The present invention relates to a method to produce milk, and especially milk having an increased unsaturated fatty acids content and a decreased trans fatty acids content. In addition, the invention relates to a method to select cows for feeding with a fat-rich feed. In another aspect, the present invention relates to a method to test a feed supplement. In yet another aspect, the invention relates to a method to reduce methane emission during milk production by cows. In a further aspect, the invention relates to a milk composition, use thereof and products based thereon.

Animal milk and especially cow's milk is an important nutrient and is widely consumed. Milk can further be processed into various milk-based products such as cream, cheese, butter, yogurt, desserts, ice-cream. Raw milk contains significant amounts of fat, protein and calcium and its composition varies widely among species.

In cow's milk, more than 70 wt.% of the total fat consists of saturated fatty acids, while unsaturated fatty acids constitute less than 30 wt.% of the milk fat. Unsaturated fatty acids, especially long-chain polyunsaturated fatty acids (LC-PUFA) are known to lower the risk of cardiovascular diseases. In prior art, it has been described that milk compositions and especially the fatty acid composition thereof can be adapted with an aim to provide compositions which are healthier to human consumers.

For instance, in EP-1 106 077 a cow's milk composition is described having a total saturated fatty acid content of between 60.30 and 68.15 g/100 g total fatty acids, a total unsaturated fatty acid content between 32.85 and 39.70 g/100 g total fatty acids. The total content of trans fatty acids (Ci8:i trans 11 and Ci8:2 trans 9,12) is from 3 to 7 g/100 g total fatty acids. This milk is obtained from cows fed with a feed supplement comprising 50-99% linseed and having a total fatty matter content between 23 and 30%.

In WO 2010/015708 cow's milk is described having 37.8-41.4 g total unsaturated fatty acids and the trans fatty acids content (C18:1 trans 10 and C18:1 trans 11) between 3.12 and 3.55 g/100 g total fat. This milk is obtained from cows fed with a feed supplement based on linseed and reducing disaccharides.

It has been observed that the unsaturated fatty acids in cow's milk mainly originate from the cow's feed. However, feeding a cow with a

supplement rich in unsaturated fatty acids such as linseed generally results in the increase of the trans fatty acids content in the milk, formed in the rumen of a cow as a result of biohydrogenation. Trans fatty acids are considered to be detrimental to human health and are associated with an increased risk of coronary heart disease. Therefore, their amount in milk should be limited.

An aim of the present invention is to provide a milk composition which is believed to be healthier for a large group of consumers. "Healthier" means having one or more of the following advantages: a higher content of unsaturated fatty acids and in particular, of ω-3 fatty acids, a lower trans fatty acids content, a lower saturated fatty acids content, a higher content of conjugated linoleic acid (CLA). In particular, an aim of the present invention is to provide a method to produce a milk composition having at the same time a relatively high amount of unsaturated fatty acids and a limited amount of trans fatty acids.

The present invention is based on the finding that the composition of cow's milk can be altered by the selective feeding based on the genotype of a cow. More in detail, the concerned gene is the bovine diacylglycerol-o- acyltransferase 1 gene, or DGAT1, which is associated with the fatty acid composition of milk. Because of polymorphism (K232A), it has two genetic variants K and A, which result in an amino acid substitution in the DGAT1 protein (lysine -alanine). Two types of alleles K and A result in three genotypes KK, AA and AK.

The inventors have surprisingly found that the cows with the AA genotype fed with a fat-rich feed produce milk having a fatty acid composition different from that of the milk obtained by genetic selection or by fat-rich diet only. In addition, the milk according to the present invention is healthier than the milk of cows having the AK or KK genotypes or the milk of cows receiving no feed supplement since it contains more unsaturated fatty acids and less saturated fatty acids than the milk obtained from AK and KK cows.

Accordingly, the present invention relates to a method to obtain milk comprising the steps of:

i) feeding of a cow having the AA genotype of the DGAT1 gene, with a feed rich in fats, and

ii) milking of the cow.

Preferably, the cows belong to the breed of Holstein or Jersey. More preferably, the cows are Holstein Friesian. In the Netherlands, the frequency of the A allele in Holstein Friesian population is 0.6 and of the K allele 0.4 which makes the frequency of genotypes AA, AK and KK to be 0.36, 0.48 and 0.16, respectively. The genotype of a cow can be determined by methods known to a skilled person, such as Polymerase Chain Reaction (PCR) using two primers and two probes per allele to be detected (so-called Taqman assay). For that, cow's blood is normally used.

Cows with the AA genotype are known to produce more milk than other cows. However, this milk has a lower content of fat and proteins than the milk of AK and KK cows. This makes the milk of AA cows less valuable for farmers to sell and also relatively expensive in transport due to a larger volume. The milk obtained from AK and KK cows, in contrast, contains relatively much fat and proteins and, thus, has historically a higher value. Therefore, the genotypes with the K allele are frequently chosen for milk production. According to the present invention, however, the milk of AA cows can offer considerable health advantages for human consumers compared to the milk of other cows.

In accordance with the present invention, the AA cows are fed with a diet rich in fats. The feed generally comprises a base roughage and/or base concentrate, and a feed supplement rich in fat. The base roughage can, for example, consist of maize silage or grass silage, yet other suitable base compositions can equally be used. Grass and maize silage are typical components of a winter diet for Dutch cows. The base concentrate can be of any type and is a conventional feedstuff typically having a dry matter content higher than 80 wt.% and a crude fat content lower than 10%.

The feed is preferably rich in fat, which means that the feed comprises more than 4 wt.% of fat, typically between 4 and 6 wt.% of fat.

Higher amounts of fat in the feed especially of unprotected oils, are less preferable as they can lead to gastric disturbances of a cow.

The feed normally comprises from 8 to 14, preferably from 10 to 12 wt.% of a feed supplement. The feed supplement suitable for use in the present invention typically comprises from 10 to 40 wt.% of fat, preferably, from 20 to 30 wt.% total dry matter. The supplement preferably contains unsaturated fatty acids in an amount of 5 to 90 wt.%, more preferably, 10 to 80 wt.% total fatty acids in the supplement. Preferably, at least 20 wt.%, more preferably, at least 30 wt.% of the unsaturated fatty acids present in the feed supplement is Ci8:3 cis 9,12,15 fatty acid.

Unsaturated fatty acids are preferably supplied in the form of triglycerides, for example, as fat or oil. Suitable sources of unsaturated fatty acids are oils containing at least 2 wt.% of unsaturated fatty acids, preferably at least 5 wt.%, for example seed oils, such as those of linseed, rapeseed, soybeans, walnuts, sunflower, cotton seed, safflower, clary sage seed, perilla, chia, and chemp. Also suitable are fish oil, algae oil, corn oil, and peanut oil. Preferably, linseed oil is used. A suitable feed supplement is, for example, Nutex® described in EP 1 106 077. In general, the feed supplement is fed to cows in an amount of 1.6-2 kg dry matter per cow per day, typically about 1.8 kg. Although the amount of the supplement given is dependent on a particular cow, the base roughage and/or concentrate used, etc., a skilled man is able to determine a suitable amount of the feed supplement in order to practise the present invention.

The unsaturated fatty acids in the feed supplement can also be protected against rumen biohydrogenation so these do not convert into trans fatty acids. Such protection is usually achieved by an additional chemical treatment and is for this reason less desired. However, an advantage of the present invention is that the chemical protection of unsaturated fats in the supplement is not needed since the low trans fatty acids content can also be achieved according to the present invention.

In another aspect, the present invention relates to a milk composition comprising fatty acids, said composition comprising per 100 g total fatty acids between 35 and 42 g unsaturated fatty acids, between 0.7 and 8.0 g Ci8:3 cis 9, 12, 15 fatty acid and less than 3.5 g trans fatty acids. Preferably, the milk of the invention originates from lactating animals, and especially of dairy cattle such as dairy cows.

Compared to regular milk, the milk composition of the invention obtained from AA cows is characterized by a high content of unsaturated fatty acids. While the content of trans fatty acids is also increased, in proportion to the unsaturated fatty acids it is less increased than in the milk of AK and KK cows. This is demonstrated by the unsat/trans ratio, which is the ratio of the total unsaturated fatty acids to the total trans fatty acids. The unsat/trans ratio is preferably higher than 12.2, more preferably higher than 13.0 and even more preferably higher than 13.5, such as for example higher than 13.7. Milk having a high unsat/trans ratio is beneficial for human health because it is associated with a lower trans fatty acids intake for the same level unsaturated fats. Accordingly, when a recommended daily amount of unsaturated fats, and in particular of Ci8:3, is taken by a consumer, the intake of trans fatty acids is much smaller in case of the milk of AA cows than of other cows.

As said above, in regular cow's milk more than 70 wt.% of the fatty acids are saturated, and hence less than 30 wt.% of the fatty acids are unsaturated. The milk composition of the present invention has a considerably lower amount of saturated fatty acids, which has a good effect on the quality of the blood lipids and a corresponding positive effect against cholesterol related diseases and other cardiovascular diseases. The saturated fatty acids in milk are here the sum of C4:o + Cs:o + Ce-.o + C _7: o + Cs:o + C&o + Cio:o + Cn _:0 + Ci2:o + Cl3:0 + Cl4:0 + Cl4:0 ISO + Cl5:0 + Cl5:0 ISO + Cl5:0 ante ISO + Cl6:0 + Cl6:0 ISO + Cl _7: 0 + Cl ₇ :0 ISO + Cl ₇ :0 ante ISO + Cl8:0 + C20:0.

In a preferred embodiment, the total amount of saturated fatty acids is between 55.0 and 64.0 g, preferably, between 56.0 and 62.0 g, more preferably between 57.0 and 60.0 g per 100 grams total fatty acids. The content of Ci2:o fatty acid is between 1.8 and 3.4 g, preferably between 2.0 and 3.0 g, more preferably between 2.4 and 2.8 g/100 g total fatty acids. The content of Ci4:o fatty acid is between 7.0 and 11.0 g, preferably between 8.0 and 9.5 g/100 g total fatty acids. The content of Ci6:o fatty acid is between 18 and 28 g, more preferably between 19 and 25 g and most preferably between 21 and 24 g/100 g total fatty acids.

C4:o (Butyrate), a fatty acid associated with anti carcinogenic effects is preferably present in an amount between 3.0 and 4.3 g/100 g total fatty acids.

A number of fatty acids may be present in the milk composition of the present invention without having a particular beneficial effect nor having any adverse effect. To this group of fatty acids, the following fatty acids belong:

C6:0; C8:0; Cl0:0; and Cl8:0.

Unsaturated fatty acids are those having at least one unsaturated bond within the fatty acid chain. The fatty acids having one unsaturated bond are called mono-unsaturated fatty acids (MUFA), those having two and more unsaturated bonds within the chain are called polyunsaturated fatty acids (PUFA). In a preferred embodiment, the milk of the invention is characterized by a total amount of unsaturated fatty acids higher than 33 g, preferably between 35 and 42 g, more preferably between 37 and 41 g/100 g total fatty acids.

The milk composition of the present invention is further characterized by a high amount of ω-3 fatty acids. The ω-3 fatty acids are in particular Ci8:3 cis 9,12,15 (ALA), C ₂ o: ₅ cis 5,8,11,14,17 (EPA), C ₂₂ :5 cis 7,10,13,16,19 (DPA) and C ₂ 2:6 cis 4,7,10,13,16,19 (DHA). Preferably, the milk composition of the invention contains Ci8:3 cis 9,12,15 (ALA) in an amount higher than 0.6 g, preferably between 0.7 g and 8.0 g, more preferably between 0.78 g and 4.0 g/100 g total fatty acids.

The term "ω-6 fatty acids" describes here Ci8:2 cis 9,12 (linoleic acid) and C22:2 cis 13,16. The content of Ci8:2 cis 9,12 is preferably between 1.0 g and 4.0 g, more preferably, between 1.5 and 2.5 g/100 g total fatty acids.

The ratio of ω-6/ ω-3 fatty acids in milk should not be too high as it may promote the pathogenesis of many diseases including cardiovascular diseases, cancer, inflammatory diseases and autoimmune diseases. In a preferred embodiment, the milk composition according to the invention has a weight ratio ω-6/ω-3 fatty acids lower than 3.0, preferably lower than 2.85, more preferably lower than 2.7.

In a further preferred embodiment, the milk composition according to the invention contains conjugated linoleic acid (CLA; Ci8:2 trans 11 cis 9) preferentially in an amount between 0.45 g and 8.0 g, preferably between 0.6 g and 4.0 g, more preferably between 0.7 g and 3.0 g/100 g total fatty acids. Although CLA contains one trans bond, in the present application it is not comprised in the trans fatty acids amount. CLA is known to have an antiinflammatory and an anti- carcinogenic effect.

Trans fatty acids are not considered particularly healthy and the content of these fatty acid should be kept limited. The term "total trans fatty acids" in the present description and the claims means the sum of Ci6:i trans 9 + Ci8:i trans (4+5+6+7+8) + Ciai trans 9 + Ciai trans 10 + Ciai trans. Total trans fatty acids is preferably low, that is, less than 4.0 g/100 g total fatty acids, preferably less than 3.5 g and more preferably less than 3 g and yet most preferably less than 2.9 g/100 g total fatty acids.

The identity and the amount of various fatty acids in milk can easily be determined using conventional gas chromatography techniques.

Particularly, the following method is used by the present inventors. Milk fat is extracted from milk samples. Fatty acid methyl esters are prepared from fat fractions as described in ISO Standard 15884 (ISO-IDF 2002b). The methyl esters are analyzed according to ISO Standard 15885 (ISO-IDF 2002a) on a

Trace GC Ultra chromatograph (Thermo Electron Corporation), using a Varian Fame Select column (100 m x 0.25 mm ID; Varian Inc.). The initial

temperature is held at 70°C for 1 minute and raised to 225°C at 3°C/min and held at 225°C for 5 minutes. A volume of Ιμΐ is injected. Each peak is identified and quantified using pure methyl ester samples (Sigma-Aldrich; Larodan). The fatty acids content is expressed as a portion of total fat weight.

In addition to an improved fatty acid composition, the milk of AA cows is also characterised by a decreased content of urea. As can be seen from Table 2, the milk of AA cows can contain 27.4 mg urea /100 g milk, while the milk of AK cows and KK cows can contain 29.1 and 31.9 mg urea /100 g milk, respectively. The milk composition of the invention preferably contains between 16.0 and 28.5 mg, more preferably between 18.0 and 28.0 urea /100 g milk.

The milk composition obtained as a result of feeding of a cow with a specific supplement, for example, such as demonstrated in the Examples, differs for individual cows and changes during the season. The fatty acid composition is also determined by the health status, lactation stage, parity and genetic background of the cow. Furthermore, the composition and quantity of the fatty acids supplied by the diet in addition to the supplements also depends on the fatty acid composition and fat percentage of the other components in the diet of the cow {e.g. the roughage or the concentrate).

Especially the composition of the roughage varies greatly during the season. With the information provided the skilled person will however be able to prepare the milk composition according to the invention.

In another aspect, the present invention relates to a method to select a cow to be fed with a fat-rich feed by choosing a cow with the AA genotype of the gene DGAT1.

In one embodiment of this method one can test cows on beforehand to determine the genotype of a cow in case the genotype is not known and further choose cows having the AA genotype to be fed with a fat-rich supplement. Such selection can easily be implemented, for example, in an automated feeding system or in a milk robot system, where cows are identified and recognized by an electronic chip. Giving the feed supplement only to the cows having the AA genotype instead of all cows also saves costs since feed supplements are generally more expensive than regular feed.

The present invention can also be practiced by feeding all the cows with a fat-rich feed, followed by milking, whereby the milk of AA cows is collected and kept separately.

In a further aspect, the present invention relates to a method to test a feed supplement for cows, comprising the steps of:

i) selecting at least one cow having the AA genotype of the gene

DGAT1,

ii) feeding the cow or cows selected in step i) with a feed supplement to be tested,

iii) milking of the cow or cows, and

iv) analysing the composition of the milk.

According to the invention, cows with the AA genotype represent the best selection for testing various feed supplements, especially those rich in fats. First, these cows produce milk with a higher content of unsaturated fatty acids and are therefore more susceptible to the fatty acids present in a feed supplement. Second, the results of the tests carried out only on AA cows are more statistically homogeneous than of the tests wherein cows with any genotype are used. Therefore, a smaller population is needed to obtain statistically reliable results.

Accordingly, the invention is further related to a herd of cows, wherein at least 60%, preferably at least 80% and more preferably at least 90% of the cows has the AA genotype of the gen DGAT1. More preferably, the herd consists of AA cows only. A herd is defined as a group of cattle or other domestic animals of a single kind kept together for a specific purpose. For the purposes of the present invention, the herd comprises at least 10 cows which are kept for the milk they give. As said above, a typical herd of Holstein Friesian cows in the Netherlands has a frequency of the AA genotype about 36%. In accordance with the present invention, the frequency of the AA cows in a herd can be increased by selection, in order to obtain a herd with mainly AA cows. Preferably, the cows in the herd belong to the breed of Holstein or Jersey. More preferably, the cows are Holstein Friesian.

Producing milk according to the invention has a further advantage of reducing the methane emission caused by cows as AA cows emit less methane than the cows of other genotypes. Therefore, the invention relates in a further aspect to a method to reduce methane emission, comprising selecting cows having the AA genotype of the gen DGAT1, and feeding those cows. The reduced methane emission is especially significant when the cows are fed with a fat-rich feed.

Methane emission is calculated per volume milk produced. It is believed that the decrease of the methane emission of AA cows is partially assigned to a relatively larger volume of milk produced and partially to the improved fatty acid composition of the milk. Estimated methane emission according to the method described in WO 2009/156453 is 9.6 g for the AA, 10.3 g for the AK and 11.6 g methane per 1 1 milk for the KK genotype. In addition to methane emission, much attention has been drawn to the ammonia emission caused by cows. One of the indicators of the ammonia emission is the urea content in cows' milk. As can be seen from Table 2, the AA cows produce milk with the lowest level of urea compared to the cows with other genotypes and hence emit less ammonia. Therefore, the AA cows are also preferable over the other genotypes because of a lower ammonia emission. Accordingly, the present invention presents a method to reduce ammonia emission caused by cows, comprising selecting cows having the AA genotype of the gene DGAT1, and feeding those cows. The reduced ammonia emission is especially significant when the cows are fed with a fat-rich feed.

In a further aspect, the present invention relates to the use of the milk composition of the invention for the preparation of a milk based composition, such as a cheese, a yogurt, a cream composition, a dessert product, also including compositions prepared from milk components, such as milk powder, whey proteins, milk fat and so on. Furthermore, the invention relates to the products produced using the milk composition, e.g. based on or comprising the milk composition of the invention. Especially, it relates to products wherein the fatty acid composition of the milk composition is essentially maintained in the final product. These products are for instance suitable for food and feed applications or for food and feed supplements.

The invention will now be illustrated by the following, non-limiting examples.

Example 1 (Comparative)

1762 cows of which 644 had the AA-genotype, 829 the AK-genotype and 289 cows had the KK-genotype of the DGAT1 gene were fed with a standard ration having a composition according to Table 1. The amounts in Table 1 are given per cow per day. The fat percentage of the feed is

approximately 3 wt.%. Table 1

¹ concentrate from linseed

2 concentrate with 960 VEM, 110 DVE, 14 RE

³ commercial product Rindavit 71 ASS-CO, containing (wt.%): 39.6% calcium carbonate, 23.6% sodium chloride, 16.5% calcium sodium phosphate, 13.7% magnesium oxide, 3% molasses, 0.55% copper sulphate, 1000000 IU vitamin A, 80000 IU vitamin D, 1350 mg copper, 100000 μg biotin, 3000 mg vitamin E

Fat percentage of regular feed as described in table 1 is about 3%.

Table 2 shows the composition of the milk obtained from the cows of different genotypes fed with the standard feed as described above.

Table 2

DGAT genotype

Component

AA AK KK

Total fat, wt.% 3.95 4.47 4.91

C4:0 g/100 g total fat 3.5 3.5 3.5

C6:0 g/100 g total fat 2.2 2.2 2.3

C8:0 g/100 g total fat 1.3 1.4 1.4 C10:0 g/100 g total fat 3.0 3.1 3.0

C12:0 g/100 g total fat 4.1 4.1 4.0

C14:0 g/100 g total fat 11.9 11.6 11.2

C16:0, g/100 g total fat 31.42 33.04 33.90

C18:0 g/100 g total fat 8.73 8.66 8.88

Total unsaturated fatty

30.0 (3.0) 28.6 (2.4) 28.0 (2.6) acids, g/100 g total fat

C18:1 cis 9, g/100 g total

24.0 21.8 20.9

fat

C18:2 cis 8, trans 11

0.42 0.38 0.37

(CLA), g/100 g total fat

C18:2 cis 9,12 (ω-6),

1.26 1.18 1.12

g/100 g total fat

C18:3 cis 9,12,15 (ω-3)

0.44 0.40 0.40

(ALA), g/100 g total fat

Total trans fatty acids,

1.48(0.46) 1.38(0.31) 1.38(0.37) g/100 g total fat

Unsat:trans 20.27 20.72 20.29

Urea, mg/100 g milk 29.1 29.3 29.8

Methane emission, g

10.7 11.3 12.5

CH ₄ /L milk

For the total unsaturated fatty acids content, the standard deviation is given in Table 2 in parentheses.

Example 2

73 cows, of which 34 of the AA genotype, 34 of the AK genotype and 5 cows of the KK genotype of the gene DGATl, were fed with a ration rich in fats having a composition according to Table 3. The amounts in Table 3 are given per cow per day. The fat percentage of the feed is 5.1 wt.%.

Table 3

Ration

Grass silage, kg dry matter 4.5

Maize silage, kg dry matter 9.34

Wheat (83% dry matter), kg product 1.5 Brewers grain (25% dry matter), kg dry matter 5

Beet pulp (27% dry matter), kg product 7

Soy (48) (87% dry matter), kg product 2.62

Rapeseed (33) (88% dry matter), kg product 0.88

Nutex® ¹ (91% dry matter), kg product 2.00

Minerals ² (88% dry matter), kg product 0.27

Total, kg dry matter 23.28

¹ Nutex® - a commercially available feed supplement based on linseed, described in EP 1 106 077.

² commercial product Rindavit 71 ASS-CO, containing (wt.%): 39.6% calcium carbonate, 23.6% sodium chloride, 16.5% calcium sodium phosphate, 13.7% magnesium oxide, 3% molasses, 0.55% copper sulphate, 1000000 IU vitamin A, 80000 IU vitamin D, 1350 mg copper, 100000 μg biotin, 3000 mg vitamin E.

Table 4 shows the composition of the milk obtained from the cows of different genotypes fed with the described supplemented feed.

Table 4

DGAT genotype

Component

AA AK KK

Total fat, wt.% 3.70 4.61 4.71

C4:0 g/100 g total fat 3.4 3.4 3.4

C6:0 g/100 g total fat 2.0 2.1 2.2

C8:0 g/100 g total fat 1.0 1.2 1.2

C10:0 g/100 g total fat 2.4 2.7 2.8

C12:0 g/100 g total fat 2.6 3.0 3.1

C14:0 g/100 g total fat 9.0 9.7 9.8

C16:0, g/100 g total fat 23.87 25.70 26.15

C18:0 g/100 g total fat 12.9 13.1 13.1

Total unsaturated

fatty acids, g/100 g 39.2 (3.5) 34.5 (2.5) 33.5 (3.2) total fat C18:1 cis 9, g/100 g

24.0 21.8 20.9 total fat

C18:2 cis 8, trans 11

0.75 0.60 0.64

(CLA), g/100 g total fat

C18:2 cis 9,12 (ω-6),

2.12 1.98 1.81 g/100 g total fat

C18:3 cis 9,12,15 (ω-3)

0.80 0.71 0.69

(ALA), g/100 g total fat

Total trans fatty acids,

2.86 (0.15) 2.54 (0.16) 2.77 (0.2) g/100 g total fat

Unsat:trans 13.71 13.58 12.09

Urea, mg/100 g milk 27.4 29.1 31.9

Methane emission, g

9.6 10.3 11.6

CH ₄ /L milk

For the total unsaturated fatty acids content, the standard deviation is given in Table 4 in parentheses. For the AA cows, it is significantly different (p<0.05) from the AK and KK groups, both during the experiment with the fat- rich supplement as during feeding with a regular ration. Also, the cows with the AA genotype fed with the fat supplement shows significantly different results (p<0.05) when compared to the results on feeding with a regular ration.

As can be seen from Table 4, the milk composition of the cows with the AA genotype has a decreased amount of fat compared to the milk of cows with other genotypes. The milk of AA cows has also a lower content of saturated acids compared to other genotypes. It has further a significantly increased amount of unsaturated fatty acids, especially of the ω-3 fatty acid Ci8:3 cis 9,12,15. The content of trans fatty acids is limited to less than 3 g/100 g total fatty acids. Further, the AA milk contains less urea relative to the other cows' milk. In addition, it can be estimated that the methane emission of AA cows is lower than of the other cows.