AU2006252054B2 - Animal feed containing plant fatty acids with conjugated double bonds - Google Patents

Description

P/00/0 Il Regulation 3.2 AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION FOR A DIVISIONAL PATENT ORIGINAL TO BE COMPLETED BY APPLICANT Name of Applicant: E.I. DU PONT DE NEMOURS AND COMPANY Actual Inventors: CAHOON, Edgar Benjamin; CARLSON, Thomas J.; HITZ, William Dean; RIPP, Kevin G. Address for Service: CALLINAN LAWRIE, 711 High Street, Kew, Victoria 3101, Australia Invention Title: ANIMAL FEED CONTAINING PLANT FATTY ACIDS WITH CONJUGATED DOUBLE BONDS The following statement is a full description of this invention, including the best method of performing it known to us: 14/12/06.atl 6237.front Da. I -2 ANIMAL FEED CONTAINING PLANT FATTY ACIDS WITH CONJUGATED DOUBLE BONDS Field of the Invention 5 This invention relates to animal feed containing plant fatty acids. Background of the Invention Fatty acids bearing chemical modifications in addition to the common double bonds are found in the storage lipids of many oilseeds (Harwood, J. L. (1980) In The 10 Biochemistry of Plants, T.S. Moore Jr., ed CRC Press, New York, pp 91-116). Some of these modifications functionalise the fatty acid to produce products that are useful in industrial applications; this is opposed to the more common usage of plant derived lipids as foods. Examples are the use of the hydroxylated fatty acid ricinoleic acid in lubricants, and the short- or medium-carbon chain length fatty acids 15 from palm oil in detergents. In some cases, fatty acid composition of the storage lipids of oilseeds produced in temperate climates can be modified by the addition of genes from exotic sources so that large amounts of unique fatty acids are produced (Ohlrogge, J. B. (1994) Plant Physiol. 104, 821-826). Fatty acids containing conjugated double bonds are major components of the 20 seed oil of limited number of plant species. For example, x-parinaric acid (9-cis, I 1 trans, 13-trans, 15-cis-octadecatetraenoic acid) and p-parinaric acid (9-trans, I I trans, 13-trans, 15-cis-octadecatetraenoic acid) compose more than 25% of the total fatty acids of the seed oil of Impatiens species (Bagby, M.O., Smith, C.R. and Wolff, I.A. (1966) Lipids 1, 263-267). In addition, a-eleostearic acid (9-cis, 1 1-trans, 13 25 trans-octadecatrienoic acid) and p-eleostearic acid (9-trans, 1 I-trans, 13-trans octadecatrienoic acid) compose >55% of the total fatty acids of the seed oil of Momordica charantia (Chisolm, M. J. and Hopkins, C. Y. (1964) Can. J. Biochem. 42, 560-564; Liu, L., Hammond, E. G. and Nikolau, B. J. (1997) Plant Physiol. 113, 1343-1349). 30 The presence of conjugated double bonds in fatty acids provides the functional basis for drying oils such as tung oil that are enriched in isomers of eleostearic acid. This is due largely to the fact that fatty acids with conjugated double 14/12/06,at13587.div spcc.2 -3 bonds display high rates of oxidation, particularly when compared to polyunsaturated fatty acids with methylene interrupted double bonds. Drying oils, such as tung oil, are used as components of paints, varnishes, and inks. Conjugated fatty acids can also be used as an animal feed additive. 5 Conjugated linoleic acids (CLAs, 18:2) have been used to improve fat composition in feed animals. U.S. Patent No. 5,581,572, issued to Cook et al. on December 22, 1998, describes a method of increasing fat firmness and improving meat quality in animals using conjugated linoleic acids. 10 U.S. Patent No. 5,554,646, issued to Cook et al. on September 10, 1996, describes a method of reducing body fat in animals using conjugated linoleic acids. U.S. Patent No. 5,519,451, issued to Cook et al. on July 6, 1999, describes a method of improving the growth or the efficiency of feed conversion of an animal which involves animal feed particles having an inner core of nutrients and an outer 15 layer containing a conjugated fatty acid or an antibody that can protect the animal from contacting diseases that can adversely affect the animal's ability to grow or efficiently convert its feed into body tissue. U.S. Patent No. 5,428,072. issued to Cook et al. on June 27, 1995, describes a method of enhancing weight gain and feed efficiency in animal which involves the 20 use of conjugated linoleic acid. The mechanism by which these effects are realized is not known. It is believed that no one heretofore has discussed the use of conjugated 18:3 fatty acids (conjugated linolenic acids or CInAs), for improving animal carcass characteristics. The biosynthesis of fatty acids with conjugated double bonds is not well 25 understood. Several reports have indicated that conjugated double bonds are formed by modification of an existing double bond (Crombie, L. and Holloway, S. J. (1985) J Chem. Soc. Perkins Trans. 11985, 2425-2434; Liu, L., Hammond, E. G. and Nikolau, B. J. (1997) Plant Physiol. 113, 1343-1349). For example, the double bonds at the 11 and 13 carbon atoms in eleostearic acid have been shown to arise 30 from the modification of the A double bond of linoleic acid (18:2A9,1 2 ) (Liu, L., Hammond, E. G. and Nikolau, B. J. (1997) Plant Physiol. 113, 1343-1349). The exact mechanism involved in conjugated double formation in fatty acids, however, 14/12/06.at13587.div spec,3 -4 has not yet been determined. Thus, while candidate enzyme classes have been suggested, no gene sequences have been isolated from those candidate classes and from tissues that are known to produce fatty acids with conjugated double bonds. 5 Summary of the Invention The present application is a divisional application of patent application No. 2003246043 which is itself a divisional application of patent application No. 56746/99 ("the parent specifications"), the contents of which are herein incorporated by reference. 10 The invention of the parent specifications concern an isolated nucleic acid fragment encoding a plant fatty acid modifying enzyme associated with conjugated double bond formation wherein said fragment or a functionally equivalent subfragment thereof (a) hybridizes to any of the nucleotide sequences set forth in SEQ ID NOS:1, 3, 19, 23, or 29 under conditions of moderate stringency or (b) is at 15 least 45% identical to a polypeptide encoded by any of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 19, 23, or 29 or a functionally equivalent subfragment thereof as determined by a comparison method designed to detect homologous sequences. The present invention relates to animal feed comprising at least one 20 conjugated linolenic acid derived from oil extracted from a natural source selected from the group consisting of bittermelon, pot marigold, jacaranda, catalpa, and pomegranate. Preferably, the conjugated linolenic acid is present in a carcass quality improving amount. 25 The invention also relates to a method of supplementing the diet of an animal with the feed of the invention. This invention also concerns animal feed comprising an ingredient derived from the processing of any of the seeds obtained from plants transformed with the chimeric genes discussed in the parent specifications as well as animal feed 30 comprising at least one conjugated linolenic acid derived from oil extracted from a natural source selected from the group consisting of bittermelon, pot marigold, jacaranda, catalpa, and pomegranate. 30/03/10ck 13 587speciclaims.doc.4 -5 Brief Description of the Figures and Sequence Descriptions The figures and the sequence listing and their descriptions in the parent specifications are herein incorporated by reference. 5 Detailed Description of the Invention The technical terms described and defined in the parent specifications are herein incorporated by reference. The term "o 6 -oleic acid desaturase" refers to a cytosolic enzyme that catalyzes the insertion of a double bond into oleic acid between the twelfth and 10 thirteenth carbon atoms relative to the carboxyl end of the acyl chain. Double bonds are referred to as "cis" or "trans" because they are chiral units that can assume the following non-equivalent structures:

-(H)

2 C C(H) 2 - H C(H) 2 \ I / C=C C=C / \ H H -(H) 2 C H cis trans The oleic acid substrate for this enzyme may be found to a glycerolipid such 15 as phosphatidylcholine. The term "o 6 -oleic acid desaturase" is used initrechangeably with the terms "ao 6 -desaturase", "A 1-oleic acid desaturase", "A 2 -desaturase", and "Fad2". The o) and A" positions are equivalent in oleic acid (C 18) because o carbons are counted from the methyl-end, while A-carbons are counted from the carboxyl-end of the fatty acid chain. The enzymes of the present invention comprise 20 activities involving desaturation of fatty acids resulting in conjugated double bond formation. The term "conjugated double bond" is defined as two double bonds in the relative positions indicated by the formula -CH=CH-CH=CH- (Grant & Hackh's Chemical Dictionary, Fifth Ed., R. Grant and C. Grant eds., McGraw-Hill, New York). The n-orbital electrons are shared between conjugated double bonds, but 25 remain relatively independent in unconjugated double bonds. This explains the greater reactivity of conjugated double bonds to oxidation. The modifying enzymes, associate with conjugated double bond formation described herein, are also referred to as "o0-oleic acid desaturase-like", or "A1-oleic acid desaturase-like. The terms " 14/12/06,at I 3587.div spec.5 -6 related" and "-like" reflect the conservation and differences in nucleic acid sequence homology between the genes encoding Fad2 enzymes versus the genes of the present invention. The invention of the parent specifications concern an isolated nucleic acid 5 fragment encoding a plant fatty acid modifying enzyme associated with conjugated double bond formation wherein said fragment or a functionally equivalent subfragment thereof (a) hybridizes to any of the nucleotide sequences set forth in SEQ ID NOS: 1, 3, 19, 23, or 29 under conditions of moderate stringency or (b) is at least 45% identical to a polypeptide encoded by any of the nucleotide sequences set 10 forth in SEQ ID NOS: 1, 3, 19, 23, or 29 or a functionally equivalent subfragment thereof as determined by a comparison method designed to detect homologous sequences. Such enzymes are normally expressed in developing seeds of Impatiens balsamina, Momordica charantia and Chrysobalanus icaco that are similar in 15 sequence to plant, membrane-bound fatty acid desaturases. However, these fatty acid modifying enzymes differ from membrane-bound fatty acid desaturases in their functionality. Specifically, these enzymes are associated with the formation of fatty acids having conjugated double bonds and, more particularly, with the formation of conjugated linolenic acids. Examples of fatty acids having conjugated double bonds 20 include, but are not limited to, eleostearic acid and/or parinaric acid. Naturally occurring plant oils containing eleostearic acid include tung oil from Aleuritesfordii or montana, which contains up to 69% a-eleostearic acid in the oil extracted from the seeds, or oils from valarian species (Centranthus microsiphon). There can also be mentioned jacaric acid (from the jacaranda tree, Jacaranda mimosifolia and 25 Jacaranda chelonia, 1 8

:

3

A

9 transI Itrans, 3eis), calendic acid (from marigold or African daisy, Calendula officinalis, and Osteospermum spinescens and Osteospermum hyoseroides, 18:3A transIOtransI2cis), catalpic acid (from the trumpet creeper, Catalpa ovata, or speciosa, or bigniniodes, 18:3A 9trans,I Irans,13cis), and punicic acid (from bittermelon and pomegranata, or Tricosanthes species, Cucurbita, and Punica 30 granatum, Tricosanthes cucumeroides, 18:3A 9 eis,II trans, I3is). These and other examples of fatty acids having conjugated double bonds may be found in "The Lipid Handbook" (Second Edition, Gunstone, F.D. et al., eds., Chapman and Hall, London, 14/12/06.at 13587div spec,6 -7 1994), Crombie and Holloway (J. Chem. Soc. Perkins Trans. 1985:2425-2434), and Liu, et al. (Plant. Physiol. [1997] 113:1343-1349). These conjugated fatty acids are also referred to as CInAs (conjugated linoleic acids) because they are all 18:3 in composition. This is in contrast to CLAs (conjugated linoleic acids) which have an 5 18:2 configuration. The nomenclature "18:3" denotes the number of carbons in the fatty acid chain (in this case "18" or stearic acid length), and the number of unsaturating double bonds (in this case "3" specifying this fatty acid as linolenic). Although 18:2 and 18:3 denote linoleic acid and linolenic acid, respectively, the positions of the double 10 bonds are not specified (i.e., they may be unconjugated or conjugated, cis or trans). The term "eleostearic acid" as used herein refers to a mixture of cis-trans isomers of A 9

'"'''

3 -octadecatrienoic acid (18:3A 9 ''''13). This mixture comprises principally ca-eleostearic acid (18:3A 9cis, Itrans,13trans) but may contain other isomers including p-eleostearic acid (1 8

:

3 A9transI itransI3trans) The term "paranaric acid" as 15 used herein refers to a mixture of cis-trans isomers of A 9 " ' 3

''

5 -octadecatetraenoic acid (18:4A 9 '1''' 3 ,1 5 ). This mixture comprises principally a-parinaric acid (18:3A9trans, I rans,I3trans,Istran) but may contain other isomers including P-parinaric acid (18:3A 9trans, IItransI3transI5trans) As those skilled in the art will appreciate, eleostearic acid and parinaric acids are separated easily by gas chromatography-mass 20 spectrometry (GC-MS, see Figure 3) and the alpha forms can be distinguished from the beta forms (marked by * in Figure 3). More details on GC-MS analyses are found in Examples 4, 5, 7, and 8 of the parent specifications. Examples of comparison methods which detect sequence homology include but are not limited to the BLAST computational method as described in the parent 25 specifications. Sequences with pLogs greater than 5, or preferably greater than 10, or more preferably greater than 15, and most preferably greater than 20, that are defined as FADs or lipid desaturases are candidates. cDNAs encoding enzymes associated with conjugated double bond formation can be identified from the candidate pools using 30 transformation screening. Individual cDNAs are inserted into expression vectors and transformed into yeast or plant host cells using methods well known to those skilled in the art (see Examples 3, 4, 5, 7, 9, and 10 of the parent specifications). Production 14/12/06.ai 13587.div spec.7 -8 of fatty acids containing conjugated double bonds is confirmed by GC-MS analyses as described in the Examples 4, 5, 7, and 8 of the parent specifications. Yeast or plant tissue culture cells are preferred for initial screening due to speed and the ease with which they can be handled when dealing with large numbers of transformants 5 and the appropriate cell biology and eucaryotic cell physiology. The instant fatty acid modifying enzymes associated with conjugated double bond formation in seeds of Impatiens balsamina, Momordica charantia, and Chrysobalanus icaco produced in heterologous host cells, particularly in the cells of microbial hosts, can be used to prepare antibodies to the fatty acid modifying 10 enzymes associated with conjugated double bond formation in seeds of Impatiens balsamina, Momordica charantia, and Chrysobalanus icaco by methods well known to those skilled in the art. The antibodies are useful for detecting the instant fatty acid modifying enzymes associated with conjugated double bond formation in seeds of Impatiens balsamina, Momordica charantia, and Chrysobalanus icaco in situ in 15 cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant fatty acid modifying enzymes associated with conjugated double bond formation in seeds of Impatiens balsamina, Momordica charantia, and Chrysobalanus icaco are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression 20 of foreign proteins are well known to those skilled in the art. Any of these could be used to construct chimeric genes for production of the instant fatty acid modifying enzymes associated with conjugated double bond formation in seeds of Impatiens balsamina, Momordica charantia, and Chrysobalanus icaco. These chimeric genes could then be introduced into appropriate microorganisms via transformation to 25 provide high level expression of the encoded fatty acid modifying enzymes associated with conjugated double bond synthesis in seeds of Impatiens balsamina, Momordica charantia, and Chrysobalanus icaco. An example of the use of the Impatiens balsamina fatty acid modifying enzyme in Saccharomyces cerevisiae for the production of parinaric acid from linolenic acid is discussed in Example 5 of the 30 parent specifications. An example of a vector for high level expression of the instant fatty acid modifying enzymes associated with conjugated double bond formation in 14/12/06.ai]3587.div spcc,8 -9 seeds of Impatiens balsamina, Momordica charantia, and Chrysobalanus icaco in a bacterial host is discussed in Example 9 of the parent specifications. It has been found that conjugated fatty acids, more specifically, conjugated linolenic acids, can also be used as an animal feed additive. The quality of meat 5 grown for consumption is dependent upon many variables that ultimately influence market demand for the product. For instance, pork quality improvement is a primary focus of the pork industry. Quality variables include pork colour, water holding capacity, size, chemical composition and firmness of lean and fat tissue. Ecxperiments have shown that the fat firmness of pork can be influenced by the 10 addition of conjugated linoleic acid (1 8

:

2

A

9 cis,i irans or A'otrans,I2cis) to swine diets (Eggert, J.M., et al. (1999) J. Anim. Sci. 77(Suppl):53; Thiel, R.C., et al. (1998) J. A nim. Sci. 76(Suppl): 13; Wiegand, B.R., F.C. Parrish Jr. and J.C. Sparks (1999) J. Anim. Sci. 77(Suppl):19; U.S. Patent No. 5,554,646; and U.S. Patent No. 5,851,572). Some experiments have also reported improved carcass leanness and the efficiency 15 of feed utilization when conjugated linoleic acid (CLA) is added as a supplement to the diet. It is not known whether feeding of different conjugated fatty acids would have similar effects. The invention of the parent specifications describe the production of conjugated double bonds in 18:3 and 18:4 fatty acids which are derived from 18:3 fatty acids in transgenic seeds that can be used as feed additives. 20 Thus, the instant invention concerns animal feed comprising an ingredient derived from the processing of any of the seeds obtained plants or plant cells transformed with any of the chimeric genes discussed herein or the animal feed can comprise at least one conjugated linolenic acid derived from oil extracted from a natural source selected from the group consisting of tung, bittermelon, pot marigold, 25 jacaranda, catalpa, and pomegranate. The ingredient or conjugated linolenic acid should be present in a carcass quality improving amount. A "carcass quality improving amount" is that amount needed to improve the carcass quality of an animal. The ingredient can be a mixture of fatty acids obtained from such seeds. This mixture can be in any form suitable for use as a feed additive. For example, the 30 mixture can be in the form of an oil whether or not it is saponified. Also of interest is animal feed comprising oil obtained from any of the foregoing seeds. This invention also includes a method of improving the carcass 14/12/06.at 13587.div spec.9 - 10 quality of an animal by supplementing a diet of the animal with any of the animal feeds discussed above. Examples 5 The present invention is further defined in the following examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can ascertain the essential 10 characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. The examples, figures and sequence listing of the parent specifications are herein incorporated by reference. 15 Example I Identification of o6-Oleic Acid Desaturase-Related Sequences from Tungp, (Aleurites fordii) Eleostearic acid composes >65% of the seed oil of tung (Aleuritesfordii) (Badami, R.C. and Batil, K.B. (1981) Prog. Lipid Res. 19:119-153). This species 20 thus represents an additional source of cDNAs or genes encoding o 6 -oleic acid desaturase-related enzymes associated with conjugated double bond formation. As described in Example 8 of the parent specifications, the coding sequences for these enzymes can be identified using PCR amplification with degenerate oligonucleotides designed from conserved amino acid sequences in o -oleic acid desaturases and 25 related enzymes. The template for PCR amplification can be first-strand cDNA or cDNA libraries prepared from tissues that accumulate fatty acids with conjugated double bonds (as described in Example 8 of the parent specifications). In addition, genes for o 6 -oleic acid desaturases do not contain introns within their open-reading frames (Okuley, J. et al. (1994) Plant Cell 6:147-158) . Therefore, genomic DNA 30 isolated from species such as tung can be used as template to amplify the coding sequences of o 6 -oleic acid desaturase-related enzymes associated with conjugated double bond formation. As a demonstration of this, genomic DNA was isolated from 14/12/06,a13587.div spec, 10 - 1I tung leaves using the method described by Shure et al. (1983) Cell 35:225-233. The resulting DNA was used as template for PCR reactions with degenerate oligonucleotide primers designed from conserved amino acid sequences in 0 6 -oleic acid desaturases and related enzymes. The sense oligonucleotide was 5 5'ttgaattcAARGCNATHCCNCCNCAYTGYTT3' (SEQ ID NO:25) which corresponds to the amino acid sequence KAIPPHCF, and the antisense oligonucleotide was 5'ttgaatTCCNCKNARCCARTCCCAYTC3' (SEQ ID NO:26) which corresponds to the amino acid sequence EWDW(L/F)RG. (Note: The base pairs in lower case were added for restriction digestion to facilitate cloning of PCR 10 amplification products and flanking EcoRI recognition sequence is underlined). Forty cycles of PCR amplification were conducted using Taq polymerase in a 100 pli reaction volume that contained 150 ng of tung genomic DNA. The annealing temperature used was 45'C. The resulting products of approximately 775 bp were subcloned into Pgem-t (Promega) according to the manufacturer's protocol and 15 transformed into E. coli DH1OB (Gibco-BRL). Nucleotide sequence was then obtained for cDNA inserts from plasmids of eleven of the resulting colonies. Homology comparisons indicated that these sequences encode 255 amino acid portions of o6-oleic acid desaturase-type enzymes. In addition, the sequences corresponded to two distinct classes of genes, which were designated Class I (SEQ 20 ID NO:27) and 2 (SEQ ID NO:29, see Figure 1 of the parent specifications). The Class I and 2 gene products share 75% amino acid sequence identity (Figure 1 of the parent specifications). Of these, the Class 2 gene product is more diverged relative to known o 6 -oleic acid desaturases. For example, the 255 amino acid sequences encoded by the Class I and 2 PCR products share 76% and 72% identity, 25 respectively, with the corresponding portion of the soybean (o-oleic acid desaturase (Figure 1 of the parent specifications). In addition, the residue immediately adjacent to the first histidine box in the Class 2 polypeptide is a glycine (as indicated by an asterisk in Figure 1 of the parent specifications). A glycine in this position is only observed in (o 6 -oleic acid desaturase-related enzymes that have diverged 30 functionality, such as the castor oleic acid hydroxylase (van de Loo, F.J. et al. (1995) Proc. Natl. A cad. Sci. US.A. 92:6743-6747) and the Crepis palaestina epoxidase (Lee, M. et al. (1998) Science 280:915-918). Given this feature of its primary 14/12/06.aW13587.div specI I - 12 structure and its more distant relation to known (o -oleic acid desaturases, it is believed that the polypeptide encoded by the Class 2 gene is the enzyme associated with conjugated double bond formation from tung. There are two other amino acid changes that are believed to be useful in identifying enzymes involved in conjugated 5 bond formation. The amino acid immediately following the first histidine, in the first histidine box mentioned above, is a conserved aspartate. The Chrysobalanus, Momordica, and Licania sequences have a glutamate substitution at this position that is not found in any other published Fad2 gene. The tung and Impatiens enzymes do not have this substitution, but they both have the glycine substitution two amino 10 acids upstream of this position. Another amino acid difference that is believed to be useful is at position 312 of the Momordica sequence. Momordica, Chrysobalanus, and Licania all contain a proline substitution at this position. None of the other published Fad2 enzymes have this substitution. The remaining portion of the tung gene is obtained using one of two 15 methods. First a cDNA library is made from mRNA isolated from tung seeds or developing seedlings as outlined in Example 1 of the parent specifications. The library clones are randomly sequenced and analyzed as outlined in Example 2 of the parent specifications. Alternatively, PCR amplification of clones is accomplished using primers selected from the sequence presented in SEQ ID NO:29 such that an 20 antisense primer is used to amplify the amino terminal portion of the gene and a sense primer amplifies the carboxy-terminal portion. The paired primers in the reaction are made to the plasmid vector sequence that resides upstream and downstream of the inserted cDNA, respectively. A second method to obtain the tung sequences involves using "inverse" PCR" on the tung genomic DNA. Briefly, tung 25 genomic DNA is fragmented using a restriction enzyme that gives a small (2-3 Kb) fragment that hybridizes to the sequence presented in SEQ ID NO:29. The digested genomic DNA is diluted and ligated in a reaction designed to favour intramolecular ligation. PCR primers are used that diverge from a sequence found in SEQ ID NO:29. The PCR fragment obtained in this reaction has the genomic sequences 30 flanking SEQ ID NO:29 and is used to construct primers that allow for the PCR amplification of the complete tung gene responsible for conjugated bond formation in the fatty acids. 14/12/06,ati 13587.div spec. 12 - 13 Example 2 Coniugated 18:3 Fatty Acids Can Improve Carcass Quality When Added to Animal Feed 5 Experiments were conducted to evaluate the effects of feeding eleostearic (18:3) conjugated fatty acids on pig growth, carcass characteristics, and fat firmness. Twenty-four pigs (barrows, castrated males, from PIC genetics) with a capacity for high rates of daily lean growth and reduced back fat were randomly assigned by litter mates, weight, and block to three dietary treatments. Group one was fed normal corn 10 feed, group two received normal corn feed supplemented with CLA, and the third group received normal corn feed supplemented with conjugated linolenic acids, i.e., ClnAs (18:3 conjugated fatty acids). Pigs were penned individually and identified by ear tattoo. The average initial weight of the barrows was 125 pounds. Pigs were placed on their respective test diets at 150 lb, after being fed a common diet. 15 Diets were fed in two phases: Phase 1 (150 to 200 lb), and Phase 2 (200 to 250 lb). Ingredient and nutrient compositions of the treatment diets are shown in Table I and Table 2, respectively. The diets were formulated to be isocaloric. Table I 20 Ingredient Composition of Diets Ingredient, % NCI NC+CLA NC+ClnA Grower Diets NC' 69.826 69.826 69.826 Soybean Meal, 48%2 25.283 25.283 25.283 A-V Fat' 2.498 2.498 2.498 L-Lysine-HC1 4 0.073 0.073 0.073 Limestone 0.838 0.838 0.838 Dical 216 0.761 0.761 0.761 Choline CH, 60%' 0.096 0.096 0.096 TM & Vitamin Premix' 0.250 0.250 0.250 Salt 9 0.300 0.300 0.300 Copper Sulfate'O 0.075 0.075 0.075 14/12/06,atl3587.div spec. 13 -14 Finisher Diets NC 75.142 75.142 75.142 Soybean Meal, 48% 20.340 20.340 20.340 A-V Fat 2.564 2.564 2.564 Limestone 0.740 0.740 0.740 Dical 21 0.525 0.525 0.525 Choline CH, 60% 0.065 0.065 0.065 TM & Vitamin Premix 0.250 0.250 0.250 Salt 0.300 0.300 0.300 Copper Sulfate 0.075 0.075 0.075 1 - normal hybrid corn, W677, from Wyffels, Atkinson, I L 2 - Perdue Farms, Inc., Greenville, NC 3 - Moyer Packing Co., Souderton, PA 4 - Archer Daniels Midland Co., Decatur, IL 5 5 - Akey, Inc., Lewisburg, OH 6 - Potash Company of Saskatchewan, Davenport, IA 7 - Akey, Inc., Lewisburg, OH 8 - Trace Minerals and Vitamin Premix, Young's, Greensboro, MD 9 - Akey, Inc., Lewisburg, OH 10 10 - Akey, Inc., Lewisburg, OH Table 2 Calculated Nutrient Composition of Treatment Diets Nutrient Phase 1 (150-200 1b) Phase 2 (200-250 1b) Energy, kcal/lb 1734 1756 Energy, kcal/kg 3823 3871 Protein, mcal % 18.00 16.00 Lysine, mcal % 1.05 0.86 Methionine+Cysteine, mcal % 0.64 0.61 Calcium, % 0.60 0.50 Total Phosphorus 0.55 0.49 15 The mixer used to prepare the diets was flushed with 300 lb corn prior to mixing and between each mix to prevent cross-contamination. Conjugated linoleic acid (CLA) was purchased from Conlinco, Inc. (Detroit Lakes, MN) as "ClareenTM". Conjugated linolenic acid (ClnA) was from a commercial source of tung oil 14/12/06.atI3587.div spec. 14 -15 (Industrial Oil Products, Woodbury, NY) that was approximately 65% a-eleostearic acid. To achieve a final conjugated fatty acid concentration of 0.50%, 0.83 lb CLA preparation/100 lb diet and 0.73 lb CLnA preparation/00 lb of diet were added. To minimise oxidation of the conjugated fatty acid, diets were prepared each 14 days 5 and refrigerated until use. Feed was added to feeders in minimal amounts daily. The antibiotic bacitracin methylene disalicylate (BMD, Alpharma, Inc., Fort Lee, NJ) was included in all diets (50 g/ton). Feed samples were collected for amino acid and fatty acid analysis. Live weights were recorded to determine average daily gains Phase 1 (150 to 10 200 lbs), and Phase 2 (200 to 250 lbs). Feed weight data were also collected to determine feed efficiency. Animals were observed 2-3 times daily for access to feeders and waterers, house temperatures, and any abnormal health conditions. Pigs were not replaced during the trial. Any animals that died were necropsied to determine the cause of death. Dead animal body weights were used to correct feed 15 efficiency. When pigs reached 250 pounds body weight they were slaughtered, processed and standard carcass measurements were collected. Because of limitations on conjugated fatty acid, pigs fed CLA and CLnA were fed a common diet four days prior to slaughter. Bellies from the eight pigs in each study group were evaluated for 20 fat firmness evaluated by measuring belly thickness before and after compression. Fat compression was achieved by placing a 50 lb weight on the fresh belly for one hour. Fat compression was quantified by subtracting the compressed belly thickness from the initial belly thickness. Belly thickness was measured using a micrometer. The results of the belly compression evaluation are shown in Table 3. Data were 25 analysed as a randomised complete block design using the GLM (General Linear Model) procedure of SAS (Statistical Analysis Systems). Table values represent the difference between compressed and uncompressed pork belly thickness. Because a pork belly is greater than 50% fat, the belly compression test is an indicator of relative firmness of pork belly fat. Addition of either CLA or CLnA to NC diets 30 resulted in greater fat firmness in pigs. The improved pork fat firmness resulting from dietary addition of CLA is consistent with results reported by others (Eggert, J.M., et al. (1999) J. Amin. Sci. 77 (Suppl):53; Thiel, R.C., et al. (1998) J. Anim. Sci. 14/12/06.at 13587.div spec,15 -16 76 (Suppl):13; Wiegand, B.R., F.C. Parrish Jr., and J.C. Sparks (1999) J Anim. Sci. 77 (Suppl):19; U.S. Patent No. 5,554,646; and U.S. Patent No. 5,851,572). Improved fat firmness resulting from dietary CLnA inclusion has not been previously reported. 5 Based on the results of this experiment, addition of conjugated linoleic acid (CLA) or conjugated linolenic acid (CInA) to pig diets results in improved fat firmness. Table 3 Results of Fat Compression Test Measurement NC NC+CLA NC+CLnA SEM Pork Belly Compression, mm 33.22 28.0 30.8 0.68 10 ' Standard Error of the Mean 2 All three test sample means were statistically different (P < 0.05). Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and 15 "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference to any prior art in this specification is not, and should not be 20 taken as, an acknowledgment or any form or suggestion that the prior art forms part of the common general knowledge in Australia. 30/03/10.ck 3587speciclaims.doc. 16

Claims (1)

1. 2. The animal feed of claim 1, wherein said conjugated linolenic acid is present in a carcass quality improving amount. to 3. A method of improving the carcass quality of an animal by supplementing a diet of the animal with the feed of claims I or 2. 30/03/10,ckl3587spciclaims.doc, 17