AU2001267960B2 - Altering milk production and milk components - Google Patents

Description

WO 02/00034 PCT/NZ01/00123 ALTERING MILK PRODUCTION AND MILK COMPONENTS The invention pertains to the field of altering the concentration and yield of milk protein produced by a pasture-fed lactating mammal. This invention also relates to a method of increasing milk yield produced from a pasture fed lactating mammal.

Milk contains, among other things, fats, proteins (casein and a variety of other proteins such as beta-lactoglobulin, alpha-lactalbumin, serum albumin, and immunoglobulins), salts, sugar (e.g.

lactose), and various vitamins vitamins A, C, D and some B vitamins) and minerals (primarily calcium and phosphorus).

Many of the components of milk have separate commercial value independent of the value of milk as a whole. It is desirable to recover such components (particularly protein) from the milk.

For example, a wide variety of whey proteins are used as functional ingredients in bakery products, confections and beverages.

Because milk protein is a desired component of milk it is desirable to have milk that is high in such protein.

The composition and yield of milk produced from lactating mammals varies with species, breed, condition of the mammal from which milk is obtained and feed. Often the feed of a lactating mammal will be either supplemented with an additional feed source or designed specifically to ensure that the lactating mammal produces the desired yield of milk. It is also known in the art that feed supplements can alter the proportions of the various components of milk.

The concept of milk fat depression (MFD) is well known to those skilled in the art. In MFD the yield of milk fat is decreased but the yield of milk and non-fat components of milk remain largely unchanged. In some cases MFD can lead to up to a 50% lowering of milk fat. Many theories were proposed as possible mechanisms by which MFD occurred. These can be divided into theories that consider MFD to result from a shortage of milk fat precursors and those that attribute MFD to a direct inhibition of milk fat synthesis. Subsequent work has dismissed many of these theories, particularly those that implicate a shortage of lipid precursors as the likely cause of MFD WO 02/00034 PCT/NZ01/00123 Other theories that relate to MFD arising from direct inhibition of fat synthesis include a number of compounds as likely candidates responsible for this inhibition. Davis and Brown (1970) first proposed that MFD arose from direct inhibition by trans-octodecenoic acids (trans fatty acids or TFA) and Pennington and Davis (1975) further proposed that TFA were involved in MFD during feeding of both high concentrate diets and diets containing polyunsaturated oils. Following studies found TFA produced in the rumen or added to the diet were associated with MFD in cows, goats and mice (Astrup et al. 1976; Selner and Schultz, 1980; Teter et al. 1990; Wonsil et al.

1994; Gaynor et al. 1994; Romo et al. 1996). The majority of these studies used partially hydrogenated vegetable oils as the dietary source of TFA and authors conclude that TFA caused the MFD.

U.S. Patent No. 5,416,115 (hereinafter '115 patent) issued to Erdman et al. (1995) describes a method for altering milkfat by administering trans-fatty acids to lactating cows, however, neither patent '115 nor the inventors own work (Erdman, 1996) were able to establish causative effects between milkfat depression and the specific fatty acid isomers responsible.

It has been shown that intermediates of fatty acid biohydrogenation in the rumen appear to play a role in the regulation of milk fat synthesis (Griinari et al. 1998, Bauman et al. 1998;,Griinari and Bauman et al. 1999). CLA and TFA are two such interrelated fatty acid groups, which are naturally occurring and are produced through ruminal biohydrogenation of unsaturated fatty acids in the diet (Griinari and Bauman et al. 1999).

The '115 patent describes a method of reducing milk fat concentration from milk produced by ruminants and enhancing milk yield from ruminants is described. The method involves administration of trans-fatty acids to ruminants. No distinction is made in this patent between specific trans-fatty acid isomers.

CLA is reported to have beneficial health effects in monogastric animal models including anticarcinogenic, antidiabetic, antiobesity and antithrombotic properties (Ip et al. 1999; Pariza PCT/NZ01/00123 Received 26 August 2002 et al. 2000). Chouinard et al. (1999a) also found that post-ruminal infusion of CLA dramatically reduced milkfat concentration and yield in lactating dairy cows. This study used a commercial source ofCLA, which contained a number of CLA isomers, consisting mainly of cis/trans-9, 11, c/t-8, 10, c/t-10, 12, and c/t-ll, 13 forms. Both the concentration and yield of milk fat were decreased by -50% following infusion of just 50 g of CLA-60 per day. A subsequent study showed milk fat and yield were reduced by 28 and 25%, respectively, following post-ruminal infusion of three different CLA mixtures of different isomer enrichment (Chouinard et al. 1999b).

Dugan et al. (1997) and Dunshea et al. (1998) demonstrated that CLA decreased body fat deposition in growing pigs, while resulting in increased rates of lean tissue accretion. Similar effects have been shown for mice (Park et al. 1997; West et al. 1998; DeLany et al. 1999) and hamsters (Gavin et al. 2000). The antiobesity effects observed in mice, rats and pigs and the factors which lead to MFD have been described as 'anti-fat synthesis' (Bauman et al. 1998). The 12 C18:1 isomer is thought to be the active isomer reducing fat synthesis and increasing muscle accretion in mice (Park et al. 1999a and Recently, the specific CLA isomer responsible for inhibiting milk fat synthesis in dairy cows has also been identified as the t/c-10, 12 isomer by Dr. Bauman and co-workers at Cornell University (Baumgard et al. 2000; Baumgard et al. 2001).

In WO 99/66922 (Bauman et al.) a method for altering the nutritional components of milk is described. Specifically, the '66922 invention describes a method of decreasing milk fat content in milk and increasing the percentage of conjugated linoleic acid isomers in milk. This is achieved by administering to a lactating mammal an effective amount of a conjugated linoleic acid (CLA) sufficient to decrease the fat content of milk produced by the lactating mammal and increase the content of CLA isomers in the milk. The CLA needs to be in a form that allows it to bypass the initial digestive processes or rumen bacterial fermentation. Lactating dairy cows are used as examples in the '66922 invention. These cows were fed a total mixed ration (TMR) diet.

Accordingly, the cows were fed an optimal diet and all the nutritional requirements of these cows would have been met.

None of the prior art, including the '66922 application and its supporting manuscripts (Chouinard et al. 1999a and b) have shown increases in the yield of milk protein or milk protein concentration.

Chouinard et al 1999a shows a significant (P<0.05) linear trend for milk yield to 3 AMENDED

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JAN-13-04 04:13PM FROM- T-292 P.005/010 F-262 decrease with increasing dose of CLA. While the applicant does not wish to be bound by any particular theory, the applicant believes that the lack of response in milk yield and milk protein concentration and yield as shown in the prior art is because the studies were all conducted on grain-based TMR diets. In such diets the cows are fed an optimal diet and their nutritional requirements are met or exceeded. A reduction in milk fat production and the additional energy this provides for the cow is therefore not likely to benefit milk yield or protein concentration and yield. Cows fed a 100% pasture diet are usually not provided with optimum levels of nutrients (especially those used to produce energy) to achieve potential milk yield and milk protein concentration and yield (Kolver and Muller, 1998). In such a situation, where cows are fed inadequate or marginal amounts of certain amino acids or the diet is not meeting the cows energy requirements, or both, the concentration and yield of milk protein and milk yield are usually increased in response to the provision of additional energy providing nutrients (Thomas, 1980; Sutton, 1989; Mackle and Bauman, 1998). This Is because milk yield and milk protein concentration and yield are most highly correlated with energy intake (Emery, 1978; Sporndly.

1989). While the applicant does not wish to be bound by any particular theory, the applicant believes that in cows being fed a sub-optimal diet that does not meet the optimal nutritional requcirement of the cow, the energy conserved through MFD is directed to increased protein yield or concentration or increased milk yield. The energy conserved could also be directed to maintaining milk yield or milk protein concentration oryield while reducing nutrient intake.

A preferred feature of the present invention is a method of increasing the milk yield or increasing roilk protein concentration oryield for dairy cows fed a predominantly pasture diet, or at least the public is provided with a useful choice.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

4 (followed by page 4a) COMS ID No: SMBI-00568673 Received by IP Australia: Time 14:16 Date 2004-01-13 JAN-13-04 04:14PM FROM-T-2P.0/1 F-2 T-292 P-006/010 F-262 SUMMAARY OF UME IVENflON In a &g aspet t present incion provides a mthod of incasing t Yield Of milk protei in ittproducedfi frw attgmanualwhilemaintainIg or impro~vingthe yied of milk Frcduced said mmmal having a sub-otia diet said method comprisin administering to a lactat mam=a a compound ftat supprese milk fat synthesis.

In a second aspec fth presetk innton provide a metIod ineain the coVnntation ofmilk 4a (follwed by page COMS ID No: SMBI-00568673 Received by IP Australia: Time 14:16 Date 2004-01-13 PCT/NZ01/00123 Received 26 August 2002 protein in milk produced from a lactating mammal while maintaining or improving the yield of milk produced said mammal having a sub-optimal diet said method comprising administering to a lactating mammal a compound that suppresses milk fat synthesis.

In a third aspect the present invention provides a method of increasing the yield of milk produced from a lactating mammal said mammal having a sub-optimal diet said method comprising administering to a lactating mammal a compound that suppresses milk fat synthesis.

In a fourth aspect the present invention provides a method of maintaining milk yield and maintaining one or both of milk protein yield and milk protein concentration while reducing the nutrient intake of a lactating mammal to a sub-optimal diet said method comprising administering to a lactating mammal a compound that suppresses milk fat synthesis.

Preferably the diet is sub-optimal because it does not meet the optimal nutritional requirements of the lactating mammal to enable it to produce one or more of an optimal yield of milk, an optimal yield of milk protein, and an optimal concentration of milk protein in the milk produced.

Preferably this is because sufficient quantities of a component of the diet that assists in the production of energy or protein or is metabolised to produce energy or protein are not available.

Preferably the diet is a predominantly pasture diet. Alternatively, preferably said diet is a mixed ration diet or insufficient quantities of a TMR diet.

Preferably the lactating mammal is a ruminant. More preferably the lactating mammal is a bovine cow, a sheep, or a goat.

Preferably milk fat synthesis is suppressed by providing the lactating mammal with a diet that suppresses milk fat synthesis. Such diets are known to those skilled in the art.

A number of edible oils, for example plant fats and oils, tallow, grease or lard can also be used to suppress milk fat synthesis.

Preferably, said compound that suppresses milk fat synthesis is an edible oil or a derivative thereof More preferably said compound is a vegetable oil or a derivative thereof Still more preferably said compound is a trans-fatty acid or a salt or ester thereof.

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JAN-13-04 04:14PM FROM-T-9 P.0/1 F-6 T-292 P.007/010 F-262 Altanativly profrmably, said campoimdihat muppiwa milk fait synthesis is aL CLA fbnuulatlou More preferably sad compound is selected from the group of coqugated linolei acid isommr coniing of.

a) olstrazw b) ciatzans 11,13 hnoldc acid; o) ci/rans 10,12 linoeic acid; adi d) a mbcture of at least two of the above compounds.

Still more PreiferblY, said omupound is dlmtrami 10, 12 CLA, or a salt or aster thereof Alteamtvoy, preferably addt compound is aistcan 8, 10 CLA, or a salt or cutwr thereo Mmspreflabbr toLA or salt or ew tlnucof is adinlnitatd to anrnut and is adminitere in a m uh that sid CLA or salt or estr ao wfis bloavallable in t&e samanm orte nmaill s infith Wic=e t r nal s ammias u=df Ch LA Io adniustered allmcnbxily, preffambly the CIA is ins aSrm protected fiannuminal bactesial tz* In aifl aspec tpresent iavaiticaprovides irdlpruduccdby a mammal gthha had smediod of topreset invaitionapplied to it. Such milk canhobbied by milk th maimmal, which wiflbowall knwn to tos akbad int&a a It wi11 boewen11awnk tos =kiled Intem at "ha much milk cu be processed into a multid ofpwdacts.. Il preast inventio tvetb also roded to pm&d dled from milk podced by at mammal tha has had aL methlod ofthe prenat invention applie to 1L The invention consist in t *siging and als mivimulgm cousktiona ofvwhich tha (blowing Throughout this spcecification the word "comprise', &r variations such as "comprises" or "6comprisin(', will be understood to imply the inclusion of a stated element, integer or step, or Z"~x of elemnts, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

6 (followed by page 6a) COMS ID No: SMBI-00568673 Received by II' Australia: Time 14:16 Date 2004-01-13 JAN-13-04 04:14PMA FROM-T-2P.0/l T-292 P-008/010 F-262 DBSCRWTLON OF DRAWINGS In01: Tanpol patltnf fatl OM tr-, (war al) aM! $ol (uWpepae =09S 6a (followed by page 7) COMS ID No: SMBI-00568673 Received by IP Australia: rime (i-tm) 14:16 Date 2004-01-13 Received29 August 2001 11-day period. Abomasal infusion of CLA-60 was performed at either 0 20(A), 40 or g/d from days 0-4 (shown by dashed lines). Values are means of four cows.

FIG. 2. Temporal pattern of milk yield across 11-day period. Abomasal infusion of CLA-60 was performed at either 0 20(A), 40 or 80 g/d from days 0-4 (shown by dashed lines).

Values are means of four cows.

FIG. 3. Temporal pattern of milk fat concentration (lower panel) and yield (upper panel). Twicedaily abomasal infusion of CLA-60 was performed at either 0 g/d for restricted and ad libitum allowance or 50 g/d for restricted and ad libitum allowances, from days 0-10 (shown by dashed lines). "Values are means of 6 cows for restricted group and 5 cows for ad libitum groups.

FIG. 4. Temporal pattern of milk protein concentration for cows offered ad libitum (lower panel) or restricted (upper panel) allowances. Twice-daily abomasal infusion of CLA-60 was performed at either 0 g/d for restricted and ad libitum allowance or 50 g/d for restricted and ad libitum allowances, from days 0-10 (shown by dashed lines). Values are means of 6 cows for restricted group and 5 cows for ad libitum groups.

FIG 5: Temporal pattern of milk fat concentration across an 8-week period. Supplementation with rumen-protected CLA was performed at either 0 or control medium or high (M) dose rates from weeks 1-6 (shown by dashed lines). Week 0 was a pre-experiment covariate where all cows received no treatment and weeks 7 and 8 were washout periods after treatments had ceased. Values are means of 12 cows per treatment group for control group and 11 cows per treatment group for the medium and high CLA dose rates. Error bars show the least significant difference (Isd) values, thus, demonstrating significant differences.

FIG 6: Temporal pattern of milk fat yield across an 8-week period. Supplementation with rumen-protected CLA was performed at either 0 or control medium or high dose rates from weeks 1-6 (shown by dashed lines). Week 0 was a pre-experiment covariate where all 7 INTELLECTUAL PROPERTY OFFICE OF N.Z.

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WO 02/00034 PCT/NZ01/00123 cows received no treatment and weeks 7 and 8 were washout periods after treatments had ceased.

Values are means of 12 cows per treatment group for control group and 11 cows per treatment group for the medium and high CLA dose rates. Error bars show the least significant difference (Isd) values, thus, demonstrating significant differences.

FIG 7: Temporal pattern of milk yield across an 8-week period. Supplementation with rumenprotected CLA was performed at either 0 or control medium or high dose rates from weeks 1-6 (shown by dashed lines). Week 0 was a pre-experiment covariate where all cows received no treatment and weeks 7 and 8 were washout periods after treatments had ceased.

Values are means of 12 cows per treatment group for control group and 11 cows per treatment group for the medium and high CLA dose rates. Error bars show the least significant difference (Isd) values, thus, demonstrating significant differences.

FIG 8: Temporal pattern of milk protein yield across an 8-week period. Supplementation with rumen-protected CLA was performed at either 0 or control medium or high dose rates from weeks 1-6 (shown by dashed lines). Week 0 was a pre-experiment covariate where all cows received no treatment and weeks 7 and 8 were washout periods after treatments had ceased.

Values are means of 12 cows per treatment group for control group and 11 cows per treatment group for the medium and high CLA dose rates. Error bars show the least significant difference (lsd) values, thus, demonstrating significant differences.

FIG 9: Temporal pattern of milk protein concentration across an 8-week period.

Supplementation with rumen-protected CLA was performed at either 0 or control medium or high dose rates from weeks 1-6 (shown by dashed lines). Week 0 was a preexperiment covariate where all cows received no treatment and weeks 7 and 8 were washout periods after treatments had ceased. Values are means of 12 cows per treatment group for control group and 11 cows per treatment group for the medium and high CLA dose rates. Error bars show the least significant difference (Isd) values, thus, demonstrating significant differences.

PCT/NZ01/00123 Received 26 August 2002 DETAILED DESCRIPTION OF THE INVENTION "Predominantly pasture diet" as used herein means a diet that consists of at least 50% pasture herbage. The diet may or may not contain a feed supplement, provided the feed supplement when in combination with the pasture diet is a sub-optimal diet.

"Protected CLA formulation" as used herein means any CLA supplement that is protected from rumen fermentation and biohydrogenation but is bioavailable in the abomasum or small intestine.

"Maintaining milk yield" or "maintaining milk protein yield" or "maintaining milk protein concentration" as used herein includes minimising the reduction of yield or concentration.

"Mixed ration diet" as used herein means a diet comprising a mixture of foodstuffs (as a TMR diet would have) only the diet is lacking in a certain food group, vitamin or mineral such that it is a sub-optimal diet.

"Sub-optimal diet" as used herein means a diet which does not meet the optimal nutritional requirements of a lactating mammal to enable it to produce one or more of an optimal yield of milk, an optimal yield of milk protein, and an optimal concentration of milk protein in the milk produced. The supply of nutrients from a sub-optimal diet is inadequate to allow a lactating mammal to achieve its genetic potential or capacity in terms of milk yield produced or the yield of milk components produced.

A diet may be sub-optimal and fail to meet the optimal nutritional requirements if it is not of a sufficient quality, or alternatively the quality of the diet may be adequate for nutritional requirements but the quantity may not be.

There are a number of methods known in the art of providing a CLA fonnulation to a ruminant so that it is bioavailable to the abomasumr. One such method is direct infusion into the abomasum.

An alternative method is to feed the ruminant a protected CLA formulation. It is also anticipated that in the future, particularly with the opportunities that the study of transgenics is providing, that other methods of administering CLA will become available.

It is anticipated that the present invention could be used to extend the lactation cycle of a cow. A cow directs a significant portion of its energy towards producing milk duringlactation. After a long period of lactation its body condition will be the poorer for it. Because of this the lactation period is usually shortened to prevent excess deterioration on body condition. It is anticipated 9 AMWOD HEE-r 1RZA,1Ag8WEU WO 02/00034 PCT/NZ01/00123 that the use of a compound that suppresses milk fat synthesis will reduce the amount of energy required for milk production and therefore reduce the impact of milk production on body condition. As a result it would be possible to milk cows for a longer duration.

Example 1 CLA infusion effects of 4 dose levels on performance of cows fed at 100% pasture diet.

TABLE 1. Fatty acid profile of CLA-60 (Natural Lipids Ltd, Hovdebygda, Norway)'.

Fatty Acid of total fatty of total CLA acids CIs: 0 2.1 Cis-9 C18: 21.4 cis-9, cis-12 C 18 :2 6.6 Conjugated linoleic acid 2 (62.3) (100.0) cis/trans 8,10 9,11 C 18 :2 22.0 35.3 cis/trans 10,12 C 1 8 2 18.5 29.7 cis/trans 11,13 C 18 2 10.1 16.2 Other CLA 11.7 18.8 Unknown 3.6 'lot no. 10685.

2 cis/trans indicates molecule has one cis and one trans double bond cis-10, trans-12 or trans-10, cis- 12 CLA) PCT/NZ01/00123 Received 26 August 2002 Referring to Tables 1 and 2 and Figs 1 and 2, the effects of CLA on milk composition and yield were studied using varied doses of a commercially available CLA product (CLA-60) which is a mixture of CLA isomers (Natural Lipids, Inc., Hovdebygda, Norway). The CLA was infused directly into the abomasum continuously (over a 4 day period) which avoids bacterial fermentation of CLA in the rumen and its subsequent breakdown. In the practical situation, cows will be fed or dosed orally with a rumen-protected CLA supplement, which is coated to escape breakdown by rumen bacteria and pass through directly into the abomasum or small intestine. The mixture contains approximately 60% CLA with the four major isomers being cis/trans 8,10, cis/trans 9,11, cis/trans 10,12, cis/trans 11,13 (Table Cows were given a diet consisting of 100% pasture. The allowance was generous but not quite ad libitum. The diet would not have met the optimal nutritional requirements of the cows due to the inability of a cow to consume optimum dry matter intake from a feed source comprising 82% water, which was the water content of the pasture. The experiment was conducted early-mid lactation cycle at the commencement of the experiment, cows were an average of 80 2 days in milk.

11 t2MEOiDED SHEET i Wi- WO 02/00034 PCT/NZ01/00123 TABLE 2. Least squares means for performance during CLA-60 infusion.

infused (g/d) 1 Variable 0 20 40 80 SED 2 DMI, kg/d 16.5 16.6 16.1 15.9 0.71 Milk yield, kg/d 19.7 21.2 21.9 20.3 0.75 Fat 3.97 2.50 2.28 1.53 0.22 g/d 784 534 501 313 51 Crude protein 3.05 2.96 2.97 2.99 0.04 g/d 598 624 650 602 Crude protein:Fat 0.78 1.23 1.32 2.01 0.19 1 Treatments involved 4-day abomasal infusion of 0, 20, 40, or 80 g CLA-60/d. Values presented represent averages from PM and AM milking for the last day of the infusion periods 2 SED standard error of the difference.

In example 1, the effects of varied dose of CLA 20, 40 or 80 g CLAd) on cow performance were examined (Table Results demonstrated a substantial reduction in milk fat and yield through abomasal infusion of 80 g of CLA-60 per day, whereas the 20 and 40 g CLA/d rates decreased milk fat by 32-36% (Table Milk fat yield was also reduced by 60 during the g CLAJday treatment. Dry matter intake (DMI) was unaffected by any treatment. Milk yield was increased during the 20 and 40 g CLA/d treatments or 1.5 kg/d; and +11% or 2.2 kg/d, respectively), but not during the 80 g CLA/d treatment. There was a quadratic trend for milk protein yield to be highest for the middle two treatments. Milk protein yield was increased by the 40 g CLA/d treatment or 52 No other milk composition variables were affected by CLA infusion. These results indicate a significant 'sparing' of energy from the reduced milk fat production and constant DMI, which may have contributed to the increased yields of milk.

WO 02/00034 PCT/NZ01/00123 Example 2 CLA infusion effects in cows offered ad libitum or restricted pasture allowance In example 2 the effects of reducing milk fat yield when cows are offered a diet consisting solely of pasture at either ad libitum or restricted allowance, on milk production (yield of milk and protein and concentration of protein) was examined (Table It was hypothesised that reducing energy expenditure by decreasing milk fat production would provide the underfed cow (restricted allowance) with additional energy for milk yield, milk protein yield and body fat accretion. Dairy cows are often exposed to periods of underfeeding in pasture-based dairying systems. Under these conditions, the provision of additional energy from reduced milk fat synthesis may spare glucogenic amino acids and allow a greater supply for milk protein synthesis.

WO 02/00034 PCT/NZ01/00123 TABLE 3. Least squares means for performance during CLA-60 infusion to cows offered restricted or ad libitum allowance'.

Restricted Ad libitum allowance allowance Variable CLA CLA CLA CLA SED 2

SED

3 Milk yield, kg/d 11.1 11.6 14.9 14.7 0.47 1.63 Fat 4.93 2.56 4.60 2.53 0.17 0.28 g/d 541 291 686 374 9.3 51.1 Crude protein 3.49 3.68 3.67 3.86 0.06 0.19 g/d 385 421 544 565 19.0 51.0 Crude protein:Fat 0.72 1.44 0.80 1.54 0.04 0.06 STreatments involved 10-day abomasal infusion of 0 CLA) or 50 CLA) g CLA-60/d for cows offered either restricted or ad libitum pasture allowance. For all milk variables, values presented represent averages from PM and AM milking for the last 3 days of the infusion periods (days 8-10).

2 Standard error of the difference (SED) when making comparison between CLA infusion treatment groups.

3 Standard error of the difference (SED) when making comparison between pasture allowance treatment groups.

A daily dose of 50 g CLA-60 (fatty acid composition as described in Table 1) per day given by abomasal infusion was effective in reducing milk fat concentration and yield by 45 for the ad lib cows, and by 48 and 46 respectively, for cows offered a restricted allowance (Table 3).

Milk protein concentration was increased by 5 (almost 0.2 units) during CLA infusion, for both ad lib and restricted cows (Table Milk protein yield was increased by 36 g/d for cows offered restricted allowance (Table There was a trend for milk protein yield to increase during PCT/NZ01/00123 Received 26 August 2002 CLA infusion for cows offered ad libitum allowance, although this trend was not statistically significant. The protein to fat ratio was increased by CLA infusion for both cows offered ad lib and restricted allowances. The experiment was conducted during late lactation cycle.

Example 3 Long-term effects of rumen-protected CLA supplementation on production In example 3, an experiment was conducted to examine the long-term effects of 3 levels of rumenprotected CLA supplement medium or high dose; fatty acid composition as described in Table 1) on dairy cow milk production (Figures The figures (Figures 5-9) show selected milk production variables for the 6 week treatment period (weeks which were preceded by the pre-experimental covariate period (week 0) and followed by 2 weeks after treatment ceased (weeks 7 and Cows were given a diet of 100% pasture. The allowance was generous but not quite ad libitum. The diet would not have met the optimal nutritional requirements of the cows due to the inability of a cow to consume optimum dry matter intake from a feed source comprising 80-87% water, which was the water content of the pasture. The CLA supplement was fed once a day. The dosage rate was 165g of CLA-60 per day for high dose and 82.5g per day for low dose from week 1 to the end of week 3. From weeks 4 to 6 the dose rate was 110 Og of per day for high dose and 55g per day for low dose. The cows were an average of 40 days inmilk early lactation cycle) at the commencement of the trial.

Results show a significant reduction in milk fat concentration (17-19 in each of the 6 weeks of treatment by supplementation of both medium and high dose of rumen-protected CLA (Figure Corresponding data for milk fat yield show a significant decrease across the first 5 of the 6 treatment weeks (Figure Milk yield was increased by an average of 9 and 12% for the medium and high CLA dose rates, respectively, across the entire 6-week treatment period (Figure Milk protein yield also increased following both medium and high CLA supplementation by an average of 5 and respectively, in all but week 5 of the 6-week treatment period. Milk protein concentration was reduced marginally during weeks 1, 2, 3 and 5 of the treatment period; this observation is likely to be a dilution effect because the rate of increase in milk yield was marginally greater than the rate of increase in milk protein yield, thereby reducing the concentration of protein in milk.

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WO 02/00034 PCT/NZ01/00123 Although the invention has been described with reference to a specific example, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms.

Literature Cited and Incorporated by Reference: Astrup, H. L. Vik-Mo, A. Ekem, and F. Bakke. 1975. Feeding protected and unprotected oils to dairy cows. J. Dairy Sci. 59:426-430.

Bauman, D. C. L. Davis, and H. F. Bucholtz. 1971. Propionate production in the rumen of cows fed either a control or high grain, low fiber diet. J. Dairy Sci. 54:1282-1287.

Bauman, D. B. A. Corl, L. H. Baumgard, and J. M. Griinari. 1998. Trans fatty acids, conjugated linoleic acid and milk fat synthesis. Pages 95-103 in Proc. Comell Nutr. Conf.

Feed Manuf, Rochester, NY. Cornell Univ., Ithaca, NY.

Bauman, McGuire, Griinari Cnouinard, Y. PCT patent application W099/66922.

Baumgard, B.A. Corl, D.A. Dwyer, A. Saebo, and D.E. Bauman. 2000. Identification of the conjugated linoleic acid isomer that inhibits milk fat synthesis. Am. J. Physiol. Regulatory Integrative Comparative Physiology 278:R179-R184.

Baumgard, L. Sangster, and D. E. Bauman. 2001. Milk fat synthesis in dairy cows is progressively reduced by increasing supplemental amounts of trans-10,cis-12 conjugated linoleic acid (CLA). J. Nutr. 131: 1764-1769.

Chouinard, P. L. Coreau, D. M. Barbano, L. E. Metzger, and D. E. Bauman. 1999a.

Conjugated linoleic acids alter milk fatty acid composition and inhibit milk fat secretion in dairy cows. J. Nutr. 129:1579-1584.

Chouinard, P. L. Coreau, A. Saebo, and D. E. Bauman. 1999b. Milk yield and composition during abomasal infusion of conjugated linoleic acids in dairy cows. J. Dairy Sci. 82:2737- WO 02/00034 PCT/NZ01/00123 2745.

Davis, C. and R. E. Brown. 1970. Low-fat milk syndrome. Pages 545-565 in Digestion and Metabolism in the Ruminant. A. T. Phillipson, ed. Oriel Press, Newcastle upon Tyne, England.

DeLany, J. F. Blohm, A. A. Truett, J. A. Scimeca, andD. B. West. 1999. Conjugated linoleic acid rapidly reduces body fat content in mice without affecting energy intake. Am. J.

Physiol. Regulatory Integrative Comp. Physiol. 276:R1172-R1179.

Dugan, M. E. J. L. Aalhus, A. L. Schaefer, and J. K. G. Kramer. 1997. The effect of conjugated linoleic on fat to lean repartitioning and feed conversion in pigs. Can. J. Anim.

Sci. 77:723-725.

Dunshea, F. E. Ostrowska, M. Muralitharan, R. Cross, D. E. Bauman, M. W. Pariza, and C.

Skarie. 1998. Dietary conjugated linoleic acid decreases back fat in finisher gilts. J. Dairy Sci. 81(Suppl. 1):131 (Abstr).

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Claims (5)

1- A methiod Ofjncpiig the Yield ofreill Proteinrinnhlk produced fomaIlttigUSWi while maintining or imfprovM Mhe yiel of Milk produced said wzmmal having I'sub-opfln diet said metthod comprising adlIkiiste~fg to a lacotatn mammal a compound tt suppesse ndilc fat synthesis.

2. A mwethod of incaing the 0cpcnfl 6 Of of mt protin in mil produced from a laCtat inIm while ,nantainiuc or improving the yield of milk produced said nmnnl havi7ng a ub optimal diet sad method cozPrlsin achinisteIuR to a lactating manmi" a compounmd that suppreses milk fat syitbss.

3. A mthod ~f morcasmg the yield Of milk Produced from a lactat nianimmal said msrnna having a ,jib-opfiIfa diet said method comprising administering to a latt inaffll a compound that suppresses Mil fat synithesis.

4. A imthod of nmintaining milk yield and maintaining one or both of nilic protein yid and milk protein concutration wbile reducing the nutient intake; of a latatin~g nrmal to a sub- optimal diet said method conzptE~g adrumutedui to a lattig mammal a coii~oliId that supprsses milk Wi sYnthess A method as claimd in aTy of t preceding 01aim wherei fth diet is a sub-optma diet because sufficienit quantities of a component of the diet that assiss in the production Of energ Or protein or is metabolized, to Produce enerm' or protein are not available.

256. A method as clamd i any o Of the- precding claims wheitin t didt is a predoImieiilY Pasture diet. 7. A method as chimd in any One of claim 1to 5 wherein said diet is a mtixed ration diet or iusumieut quantities of a TMR diCL a. A mecthod as cilinid inaay One Of the- precedig clams wbmeei the lactating n==12l Is a ruminant. 21 COMS ID No: SMBI-00568673 Received by IP Australia: Time 14:16 Date 2004-01-13 WO 02/00034 PCT/NZ01/00123 9. A method as claimed in any one of the preceding claims wherein the lactating mammal is selected from a group comprising a bovine cow, a sheep, or a goat. A method as claimed in any one of the preceding claims wherein milk fat synthesis is suppressed by providing the lactating mammal with a diet that suppresses milk fat synthesis. 11. A method as claimed in any one of claims 1-9 wherein said compound is an edible oil or a derivative thereof. 12. A method as claimed in any one of claims 1-9 wherein said compound is a vegetable oil or a derivative thereof. 13. A method as claimed in any one of claims 1-9 wherein said compound is a trans-fatty acid or a salt or ester thereof. 14. A method as claimed in any one of claims 1-9 wherein said compound is a CLA formulation. A method as claimed in any one of claims 1 to 9 wherein said compound is selected from the group of conjugated linoleic acid isomers consisting of: a) cis/trans 8,10 linoleic acid; b) cis/trans 11,13 linoleic acid; c) cis/trans 10,12 linoleic acid; and d) a mixture of at least two of the above compounds. 16. A method as claimed in any one of claims 1 to 9 wherein said compound is the cis/trans 10,12 isomer of CLA or a salt or ester thereof. 17. A method as claimed in any one of claims 1 to 9 wherein said compound is the cis/trans 8, 10 isomer of CLA or a salt or ester thereof. 18. A method as claimed in any one of claims 14-17 wherein said compound is administered 22 JAN-13-04 04:14PM FROM- T-292 P.010/010 F-262 to a nruminat and is admnistered in a fbrm such that said CLA or salt or aster thereof is bioavailable In the abomaum or small intestine. 19. A method as claimed i any one of claims 14-17 whreln said coapound is adminlstaed to a u iant and wherein the CLA foArulation Is administend in a form such that the CLA is protected fin ruminal bacteial ftmaefio. A method as clulmed in any one of claims 1-4 mubstantially as herein dascribed with rafbrnce to any example threof 21. Milk producedby an imal that hba had tbo mahod of any ow of clams 1 -20 applied to t. 22. Aproduct derived ftomthe milk of olaim 21. 23. A method of increasing the yield of milk protein in milk substantially as hereinbefore described with reference to the Examples 1 to 3. 24. A method of increasing the concentration of milk protein in milk substantially as hereinbefore described with reference to the Examples 1 to 3. A method of increasing the yield of milk substantially as hereinbefore described with reference to the Examples 1 to 3. 26. A method of maintaining milk yield or maintaining milk protein yield or maintaining milk protein concentration substantially as herenbefore deserlbed with reference to the Examples I to 3. 23 COMS ID No: SMBI-00568673 Received by IP Australia: Time 14:16 Date 2004-01-13