AU2013265950A1 - Method for improving growth performance and/or bone performance of animal - Google Patents

Abstract

The invention provides a method for improving growth performance and/or bone performance of an animal. In particularly, the invention provides a method for improving growth performance and/or bone performance of an animal by administering to the animal 25-Hydroxy Vitamin D3 and phytase. The invention also provides use of a composition comprising 25-Hydroxy Vitamin DA and phytase as an animal feed or feed additive.

Description

WO 2013/174148 PCT/CN2013/000604 METHOD FOR IMPROVING GROWTH PERFORMANCE AND/OR BONE PERFORMANCE OF ANIMAL Technical Field The invention is related to a method for improving growth performance and/or bone performance of an animal. In particularly, the invention is related to a method for improving growth performance and/or bone performance of an animal by administering to the animal 25-Hydroxy Vitamin D 3 and phytase. Background of invention Phytase is a type of enzyme that catalyzes the hydrolysis of phytic acid, which is indigestible for organisms, to phosphorus which is digestible for organisms, including 3 phytase and 6-phytase. Phytase has been widely used as anirnal fee additive, which may increase use efficiency of phosphorus in plant feed by 60% and decrease phosphorus excreted from feces by 40% so it is significantly useful to improve production benefit in livestock agriculture and reduce pollution of phosphorus from phytic acid. (see US 3,297.548; Nelson T. S. et al, J Nutrition, 101: 1289-1294, 1971; and Nelson T. S, et al.. Poult Sci. 47:1!842-1848, 1968) 25-Hydroxy Vitamin D 3 has also been used widely as an animal feed additive. For example, US 2003/01170324 discloses a feed premix comprising 25-Hydroxy Vitamin D 3 which may be added to poultry, swine, canine, or feline food; US 2005/0064018 discloses adding a combination of 25-Hydroxy Vitamin D 3 and vitamin D 3 to animal feed to improve swine's bone strength; and US 5,695,794 discloses adding a combination of 25 Hydroxy Vitamin D 3 and vitarnin D 3 to poultry feed to ameliorate the effects of tibial dyschondroplasia. Surprisingly, the inventors of the invention found that a combination of 25-Hydroxy Vitamin D 3 and phytase can improve growth performance and/or bone performance of an animal and provide synergistic effect. Content of invention Therefore, the invention provides a method for improving growth performance and/or bone performance of an animal, comprising administering to the animal an effective amount of 25-Hydroxy Vitamin D 3 and phytase. In the invention, the term "animal" includes all animals including human. The examples of the animals include non-ruminants and ruminants. Ruminants include but are not limited to sheep, goat and cattle; and non-ruminants include but are not limited to horse; rabbit; pig including but not limited to infant pig, piglet growing-fattening pig, sow and boar; and poultry such as turkey, duck and chicken (including but not limited to broiler chicken, egg-laying chicken) etc-. 1 WO 2013/174148 PCT/CN2013/000604 in the invention, the term "growth performance" means average daily weight gain (ADWG), average daily feed intake (ADFI) and/or feed conversion rate (FCR) of the animal, In the invention, the term "bone performance" means fresh weight, length. radius, breaking strength, and/or bone mineral density (BMDb, and/or ash, Ca and/or P contents of the bone of the animal According to the method of the invention, administration of 25-Hydroxy Viamin D 3 and phytase to an animal can provide synergistic effects to improve growth performance and/or bone performance of the animal. The term "synergistic effect" means the phenomenon of 1±1>2, i.e. two or more substances mutualy provide better effect than the summation of effects produced by each one of the substances, or the phenomenon of "mutualy promotive effect" i.e. the effect produced by one substance is reinforced in the presence of the other substances. In the invention, 25-Hydroxy Vitamin D 3 may come from any source, including commercial routes or direct synthesis. For example, 25-Hydroxy Viamin D 3 of the invention is synthesized according to the method described in US 4,310,467 and US 3,565,924. In the invention, the phytase may be any type of phosphatase enzyme that catalyzes the hydrolysis of phytic acid (myo-inositol hexakisphosphate) and releases a usable form of inorganic phosphorus. The phytase of the invention may be 3-phytase, 6-phytase or a combination thereof. The phyrase of the invention may come from any source including plant and microbiological source. A preferable plant source for phytase is malt. Preferred microbiological sources for ph ytase are Aspergillus oryzar, Aspergillus niger, Aspergillus pieuum and Peniophora lycli, and the phytase from Aspergillus niger has been commercialized for use in animal feed. Phytase has also been isolated from E&oli, B. subtilis and other thermostable microbes. Genes encoding phytase have been cloned from many microorganisms, and phytase can easily be produced and purified in industrial scale. Moreover, phyrase can be genetically engineered such that it is more resistant to thermal, light, and/or chemical degradation. Examples of commercial phytase are Natuphos 5000 L NatuphosrM 5000 G, Ronozyme® Hiphos M/L etc. A preferred phytase of the invention is 6-phytase (EC 3.i.326), preferably produced by a genetically modified strain Aspergillus oryzac (DS MZ 22594). The activity of phytase is measured in FTU units: 1 FTU (also-caled FYT) is the amount of phytase that liberates I pimole phosphate per minute at pH 5.5 and 37 0 C. The amount of 25-Hydroxy Vitamin D 3 and phytase for administration depends on the particular conditions of the animal Usually, based on the body weight of the animal to be 2 WO 2013/174148 PCT/CN2013/000604 administered, 25-Hydroxy Vitamin D 3 is administered in an amount of 0.2-20 pg/kg body weight per day, preferably 0.5-15 pg/kg body weight per day, more preferably 1-10 pg/kg body weight per day, even more preferably P.5-5 gkg body weight per day, and most preferably 2 pg/kg body weight per day; and the phytase is administered in an amount of 15-150 FTU/kg body weight per day, preferably 20-120 FTU/kg body weight per day, more preferably 30-100 FTU/kg body weight per day, even more preferably 50 80 FTU/kg body weight per day, and most preferably 60 FTU/kg body weight per day. The administration may be 'drried out once a day, or two or more times within one day to the animal In addition, 25-H-ydroxy Vitamin D 3 and phytase may be administered simultaneously, or separately and successively, for example, administered in an interval of 10) mins, 20 mins, 30 mins. 40 mins, 50 mins, 1 h, 2 h, 3 h or 4 h. The 25-Hlydroxy Vitamin D 3 and phytase may be directly administered to the animal as a feed composition, or as components of an animal feed or in drinking water of the animal Accordingly, in one embodiment, the invention provides use of a composition comprising 25-Hydroxy Vitamin D 3 and phytase as an animal feed or feed additive. Preferably, the animal feed or the animal feed with addition of the feed additive comprises 5-500 pgkg, more preferably 10-300 pg/kg, even more preferably 20-200 pg/kg, even more preferably 40-100 pg/kg, and most preferably 50 pg/kg of 25-Hydroxy Vitamin D 3 ; and 500-4000 FTU/kg, more preferably 800-3000 FTU/kg, even more preferably1000-2500 FTU/kg, even more preferably 1200-2000 FTU/kg, and most preferably 1500 FTU/kg of phytase The invention will be further described with reference to the following non-limiting examples. Examples Example 1 Piglets of an initial body weight of 710.5 kg were fed a basal diet ad libitum until 1010,5 kg. Groups of 8 piglets each were fed as follows: A: basal diet, neither 25-Hydroxy Vitamin D 3 nor 6-phytase was added; B: basal diet t50 pg/kg 25-Hydroxy Vitamin D 3 , and no 6-phylase was added; C: basal diet + 1500 FlU/kg 6-phytase, and no 25-Hydroxy Vitamin D 3 was added; D: basal diet + 50 pg/kg 25-Hydroxy Vitamin D 3 + 1500 FTU/kg 6-phytase. The formulation of the basal diet was as follows: Component Con tent(%) Maize 51L07 Soybeatnmeal 20.00 WO 2013/174148 PCT/CN2013/000604 Whey Powder 12:00 Fish meal 3 30 Wheat Bran 3 50 Plasrna Protein 5.00 Limestone 0.80 Soy Oil 1.30 L-Lysine HCi 0.48 L-Methionine 0.10 L-Threonine 0.10 L-Tryptophan 0.05 NaCi 0,20 Premnix* 1LO0 Zeolite Powder 1L10 In total(%) 100 *~ Premix provide in 1000 g of the basal diet: VA, 6600 lU: VD 3 , 2000 IU; VE, 50 IUl;

VK

3 , 2 mg: VB 1 , 4,5 mg; VB 2 , 8 mg: VB 6 . 6 mg; VBe 2 , 60 pig; Niacin, 20 mg; pantothenicacid. 32 mg; Folic acid, 0.8 mg; Biotin. 0.23 mg; Choline, '750 mg; Fe, 150 mg; Zn, 2340 mg; Cu, 120 mg: Mn, 50 mg: 1, 0.6 mg; and Se, 0.3 mg The 25-Hydroxy Vitamin D 3 used herein was obtained from DSM Nutritional Products Ltd.( Ily .DR 1.25% Beadlet, Basel, Switzerland), and the 6-phytase used herein was also obtained from DSM Nutritional Products Ltd.(Ronozyme® Hiphos GT, BaseL Switzerland). After the feeding term ended, the piglets were not fed for 12 hours and then weighed. Average daily weight gain (ADWG) during the term was calculated, average daily feed intake (ADFI) was calculated based on the feed weight before the feeding and rest feed weight after the feeding, and feed conversion rate (FCR) was also calculated based on the average daily weight gain and average daily feed Intake (ADFI/ADWG)). The experiment was run 6 times in parallel and averages were obtained as follows. Variables A B C D ADWG(g) 208.33±12.95 226.64±9.68 227.39± 1.59 244.48±16.22 ADFI(g) 341.64±28.97 335.61±21.64 303.58±8.54 j317.11±17.85 FCR 1.64±0.06 1.48±0.08 P 35±0 06 1.3 1±0.07 The data indicate that, compared with the piglets administered with neither 25-Hydroxy Vitamin D 3 nor 6-phytase or with any one of 25-Hydroxy Vitamin D 3 and 6 phytase, the piglets administered with both 25-.Hydroxy Vitamin D 3 and 6-phytase had quicker weight increase and lower FCR, so 25-Hydroxy Vitamin D 3 and 6-phytase had synergistic effect in improving growth performance of the piglets. Example 2 4 WO 2013/174148 PCT/CN2013/000604 Piglets of an initial body weight of 1 O.5 kg obtained from Example 1 were fed a basal diet ad libitum until 20 0.5 kg. Groups of 8 piglets each were fed as follows: A: basal diet, neither 25-Hydroxy Viamin D 3 nor 6-phytase was added; B: basal diet + 50 pig/kg 25H Hydroxy Vitamin D 3 , and no 6-phytase was added; C: basal diet + 1500 FrU/kg 6-phy tase, and no 25-H ydroxy Vitamin D 3 was ad ded; D: hasal diet - 50 pg/kg 25-Hyd doxy Vitamin D 3 1500 FTU/kg 6-phytase. The fornmulation of the basal diet was as follows: WO 2013/174148 PCT/CN2013/000604 Component Content (%) Maize 61.24 Soybean Meal 24.47 Whey Powder 4.00 Fish Meal 2.40 Wheat Bran 4.00 Limestone 0.79 CaHPO 4 0.06 Soy Oil 0.15 L-Lysine HICi 0.37 LDMethionine 0.05 L-.hreorine 0.10 L-Tryptophan 0.05 NaCT 0.20 Premix* 1.00 Zeolite Powder 1L12 In total(%) 100 * Premix provide in 1000 g of the basal diet; VA, 6600 LU; VD 3 , 2000 IU; VE, 50 LU;

VK

3 , 2 mg; VB 1 , 4.5 mg; VB 2 , 8 mg; VB 6 , 6 mg: VB 12 60 prg; Niacin, 20 mg; pantothenicacid. 32 mg; Folic acid, 0.8 mg: Biotin, 0.23 mg: Choline, 750 mg: Fe, 150 mg: Zn, 2340 mg; Cu, 120 mg; Mn, 50 mg: I, 0.6 mg; and Se, 0.3 mg The used 25-Hydroxy Viamin D 3 as obtained from DSM Nutriinal Product L td( Hy DR. 1 .25% Beadlet, Basel, Switzerland), and the used 6-phytase was also obtained from DSM Nutritional Products Ltd.(RonozymeR Hiphos GT, Basel Switzerland). After the feeding term ended, the piglets were kept in limosis for 12 hours and then weighed. Average daily weight gain (ADWG) during the term was calculated, average daily feed intake (ADFI) was calculated based on the feed weight before the feeding and rest feed weight after the feeding, and feed conversion rate (FCR) was also calculated based on the average daily weight gain and average daily feed intake (ADFI/ADWG). The experiment was run 6 times in paralel and averages were obtained as follows. [Variables A B C D ADWG(g) I354.30±10.63 380,62±6.75 394.22±12A43 399.29±6.68 ADFI~g) 72L.45±21.32 738.22±33.44 730.83±37.70 7.89±29.03 FCR 2.04±0.09 1,95±0.10 1.86±0.09 1.80±0.08 The above experimental data also indicate that the simultaneous administration of 25 Hydroxy Vitamin D 3 and 6-phytase improved the weight gain and reduced FCR of the piglets and provided synergistic efEfects. 6 WO 2013/174148 PCT/CN2013/000604 Example 3 After the feeding of example 2 ended, fresh weight, length, radius, breaking strength, bone rnineral density (BMD), and ash, Ca and P contents of the piglets' femurs were measured as below: Femur fresh weight: left femur was drawn out, muscles and hamstrings were stripped off to separate the femur (no injury of periosteum and cartilage cap), and then the femur fresh weight was measured. Femur length: Femur length was measured by Vernier Caliper after the femur fresh weight was measured. Femur radius: femur radius (cannon circumference of femur) was measured at a middle position after the femur fresh weight was measured. Femur breaking strength: femur breaking strength was measured by Electronic Universal Material Mechanics Testing Machines (South China University of Technology, Guangzhou, China) to show the tibial strength. The span was set as 5 cm and the loading speed was set as 3 min/min. The bone was broken at a middle point The sensor indicated the biggest force loaded during the breaking tern, which was expressed as breaking strength (N). Femur bone mineral density: femur bone mineral density was measured by Bone Densitometer SD-20l0 (Beijing Search Institute of Nuclear Industry, Beijing, China) with a radioactive source of low energy nucldes (241 Am). The bone for measuring (fresh sarnple) was positioned under the defector, and the driving device was started to synchronously move the radioactive source and detector. The amount of radiation penetrating through the bone was detected and bone mineral content (BMC, gecm) per unit of length, was calculated based on the permeation rate. Femur bone mineral density (BMD, g/cm~) was calculated based on the scan length and the bone mineral content (BMC). Femur ash content After wrapped with filter papers, the femur was dipped in anhydrous ethanol for dehydration for 24 h and then extracted by ether for degrease for 24 h. After dried to constant weight at 100-105 ">C, the dry weight of the dehydrated and degreased femur was recorded. ILater, the femur was crushed, carbonized under low temperature until there was no smoke, transferred to a high temperature furnace for burning at 550 "C for 3 h, taken out into a drier for half an hour, and then weighed. It was put into the high temperature furnace for burning for 3 h. into a drier for cooling for half an hour, and then weighed again until the difference between two adjacent weighings was less than 1%. Finaly, the ash content of the dehydrated and degreased femur was calculated by: Ash Content (%)= [ash content after ashed(g)j / [dry weight of degreased bone before ashed (g~j x 100% 7 WO 2013/174148 PCT/CN2013/000604 Femur ash Ca content: measured by EDTA direct volumetric method (see China State St andard GB/F 6436-92). Femur ash P content: measured by P Mo.V acid colorimetric method (see China State St andard GB/T6437-92). Each group was measured twice and averages were obtained as follows: Variables A B C D Femr reh wigt g) 81.07±4.1 85.55±1.62 85.734.75 85.45±3.01 Femu legth mm) 111.45±1.80 113.14±0.81 113.7±1.49 113.74±1.61 Femr rdis (m) 8.43±0.13 8.61±0.09 8.66±0.22 8.69±0.09 m0.180±0.005 0.1840.002 0.237±0.004 0.251 0.003 553.67±31.55 649.83±27.66 844.58±34.89 940.17±29.83 Ca(%)11.68±0.20 12.70±0.18 12.58±0.65 13.74±0.31 P(%)5.90±0.32 6.26±0.18 6.62±0.62 6.91±0.31 Ash (%35.63±0.41 36.57±0.61 40.95±0.40 40.69±0.26 1 The data indicate that, the administration of 25-H-ydroxy Vitamin D 3 and 6-phytase improved bone performance of the piglets. Specialy, the simultaneous administration of 25-Hydroxy Vitamin D 3 and 6-phytase provided synergistic effects in improving bone mineral density and breaking strength, and increasing Ca and P contents of the bone. The above examples illustrate the invention without limitaion. 1t should be understood that any person skiled in the art may make obvious amendments and modification to the invention, which should be covered within the protection scope of the present invention. .. . . . . . . . . . . . . .. . . . . . . . . . . .. .

Claims (14)

1. A method for improving growth performance and/or bone performance of an animal comprising administering to the animal an effective amount of 25-H ydroxy Vitamin D 3 and phytase.

2. The method of claim 1, wherein the animal is selected from the group consisting of non-ruminants and ruminants, ruminants include but not limited to sheep, goat and cattle, and non-ruminants include but not limited to horse; rabbit; pig including but not limited to infant pig, piglet, growing-fattening pig, sow and boar; and poultry such as turkey, duck and chicken,

3. The method of clim 1, wherein the growth performance is average daily weight gain (ADWG), average daily feed intake (ADFI) and/or feed conversion rate (FCR) of the animal.

4. The method of claim 1, wherein the phytase is 3-phytase, 6-phytase or a combination thereof.

5. The method of claim 4, wherein the 6-phytase is derived from a genetically modified strain Aspergillus otyzac (DSMZ 22594).

6. The method of any one of claims I to 5, wherein, based on the body weight of the animal to be administered, the 25-Hydroxy Vitamin D 3 is administered in the amount of 0.2-20 pgkg body weight per day, preferably 0.5-15 gkg body weight per day, more preferably 1-10 psg/kg body weight per day, even more preferably 1.5-5 g/kg body weight per day, and most preferably 2 pig/kg body weight per day; and the phytase is administered in an amount of 15-150 FfTU/kg body weight per day, preferably 20-120 FTU/kg body weight per day, more preferably 30-100 FTU/kg body weight per day, even more preferably 50-80 FTU/kg body weight per day, and most pref'erably 60 FTV/kg body weight per day.

7. The method of any one of claims 1 to 5, wherein the 25-Hydroxy Vitamin D 3 and the phytase are administered to the animal simultaneously.

8. The method of any one of claims 1 to 5, wherein the 25-Hydroxy Vitamin D 3 and the phytase are administered to the animal separately and successively.

9. The method of claim 8, wherein the 25-Hydroxy Vitamin D 3 and the phytase are administered to the animal separately in an interval of 10 mins, 20 mins, 30 mins, 40 mins, 50 mins, 1 h, 2 h, 3 h or 4 h.

10. The method of any one of claims 1 to 9, wherein the 25-Ilydroxy Vitamin D 3 and the phytase are administered to the animal as a feed composition, or as components of an animal feed or in drinking water of the animal. 9 WO 2013/174148 PCT/CN2013/000604

11. Use of a composition comprising 25-Hydroxy Vitamin D 3 and phytase as an animal feed or feed additive.

12. The use of claim 11, wherein the phytase is 3-phytase, 6-phytase or a combination thereof.

13. The method of claim 12, wherein the 6-phytase is derived from a geneticaly modified strain Ahpergillus oryzac (DS MZ 22594).

14. The use of any one of claims 11i to 13, wherein the animal feed or the animal feed with addition of the feed additive comprises 5-500 pig/kg, preferably 10-300 pg/kg, more preferably 20-200 pg/kg. even more preferably 40-100 pg/kg, and most preferably 50 gg/kg of 25-H ydroxy Vitamin Db; and 500-4000 FTU/kg, preferably 800-3000 FTU/kg, more preferablyl000-2500) FTU/kg, even more preferably 1200-2000 FLU/kg, and most preferably 1500 FTU/kg of phytase, 10