the in vitro data demonstrating that zinc butyrate effectively inhibits collagen production is a strong indicator that this is the mechanism occurring in vivo.

Combining chromium propionate with zinc and butyric acid salt in vivo reduced woody breast disease incidence and severity without negatively impacting broiler performance or breast meat yield.

The present invention may be used to treat any type of“woody breast” condition or syndrome in birds and, specifically, poultry. In this regard, woody breast is conventionally described as a muscle myopathy that results in necrosis of muscle fibers with macrophage infiltration. In response to the necrosis, fibrosis takes place, leading to the synthesis of connective tissue and the replacement of muscle-specific proteins with highly cross-linked collagen. The highly cross-linked collagen gives the muscle its woody phenotype. While the meat is not harmful to the consumer, the occurrence of the woody breast tends to impair the visual appearance and reduces consumers’ willingness to buy chicken breast fillets.

According to at least one embodiment of the invention, chickens or other poultry are provided through their food or water source a composition containing one or more forms of zinc and butyric acid salt and chromium. Any forms or sources of these compounds are suitable for this purpose, and can include mixtures of the components, including zinc butyrate, as well as the zinc, butyric acid, and chromium components individually. In this regard, the components can be provided individually from conventional food grade/pharmaceutical sources known in the art including, but not limited to, zinc oxide, zinc sulfate, zinc gluconate, zinc acetate, chromium picolinate, chromium nicotinate, chromium polynicotinate, chromium chloride, chromium propionate, sodium butyrate, magnesium butyrate, etc. The feed compositions of the invention should preferably include at least about 50 grams of butyric acid/metric ton of feed, at least about 10 grams of zinc/metric ton of feed and at least about 50 ppb of chromium/metric ton of feed, with about 80-180 grams of butyric acid/metric ton of feed, about 20-80 grams of zinc/metric ton of feed, and about 100-2000 ppb

chromium/metric ton of feed being preferred, and about 120-140 grams of butyric acid/metric ton of feed, between about 40-60 grams of zinc/metric ton of feed, and between about 150-200 ppb chromium/metric ton of feed being most preferred.

In one embodiment of the invention, the composition includes the commercial formulation ButiPEARL Z which contains 10.9% zinc and 29.1% butyrate. In another embodiment, the formulation includes ButiPEARL, a commercial formulation of at least 45% calcium butyrate. In another embodiment of the invention, the composition includes the commercial formulation KemTRACE Cr, an organic source of chromium containing between

0.04-0.4% chromium. According to at least one embodiment of the invention, one or more of the ingredients are provided in encapsulated form to provide a slower, more controlled release of ingredients in the animal.

As described herein, the combination of zinc and chromium trace minerals and butyric acid provided by the present invention supports healthy muscle tissue development without negatively affecting broiler growth, feed conversion efficiency of breast meat yield. Prior solutions have not definitively shown this improvement in meat quality and without negatively affecting other performance parameters. The applicant’s unique combination optimizes muscle cell development and function while simultaneously reducing the secretion and/or signaling of pro-fibrotic molecules. Furthermore, the applicant has demonstrated that the combination of zinc ion and butyric acid is critical for reducing collagen deposition by myofibroblasts. The compositions of the invention may optionally include one or more antioxidants to help reduce reactive oxygen radical species in the blood and/or prevent oil oxidation. However, the inventors have found that antioxidants may increase the incidence of woody breast. As such, preferred compositions of the invention do not include detectable levels of antioxidants, i.e. are

“substantially free” of antioxidants. Antioxidants are well known in the art and include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, lycopene, lutein, selenium, manganese, zeaxanthin, flavonoids, flavones, catechins, polyphenols, phenol derivatives, and phytoestrogens. In one embodiment, the composition includes ethoxyquin to prevent oil oxidation.

The compositions of the invention can further include other ingredients or compounds that may be beneficial for poultry including, but not limited to, carbohydrate, protein, fat and oil, vitamins, minerals, probiotics, medicines, flavors, colors, etc. The compositions may also be combined with a pharmaceutically acceptable carrier that may include one or more carriers or excipients, such as fillers, diluents, binders, lubricants, and disintegrants. Such ingredients and their relative amounts to be included are well known to persons skilled in the art.

While the compositions of the invention are described in particular for administration in the animal’s feed, the compositions may likewise be administered in the animal’s water source.

In addition, the compositions may be administered via conventional pharmaceutical routes including, but not limited to, inhilation, intravenously, rectally, sublingually, etc. via a pharmaceutical carrier appropriate to the selected route of administration.

The ingredients of the formulation may be combined by simply mixing at room temperature (25-30°C) with agitation. The ingredients of the invention can either be mixed sequentially or can be added all at once to achieve the unique composition of the invention. In preferred embodiments the ingredients are mixed with agitation to improve miscibility, whereby smaller batches are mixed using a magnetic stir bar at 400-600 rpm, and larger batches are mixed using a mechanical agitator at a speed of less than 300 rpm. The ingredients can also be mixed without agitation. The composition can in turn be simply combined with the animal feed prior to administration to the animals. Animal feed formulations may be provided to the animals through any convention means well known to persons skilled in the art, include but not limited to top- dress, mixed in by hand, pelleted, mixed with crumbles, etc.

Once combined with the animal feed, the composition of the invention is preferably provided to the animals ad libitum, and preferably for a time period of 7 days or more and optimally through the life of the bird. If the composition is administered by a route other than via the animal’s feed in quantities that are equivalent to the dose levels of ingredients achieved by providing the ingredients through the animal’s water or feed. In general, composition should be administered to the animal in an amount sufficient to achieve an equivalent dose of from about 2 to 220 g zinc, about 60 to 600 g butyric acid, and about 50 to 2000 ppm chromium/metric ton.

The following examples are offered to illustrate but not limit the invention. Thus, it is presented with the understanding that various formulation modifications as well as method of delivery modifications may be made and still are within the spirit of the invention.

EXAMPLE 1

Zinc Butyrate and the combinations of zinc and butyrate were tested in a cell culture model to examine their effects on collagen production in cultured chicken myofibroblasts.

MATERIALS AND METHODS

Chicken embryo dermal myofibroblast (CEDM) media was comprised of DMEM with L- glut and high glucose (Sigma, cat. #D5648, pH 7.2) supplemented with 3.7 g/L sodium bicarbonate, 6% heat-inactivated fetal bovine serum (FBS, Neuromics, Edina, MN, USA), 2% chicken serum (Fisher, cat. #16110-082), and lx antibiotic-antimycotic (Millipore Sigma, cat.

#A5955). Ten (10) pg of lyophilized recombinant TGF-B1 (referred to as TGFb, Fisher, cat

#NC0526573) was dissolved in 1 mL of 10 mM citric acid (pH 3.0). Aliquots of this stock solution was diluted 1:10 in DMEM media prior to use in cell culture assay. Final effective concentration of TGFb used in all experiments was 1.0 ng/mL (~ 20 times the manufacturer’s reported EDso).

CEDM cell culture and stimulation. CEDM cell derivation was replicated from Kosla et al. All cultured and stimulated cells were incubated at 37°C in 5% CCh. CEDM cells were seeded into 384- or 96-well plates and rested overnight. Cells were stimulated with ±TGFb and ± test compounds for 48 hr then fixed and stained for collagen.

Collagen staining and quantification. Media from CEDM cell culture plate was removed and cells were gently washed twice using room temperature phosphate buffered saline (PBS, pH

7.0). Cells were fixed in methanol at room temperature overnight, washed twice with PBS and stained with picrosirius red stain (Fisher, cat. #NC1114978). Stained cells were washed three times with acidified water and picrosirius red stain was eluted from the plate with 1 M NaOH.

Absorbance was measured at 540 nm and normalized to the average absorbance of the untreated control wells.

SYTOX Green cell death assay. SYTOX™ Green (Fisher, cat. #87020) was included in

CEDM cell culture media at 0.16 mM at the time of seeding. Prior to administration of TGFb or compounds, green fluorescent dead cells were counted on the SpectraMax^® i3x MiniMax 300

Imaging Cytometer (Molecular Devices, San Jose, CA, USA) at 488 nm wavelength using the

SofiMax Pro 7 software (Molecular Devices). The same software settings were used to analyze the increase in cell death after two days in culture with cytokine and compounds. RESULTS

Zinc butyrate inhibits collagen production. The cell culture experiments demonstrate that zinc butyrate is an inhibitor of collagen production (FIG. 1 A). Zinc butyrate does not increase cell death in CEDMs treated with TGFb vmtil the concentration reaches 50 mM (FIG. IB). Although this increase in cell death at 50 mM is greater than CEDMs without any zinc butyrate, a total of

1,500 dead cells is relatively low when compared to the 25,000 cells that were seeded into the well.

To determine which of the active components of zinc butyrate, either zinc ion or butyric acid or both, are responsible for inhibiting collagen production, CEDM cells treated with or without TGFb were stimulated with ZnSC>4, butyric acid, or a combination. ZnSC>4 or butyric acid treatments alone or mixed together do reduce collagen production in CEDM. However, these individual components are not as effective as the zinc butyrate at reducing collagen production

(FIGS. 1C & ID).

EXAMPLE 2

Synergistic Effect of Butyric Acid, Zinc and Chromium

The objective of the study was to evaluate the effects of KemTRACE® Chromium and

ButiPEARL™ Z alone or in combination on growth performance and processing yields during with broiler chickens grown to 52 days of age during summer heat.

The treatment groups included KemTRACE® Chromium (200 ppb), ButiPEARL™ Z (1 lb/ton) and a ButiPEARL™ Z (1 lb/ton), KemTRACE® Chromium (200 ppb) combination. At day of hatch, Cobb x Cobb 700 male broiler chicks were randomly assigned to the four treatments

(15 reps/treatment; 28 birds/rep): control (no additives). Chicks were exposed to summer heat from dlO until the end of the trial (d52), during the day. No antibiotic growth promoters (BMD) were included in the basal diet. Feed intake, body weight (BW), mortality and feed conversion were determined at 52 d. In addition, meat quality parameters were determined.

Table 1. Dietary formulations (lb/ton) and calculated nutrient content of basal diet.

The addition of ButiPEARL™ Z and KemTRACE® Chromium, or com starch were added to achieve final experimental treatments.

Table 3. Performance results after 52 days and woody breast scores.

At the end of the trial, feed conversion was improved by 4 points with the combined chromium and ButiPEARL Z treatment (Table 3). Therefore, the combination of the two products did not negatively affect performance. The combined treatment (Treatment. 4) did not negatively affect the average breast weight of the birds (FIG. 2A), but it did have a lower average woody breast score (Table 3). In addition, the combined treatment 4 reduced the frequency of moderate to severe woody breast scores (FIG. 2B).

EXAMPLE 3

Effect of ButiPEARL™ Z, KemTRACE^® Chromium and RENDOX^® EQ on

Growth PerformanceWoody Breast Incidence

Treatments and diets

The experimental design for this trial consisted of three additives:

ButiPEARL™ Z, KemTRACE^® Chromium and RENDOX^® EQ. The active ingredient in RENDOX^® EQ is ethoxy quin. These three additives were arranged into

8 treatments (Table 4) replicated in 16 pens, with 16 day of hatch ROSS 708 chickens per pen (total 2,048 birds).

The basal diets were formulated to meet or exceed the nutrient requirements of broiler chickens. Diets were made for four growth phases, starter, grower, finisher, and withdrawal and are detailed in Table 6. In the starter phase the feed was crumbled for smaller feed particle size and was fed from

Day 0 to 14. Grower feed was fed from Day 15 - 28. Finisher feed was fed Day 29 - 42 and

Withdrawal feed was fed Day 43 - 53 of the study.

Processing and performance parameters. Pen weights and feed consumption were recorded at end of study for calculating Feed Conversion. At day 50 four birds from each pen were processed. Breast fillets were weighed and scored for woody breast.

Results

To evaluate the effect of KemTRACE Cr, ButiPEARL Z and RENDOX^® EQ, alone or in combination, on woody breast and performance male broilers were reared to 52 days of age. Birds were weighed, and breast fillets were weighed and scored for woody breast. There were no differences in body or breast fillet weight (Table 5) or breast fillet weight yield (FIG. 3A) in any of the treatment groups. Co-treatment with KemTRACE Cr and ButiPEARL Z reduced the average woody breast score (Table 5). Co-treatment with both KemTRACE Cr and ButiPEARL Z also reduced the frequency of breast fillets with moderate to severe woody breast (FIG. 3B).

T

It should be appreciated that minor dosage and formulation modifications of the composition and the ranges expressed herein may be made and still come within the scope and spirit of the present invention. Having described the invention with reference to particular compositions, theories of effectiveness, and the like, it will be apparent to those of skill in the art that it is not intended that the invention be limited by such illustrative embodiments or mechanisms, and that modifications can be made without departing from the scope or spirit of the invention, as defined by the appended claims. It is intended that all such obvious modifications and variations be included within the scope of the present invention as defined in the appended claims. The claims are meant to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates to the contrary.

The foregoing description has been presented for the purposes of illustration and description. It is not intended to be an exhaustive list or limit the invention to the precise forms disclosed. It is contemplated that other alternative processes and methods obvious to those skilled in the art are considered included in the invention. The description is merely examples of embodiments. It is understood that any other modifications, substitutions, and/or additions may be made, which are within the intended spirit and scope of the disclosure. From the foregoing, it can be seen that the exemplary aspects of the disclosure accomplishes at least all of the intended objectives.

Claims

1. A composition for treating woody breast disease in poultry comprising: butyric acid; zinc; and chromium.

2. The composition of claim 1, wherein the composition comprises zinc butyrate or a salt of zinc and butyric acid.

3. The composition of claim 1, wherein the composition comprises chromium propionate.

4. The composition of claim 1, further including a pharmaceutically acceptable carrier.

5. The composition of claim 1, further including animal feed.

6. The composition of claim 5, wherein the composition is added to animal feed and includes at least 50 g of the butyric acid/metric ton of the animal feed, at least 10 g of the zinc/metric ton of the animal feed, and at least 100 ppb of the chromium/metric ton of the animal feed.

7. The composition of claim 6, wherein the composition includes between about 80-180 g of the butyric acid/metric ton of the animal feed, between about 20-80 g of the zinc/metric ton of the animal feed, and between about 100-200 ppb/metric ton of the animal feed.

8. The composition of claim 1 wherein the zinc and the butyric acid are encapsulated.

9. The composition of claim 1, wherein the composition is substantially free of antioxidants.

10. A method of preventing or treating woody breast disease in poultry comprising:

administering to poultry a composition comprising butyric acid; zinc; and chromium.

11. The method of claim 10, wherein the composition comprises zinc butyrate or a salt of zinc and butyric acid and chromium propionate.

12. The method of claim 10, wherein the composition is administered to the poultry by providing the composition in animal feed.

13. The method of claim 10, wherein the poultry is a chicken.

14. The method of claim 10, wherein the composition includes at least 50 g of the butyric acid/metric ton of the animal feed, at least 10 g of the zinc/metric ton of the animal feed, and at least 100 ppb of the chromium/metric ton of the animal feed.

15. The method of claim 14, wherein the composition includes between about 80-180 g of the butyric acid/metric ton of the animal feed, between about 20-80 g of the zinc/metric ton of the animal feed, and between about 100-200 ppb/metric ton of the animal feed.

16. The method of claim 10, wherein the composition is administered to the poultry with a pharmaceutically acceptable carrier.

17. The method of claim 10, wherein the composition is administered to the poultry through the poultry’s water source.

18. The method of claim 10, wherein the composition is substantially free of antioxidant.

19. A method of manufacturing a composition for treating woody breast disease in poultry comprising: combining butyric acid; zinc; and chromium to form a composition.