Background Of The Invention: The omega-3 (or n-3) polyunsaturated fatty acid known as docosahexaenoic acid (DHA) is found in abundance in the brain and retina. DHA has been found to be required at high levels in the brain and retina for optimal mental functioning (learning ability, etc.) and visual acuity (visual performance), respectively. Therefore, dietary DHA is regarded as an essential dietary nutrient for infants (both preterm and term) and children on mixed diets ( (British Nutrition Foundation: Unsaturated fatty acids: Nutritional and Physiological Significance. Andover, England: Chapman and Hall, 1992, and British Nutrition Foundation; Recommendations for Intakes of Unsaturated Fatty Acids, 1992).

Studies have shown significant alleviation of various risk factors for cardiovascular disease (CVD) when fish and fish oils containing the omega-3 polyunsaturated fatty acids DHA and eicosapentaenoic acid (EPA) are consumed. Epidemiological studies have also shown an inverse relationship between DHA levels in the population (diet/blood) and the risk of CVD (Leng, G. C. et al., Arterioscler. Thromb. 1994; 14: 471-478, Simon, J. A., et al. Am. J. Epidemiol. 1995; 142: 469-476). Therefore, part of the cardioprotective effect of fish/fish oils containing omega-3 polyunsaturated fatty acids is due to DHA, in addition to EPA. The cardioprotective effects of omega-3 fatty acids are considered to be mediated by a number of physiological/biochemical mechanisms. Studies have shown that the enrichment of heart tissue in DHA provides an antiarrhythmic effect (Pepe, S. and McLennan, P. L. J. Nutr. 1996; 126: 34-42) which may

account for the reductions in cardiac arrest and sudden cardiac death in those having a higher DHA status (in diet/body). In addition, dietary DHA intakes and increased status in the body have been implicated in favourable effects on attention-deficit disorders (Stevens, L. J. et al Am.

J. Clin. Nutr. 1995; 62: 761-768), depression and anxiety disorders (Hibbeln, J. R. and Salem, N. Am. J. Clin. Nutr. 1995; 62: 1-9.), as well as protection against breast cancer in postmenopausal women (Zhu, Z. R., et al., Nutr. Cancer. 1995; 24: 151-160).

DHA is found in abundance in fish and fish oils (with very minor amounts in eggs and some meats), but it is absent from all plant-derived food products including vegetable oils. Certain plant oils (eg., soybean oil) contain moderate amounts of another omega-3 fatty acid, alpha-linolenic acid (LNA), which can, to an extremely limited extent (Emken, E. A., et al. BBA. 1994; 1213: 277-288 and Salem, N., et al., Proc. Natl. Acad. Sci. 1996; 93: 49-54.) be metabolically converted in the body to provide sub-optimal amounts of DHA.

Mothers'breast milk in North America and elsewhere typically contains approx. 0.14 to 0.2% by wt. of the fat/fatty acids as DHA (Chen, Z.-Y., et al., Lipids. 1995; 30: 15-21 and Makrides, M., et al., Am.

J. Clin. Nutr. 1995; 61: 1231-1233). Infants on breast milk (a source of DHA) perform better in visual acuity testing than those on formulae typically used in North America lacking DHA (Nettleton, J. A. J. Am.

Diet Assoc. 1993; 93: 58-64 and Makrides, M., et al., Lancet. 1995; 345: 1463-68). Intelligence scores were also found to be higher in children receiving breast milk containing DHA when young (Lucas, A., et al., Lancet. 1992; 339: 261-264). DHA has been added to selected infant formula products in Japan and recently in Europe to provide a direct dietary source of DHA in infant formula thereby providing optimal DHA levels and its associated benefits to infants. (JP 043411; WO 9212711; EP 404058; US 4670285). Unfortunately, cow's milk is devoid of DHA and has only very modest amounts of LNA.

Consequently, the recommended dietary levels of DHA for children

on mixed diets (see recommendations of the British Nutrition Foundation; Recommendations for Intakes of Unsaturated Fatty Acids, 1992) cannot be fulfilled with cow's milk and associated dairy products (cheeses, ice-creams, etc.). This is particularly important at a development stage where there is active learning, information processing, and intellectual development.

Efforts have been made to increase dietary intake of DHA by adding or incorporating DHA into various foods. Methods have been developed to increase the level of omega-3 fatty acids in the flesh of beef cattle (US 5290573), sows (US 5106639; DE 3808885; Taugbol, O. et al., Zentralbl. Veterinarmed. A., 40 (6): 437-443,1993), poultry (US 5012761; JP 04271754; US 5133963; US 5069903), and eggs (KR 9311396; US 5069903). DHA has also been added as a dietary supplement to infant formula as discussed above, and milk. Sources of DHA for supplementing milk or infant formula include fish products, fatty acid containing microbial oils (US 5374657; US 5397591; US 5407957), or fatty acids extracted from a mixture of egg yolk and coconut oil (US 4670285).

Researchers have been able to increase DHA content in the expressed milk of humans (Harris, W. S. et al., Am. J. Clin. Nutr. 40 (4): 780-785,1984; Henderson, R. A., Lipids, 27 (11): 863-869,1992; US 5069903), sows (Taugbol, O. et al., Zentralbl, Veterinarmed. A. 40 (6): 437-443,1993), and rats (Yonekubo, A., et al. J. Nutr. 123 (10): 1703-1708, 1993). However, researchers have had difficulty obtaining significant levels of DHA in cow's milk. (Hebeisen, D. F., et al. Int. J. Vitam. Nutr.

Res., 63 (3): 229-233,1993).

A method, a feed additive and a feed to increase DHA content in expressed milk of dairy cattle has been disclosed by the present inventors in PCT Patent Application PCT/CA97/00430 published as WO 97/49297 and in Wright, T.; McBride, B.; and Holub, B, World Rev. Nutr. Diet 83: 160-165,1998. The disclosed feed additive included blood meal, however, there is a perception by some in the

public and industry that blood meal in cattle feed could be linked with mad cow disease or bovine spongiform encephAlopathy (BSE).

Therefore, there is a demand for a feed additive which increases DHA content in expressed milk in dairy cattle, but does not contain blood meal.

SUMMARY OF THE INVENTION The present inventors have found that DHA (docosahexaenoic acid) is expressed in the milk of dairy cattle fed a novel feed additive containing DHA, and inhibitors of microbial degradation of DHA in the rumen of the cattle and which does not contain blood meal. The feed additive does not affect the ability of the cattle to digest the feed by normal symbiotic digestion. The feed additive is also palatable to the cattle, and therefore food consumption is not decreased. As a result, the health of the cattle is maintained and their productivity is not reduced.

The present inventors found that when dairy cattle are fed the feed additive throughout lactation, the levels of DHA in the expressed milk are between 0.14% and 0.99% of the long chain fatty acids in the milk. These DHA levels are as high as, or exceed the World Health Organization recommended levels of DHA in milk (0.2 to 0.3%). A feed additive containing only fish meal with DHA resulted in amounts of DHA far below the levels obtained with the novel feed additive of the present invention.

Broadly stated, the present invention relates to a feed additive for dairy cattle and which comprises a source of DHA and inhibitors of microbial degradation of DHA in the rumen of dairy cattle and does not contain blood meal. The source of DHA and the inhibitors of microbial degradation of DHA are present in the feed in an amount sufficient to enhance the concentration of DHA in the milk of dairy cows fed with feed containing the additive. In an embodiment of the invention, the source of DHA is fish meal. The inhibitors of microbial degradation comprise feather meal. In one embodiment the inhibitors

of microbial degradation consists of feather meal. Preferably the feed additive comprises an amount of feather meal sufficient to increase the concentration of DHA in milk from cattle consuming the feed additive. The invention also contemplates a feed containing the feed additive.

The invention also relates to a method of producing milk in dairy cattle which is enriched for DHA comprising feeding dairy cattle a diet containing a feed additive of the invention for a period of time longer than one day and preferably for at least about 14 days and milking the dairy cattle to obtain milk enriched for DHA.

The invention further relates to expressed milk from dairy cattle enriched for DHA which is produced by feeding cattle a diet containing a feed additive of the invention for a period of more than one day, preferably at least about 14 days, and milking the dairy cattle to obtain milk enriched for DHA. The expressed milk of the invention preferably containing about 0.2% to 0.5% of DHA. The invention further relates to a DHA-enriched dairy product produced using the expressed milk of the invention. The DHA-enriched dairy product is preferably selected from the group consisting of cheese, yogurt, cream, ice-creams, powdered milk, evaporated milk, infant milk and butter.

In one embodiment, the invention relates to the use of feather meal as an inhibitor of microbial degradation of DHA in the rumen of dairy cattle in an amount sufficient to increase the concentration of DHA in milk. In a preferred embodiment the feather meal is added to the feed for dairy cattle.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become

apparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT As hereinbefore mentioned, the present invention relates to a feed additive for dairy cattle which comprises a source of DHA, and inhibitors of microbial degradation of DHA in the rumen of dairy cattle with no blood meal. Preferably, these components are present in an amount sufficient to increase the concentration of DHA in the milk of dairy cows fed a diet containing the additive."An amount sufficient"as used herein would be understood by a person skilled in the art to be the amount which results in an increased concentration of DHA in milk.

The DHA in the feed additive may be a DHA concentrate containing 5 to 98% DHA, or it may be a component of an extract from a source known to contain DHA; for example, it may be a component of an extract derived from fish, such as fish meal. DHA concentrates may be obtained from commercial sources for example, from Life Plus International, Inc. (Batesville, Arkansas), Norsk Hydro As, and Nippon Oils and Fats KK. Alternate sources of DHA could also include algal biomass or bioengineered oil-seed corps containing DHA.

Indeed any source of DHA which is palatable to cattle and which does not compromise the taste qualities of milk produced may be used.

According to an embodiment of the invention, the feed additive comprises feather meal and fish meal containing DHA. The fish meal may be made from mackeral, caplin, talapia, manhaden, or herring, preferably herring, and contains an amount of DHA which will provide about 5 to 12% DHA by weight of the total fatty acids in the feed as described herein. Fish meal may be selected which has 76.7% protein and total fat content of 8.2% (dry measurement basis). The feather meal may be made from the feathers of broiler chickens, although the feathers of any foul including turkeys and geese, may be used. Feather meal may be selected which has 93.9% protein, and a total fat content of 2.6% (dry measurement basis). It will be appreciated

that the fish meal and feather meal may be obtained from commercial sources for example, Ralston Purina, Shurgain, Masterfields, ADM & Purina Mills.

In a preferred embodiment, the feed additive contains 15 to 60% feather meal, preferably 15 to 20% by weight of the total feed additive; and 15 to 60% fish meal, preferably 50 to 55% by weight of the total feed additive.

The feed additive may contain a carbohydrate fraction such as soft white wheat or corn. Preferably the feed additive contains soft white wheat. However, the feed additive may not contain blood meal.

The feed additive may be added to a basal feed which may contain a carbohydrate fraction, a protein fraction, a lipid fraction and/or a vitamin/mineral fraction. Examples of components in carbohydrate fractions include corn silage, alfalfa hay, Timothy hay, wheat straw, barley grain, canola meal, oat grain, mixed straw, and corn. Typical components in the vitamin/mineral fraction include magnesium oxide, limestone, potassium chloride, sodium chloride, and a trace mineral supplement, containing zinc, copper, manganese, selenium, vitamins A, D and E. Commercial sources of these components are Ralston Purina, Shurgain, Masterfields, ADM & Purina Mills. Typical protein feeds include soybean meal, corn gluten meal, distillers dried grains, and meat meal. Typical lipid feeds including tallow, fast food waste, and megalac (Church & Dwight Co.).

The feed containing the feed additive may be pelleted for feeding to dairy cattle, or the feed additive and basal feed may be fed to the cattle in a total mixed ration or as separate ingredients.

In an embodiment of the invention a feed is provided comprising (a) a basal feed containing 2 to 10%, preferably 5-7%, mixed straw; 40 to 55%, preferably 45 to 47% corn silage; 35 to 50%, preferably 42 to 45% high moisture corn, and 2-4% of a vitamin/mineral fraction, each percentage being a percentage of the total weight of the basal feed; and (b) a feed additive comprising 1 to 7% feather meal, and 1 to 13%

herring meal, each percentage being a percentage of the total weight of the feed (dry weight basis). In a preferred embodiment of the invention, the feed additive comprises about 3.4%, feather meal and about 6.2% herring meal, each percentage being a percentage of the total weight of the feed (dry weight basis). In a more preferred embodiment, the feed additive comprises about 1.0% feather meal and about 1.9% herring meal, each percentage being a percentage of the total weight of the feed (dry weight basis).

As illustrated in the examples herein, the use of a feed additive comprising feather meal and a source of DHA such as fish meal provides milk with an increased concentration of DHA thereby avoiding the concerns (mad cow disease, BSE) and further cost associated with blood meal.

Accordingly, the invention also relates to the use of feather meal as an inhibitor of microbial degradation of DHA in the rumen of dairy cattle. The use of feather meal is characterized in that the feather meal is added to the feed for dairy cattle. According to a preferred embodiment, the use is characterized in that an amount sufficient to enrich milk from dairy cattle with DHA is added to feed additive, which itself may be added to cattle feed.

The invention also relates to a method of producing milk in dairy cattle which is enriched for DHA comprising feeding the dairy cattle a diet containing the feed additive of the invention for a period of at least about 7 days, and milking the dairy cattle to obtain milk enriched for DHA. The cattle may be fed a basal feed containing the feed additive, or the feed additive and basal feed may be fed to the cattle in a total mixed ration or as separate ingredients.

The cattle are preferably fed throughout lactation, and for a time longer than one day, preferably for at least 14 days, and more preferably 17 days, in order to obtain expressed milk with a DHA content which is greater than 0.2 % of long chain fatty acids in the milk. Typically the cattle are fed the feed additive (which may be part

of a basal feed) twice daily. The amount of feed additive given to the cattle ranges from 600 grams to 7.5 kg per animal per day.

The method of the invention for producing expressed milk enriched for DHA may be applied to any breed of dairy cattle, for example, Ayshire, Guernsey, Holstein, Jersey, Brown Swiss, Dutch Belted, Canadienne and Milking Shorthorn. It will be appreciated that the method may also be applied to other ruminant species such as sheep and goats to produce expressed milk enriched for DHA.

The expressed milk from dairy cattle enriched for DHA produced by a method of the invention contains levels of DHA typically in the range of 0.14 to 0.99% of long chain fatty acids in the milk. These levels are as high as, or higher than found in human expressed milk. The DHA content of expressed milk obtained using the method of the invention meets or exceeds the World Health Organization recommended levels of DHA in milk (0.2 to 0.3%). The taste of the milk enriched for DHA produced by the method of the invention is not altered and it is therefore suitable for human consumption and concern about transference of mad cow disease and BSE are avoided by not including blood meal. Further, the method achieves these concentrations of DHA in milk using only feather meal as the inhibitor of DHA degradation in the rumen.

It will be appreciated that other DHA-enriched dairy products can be produced by using the method described herein. For example, cheese, yogurt, cream, ice-creams, powdered milk, evaporated milk, infant formula, and butter enriched for DHA may be produced using the method of the invention.

The milk and dairy products of the present invention enriched for DHA are nutritionally superior products to conventional milk products. The milk and dairy products may be of particular benefit with respect to the various factors for brain development, visual acuity, and cardiovascular disease. The benefits of the invention also extend beyond the production of DHA-enriched food products for

human consumption. For example, dairy cattle that are fed with a feed of the invention can be expected to exhibit improved health effects associated with omega-3 fatty acids since DHA is an essential nutrient for growth, development, and neuronal functioning in the animals.

The following non-limiting examples are illustrative of the present invention: EXAMPLES Example 1 DHA Production with Three Different Levels of Supplement Containing Fish Meal, Feather Meal and Blood Meal Six multiparous Holstein dairy cows post-peak lactation were assigned at random to a Latin square design, with a 3 x 2 factorial arrangement of treatment. The Latin square was balanced for residual effects. The treatments were: Three dietary levels of a custom rumen- undegradable protein supplement, and two different feed intake levels of a custom rumen protein basal ration. The length of each of the six periods was 21 days. Each 21 day period was divided into three phases: (1) 7 days for treatment adaptation and ad libitum intake, (2) 14 days for restricted or ad libitum feed intake (depending on treatment), and (3) the final five days of phase (2) were used for data collection, when relevant measures were recorded.

The cows were fed twice daily in equal amounts at approximately 0600 and 1400 hours. The milking was done twice daily at 0500 and 1600 hours. Individual milk samples were taken from both the morning and afternoon milking, and later pooled based on production, to obtain a representative daily sample from each cow.

Tables 1 and 2 show the composition of the basal ration and the custom feed supplement respectively. The protein supplement was added to the basal ration at three levels of total intake (dry matter basis): Low 4.5%, Medium 14.9%, and High 29.1%, to obtain the three levels of protein supplementation. Table 3 shows the amount of Feather meal, Blood meal, and Herring Meal in the three supplement levels, as a percent of feed.

Table 1. Low Crude Protein Ration

Ingredient % of Ration DM Basis MixedStraw 6.3 CornSilage 46. 0 High Moisture Corn 44.3 Mineral and Vitamin Mix 3.1 Table 2. Ingredients in Protein Supplement Component Percent of Supplement (As Fed) Soft White Wheat 25 Feathermeal 22. 8 Bloodmeal 9. 5 HerringMeal 42.7 Table 3. Ingredients in Protein Supplement as a Percent of Diet. Supplement Percent of Percent of Percent of Herring Level Feather Meal Blood Meal Meal In Feed In Feed In Feed LOW 1. 03 0. 43 1.92 MEDIUM 3. 36 1. 40 6.23 HIGH 6. 63 2. 76 12.56 No ill effects were observed in animals consuming the feed supplement. Table 4 shows the DHA content (% long chain fatty acids in the milk) in expressed milk two weeks after the cows first started consuming the feed. Milk production and feed consumption were similar to production/consumption observed when the cows were fed conventional feeds.

An omega-3 fatty acid analysis was carried out on the feed containing the protein supplement. DHA, EPA, DHA+EPA, and total omega-3 in the feed were 6.5,4.3,10.8, and 12.9 %, respectively, of total fatty acids in the feed.

Table 4. Effects of Custom feed supplement on DHA levels in milk.

Supplement DHA (ls mean; % Standard Error Main Effect P- Level of long chain fatty Value acids) LOW 0. 15 0. 02 0.0001 MEDIUM 0. 28 0. 02 0.0001 HIGH 0. 35 0. 02 0.0001 Example 2 Fish meal Supplement Alone Pelleted fish meal was fed to 5 Holstein cows for three weeks.

An identical ration, but without fish meal, was fed to 5 control cows for comparison purposes. The ration was the low crude protein ration of Table 2. Fish meal was fed to the cows at a level of 1.92% of the diet.

No blood meal or feather meal were fed to the treatment cows.

Milk samples were taken for fatty acid analysis on day zero from all cows, when no fish meal had been fed to either group. Milk samples were subsequently taken on day 21 from all cows when five had consumed fish meal. Results were compared between groups for both days. Results for EPA and DHA are expressed on a percent fatty acid basis of all long chain fatty acids, and are shown in Table 5.

Table 5. Results of Fish meal Experiment. % of long chain Fish meal No Fish meal Significance FA Day 0 EPA 0. 032 0. 033 NS DHA 0. 020 0. 020 NS Day 21 EPA 0. 041 0. 032 N S DHA 0. 061 0. 036 0.

There was no significant difference between control and fish

meal groups for concentration of EPA, but there was a significant difference between the groups for DHA concentration in milk at day 21.

The mean concentration of DHA from the fish meal supplemented cows in this experiment is significantly less that the 0.15 level reported in Example I for the equivalent level of fish meal supplementation. This is presumably due to the lack of inhibitors in the feed of microbial degradation of DHA in the rumen of the dairy cattle.

Example 3 DHA Production with Three Different Levels of Supplement Containing Fish Meal and Feather Meal This experiment was designed as a 6 x 6 Latin square (six cows x six periods), balanced for residual effects, with 21 day periods. There was a 3 x 2 factorial arrangement of treatment. The treatments were: three dietary levels each for two different rumen-undegradable protein (RUP) supplements. Each 21 day period was divided into three phases: (1) 7 days for treatment adaptation and ad libitum intake of prescribed dietary treatment; (2) days 8-21 for ad libitum feed intake and (3) days 17-21 of phase (2) were used for data collection, when relevant measures were recorded.

The cows were fed twice daily in equal amounts at approximately 0600 and 1400 hours. The milking was done twice daily at 0500 and 1600 hours. Individual milk samples were taken from both the morning and afternoon milking, and later pooled based on production, to obtain a representative daily sample from each cow.

Table 6 shows the composition of the basal ration which all cows received. Tables 7 and 8 show the composition of the respective rumen-undegradable protein feed supplements used in the treatments. The protein supplements were added to the basal ration at three levels of total intake (dry matter basis): Low 3.75%, Medium 11.75% and High 27.0%, to obtain three levels of protein supplementation.

Table 6. Basal Diet Ingredient % As Fed Straw 4.0 Corn Silage 60.6 High moisture corn grain 33.6 Mineral/Vitamins 0.8 Salt 0. Ground Lime0. 7 Table 7. Supplement: Feather Meal Based Ingredient % As Fed Wheat 25 FeatherMeal 60 Herring Meal I 15 Table 8. Supplement: Fish Meal Based Ingredient % As Fed Wheat 25 Feather Meal 15 Herring Meal 60

No ill effects were observed in animals consuming the feed supplement. Table 9 shows the milk and milk solids yield (kg/day) for milk, protein, fat and lactose, for each of three different supplement levels. Tables 10 shows the percent of protein, fat, lactose and DHA in the respective expressed milk samples. DHA levels are given as a percentage of long chain fatty acids.

Table 9. Milk and Milk Solids Yield (Kg/d) Fish Meal Supplement Feather Meal Supplement P-Value 3.75 11. 75 27.0 3.75 11. 75 27, 0 SE Suppl. Level Milk 23.7 22.8 21.3 27 Protein 0.72 0.73 0.78 0.66 0.64 0.70.03.002.12 Fat 0.70 0.59 0.64 0.57.04.44.21 Lact 1.07 1.01 1. 051.06 0.96 1. 01. 04. 43.16 1 Table 10. Milk Composition (%) Fish Meal Supplement Feather Meal Supplement P-Value 3. 75 11. 75 27. 03. 75 11. 75 27. 0 SESuppl. Level Protein 3.11 3.29 3.02 3.15.06.001.01 Fat 3.00 2.63 3.00 2.51.18.99.13 Lact 4.51 4.37 4.51 4.49.04.06.10 DHA* 1 0.38 0.68 0.73 0.28 0.30 0. 28. 04. 0001.0005 * °o o Long Chain Fatty Acids

The feather meal based supplement resulted in DHA transfer from diet to expressed milk but changing dietary levels of the supplement did not result in an increased level of DHA in the expressed milk of dairy cattle. The fish meal based supplement, however, did result in a much increased level of DHA in the expressed milk over the three dietary levels. The above example shows that a feed additive comprising a source of DHA and just feather meal, is sufficient to enhance transfer of DHA from diet to the expressed milk of dairy cattle.

Table 11. Comparison of Data for Examples I and III Supplement Level Example I Fish-III Feather-III SE P-Value Low 34.4 39.6 86.6 2.9 0.0001 Medium 20.2 17.5 44.1 7.4 0.0001 High 10.9 8.9 14.5 7.4 0.0001

For comparison purposes, these data effectively demonstrate the abilities of the three supplements. Example I and the fish-based supplement of Example III are reasonably similar in transfer efficiency.

The high levels of DHA in the milk from the fish-based supplement of <BR> <BR> <BR> Example III are a reflection of greater DHA present in the supplement compared to the similar levels of Example I. The feather-based <BR> <BR> <BR> supplement of Example m has excellent transfer efficiency (87%) at the low level which is probably a reflection of the inhibitory nature of the feather meals.

Having illustrated and described the principles of the invention in a preferred embodiment, it should be appreciated to those skilled in the art that the invention can be modified in arrangement and detail without departure from such principles. We claim all modifications coming within the scope of the following claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.