EP2121976A2 - Method for selection of non-human mammal producing milk with improved fatty acid composition - Google Patents
Method for selection of non-human mammal producing milk with improved fatty acid compositionInfo
- Publication number
- EP2121976A2 EP2121976A2 EP08712611A EP08712611A EP2121976A2 EP 2121976 A2 EP2121976 A2 EP 2121976A2 EP 08712611 A EP08712611 A EP 08712611A EP 08712611 A EP08712611 A EP 08712611A EP 2121976 A2 EP2121976 A2 EP 2121976A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- milk
- fatty acids
- dgatl
- allele
- cis
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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Classifications
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- A—HUMAN NECESSITIES
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- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/20—Dietetic milk products not covered by groups A23C9/12 - A23C9/18
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/115—Fatty acids or derivatives thereof; Fats or oils
- A23L33/12—Fatty acids or derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/101—Bovine
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C2230/00—Aspects relating to animal feed or genotype
- A23C2230/10—Animal milk with modified composition due to a specific feed
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
Definitions
- the invention relates to the use of means that detect the presence of a SCD 878V and/or of additional means that detect the presence of a DGATl 232A allele in a method for identifying a non-human mammal which produces milk with an improved fatty acid composition.
- milk and milk-derived foods including cheese and butter
- milk and milk-derived foods were the main source of dietary saturated fatty acids across Europe, ranging between 27% and 58%.
- the contribution from milk and milk-derived foods to dietary trans fatty acids was between 17% and 72%.
- Milk- fat is relatively high in saturated fatty acids, especially C14:0 and C16:0, and low in polyunsaturated fatty acids.
- Milk-fat composition can be altered through the nutrition of dairy cows (12), and possibly by selective breeding, although prospects for the latter have not been studied extensively.
- the major prerequisite for selective breeding is existence of genetic variation in milk-fat composition among cows.
- For milk-fat percentage around half the observed variation is estimated to be due to genetic variation (13-16). Phenotypic variation in milk-fat composition has been reported as well, both between and within breeds, although the number of reports on genetic variation is limited and recent publications are lacking (17-21).
- DGATl acyl CoA:diacylglycerol acyltransferase 1
- WO02/36824 discloses that bovine carrying the DGATl 232A allele produce more milk, said milk containing more milk-protein and less milk-fat than milk from cows that do not carry the DGATl 232A allele. The effect of the DGATl 232A allele on milk fatty acid composition is not known.
- the present invention establishes that the DGATl 232 A allele is further associated with at least one of: a reduced content of saturated C 16 fatty acids, and an increased content of unsaturated Cl 8 fatty acids in the milk- fat composition.
- SNP single nucleotide polymorphism
- This gene codes for the Stearoyl-Coenzyme A desaturase (SCD).
- SCD Stearoyl-Coenzyme A desaturase
- the mutation A878V in the SCD enzyme was found particularly attractive for conferring healthier fatty acid composition to the milk.
- the inventors established that the presence of both a SCD 878 V and a DGATl 232 A allele is associated with a milk having an even more healthy fatty acid composition.
- a SCD 878 V allele detects the presence of a SCD 878 V allele and/or of additional means that detect the presence of a DGATl 232A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a different milk-fat composition than the one from a milk produced by a non-human mammal not having a SCD 878 V and/or DGATl 232 A allele.
- milk or milk- fat could be replaced by milk or milk- fat composition.
- a milk or milk-fat composition is preferably defined by a fatty acid composition.
- "different” preferably means that using any of the means as described herein the milk-fat composition of a milk produced by a non-human mammal having a SCD 878V and/or a DGATl 232 A allele is analyzed as being different (i.e. distinct) from the milk-fat composition of a milk produced by a non- human mammal not having a SCD 878 V and/or DGATl 232 A allele.
- a "different" milk-fat composition more preferably means a healthier milk-fat composition.
- a "healthy" milk-fat composition is further herein defined by reference to its fatty acid composition.
- a SCD 878 V allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a different milk-fat composition than the one from a milk produced by a non- human mammal not having a SCD 575FaIIeIe. More preferably, in this embodiment, the milk has a milk-fat with an increased desaturation index for at least one of C16, C17,C18 and CLA fatty acids.
- unsaturated Cl 8 fatty acids are preferably selected from the following group: C18:l cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid), CLA (conjugated linoleic acid) and C18:3 cis 9,12,15 (linolenic acid). All these unsaturated C18 cis-isomers C18: 1 cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid), CLA and C18:3 cis 9,12,15 (linolenic acid) are known to have a positive effect on human health.
- the CLA is selected from the following isomers consisting of: C 18:2 cis 9, trans 11 and C18:2 translO, cis 12. Even more preferably, the CLA is the C18:2 cis9, transl l isomer.
- a desaturation index is defined as being the ratio between the product and the substrate plus the product, said ratio being expressed as a percentage.
- Substrate and product mean substrate and product for a SCD enzyme.
- a SCD enzyme specifically introduces a cis double bond at the 9 position of a fatty acid.
- the substrate and product are expressed as content of substrate and product as later defined herein.
- the C16 index is: (C16:lcis9 / (C16:0 + C16:lcis9))xl00.
- the C17 index is: (C17:lcis9 / (C17:0 + C17:lcis9))xl00.
- the C18 index is: ((C18:lcis9 + C18:ltransl2) / (C18:0 + C18:lcis9 + C18:ltransl2))xl00.
- the Cl 8 index is the index of C18: lcis9.
- the index of C18:lcis9 is as follows: (C18:lcis9 / (C18:0 + C18:lcis9))xl00.
- the index of CLA is as follows: (CLA/ (C 18: 1 transl l + CLA))xlOO, which is (C 18:2cis9transl 1/(C 18:1 transl l + C18:2cis9transl l))xl00. Therefore, the C18 index does not include the CLA index.
- the CLA index is calculated as an index which is separate from the Cl 8 index.
- the invention relates to a use of means, that detect the presence of a SCD 878V allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk-fat composition with at least one of an increased content of C16:lcis9, C17:lcis9 and CLA and a decreased content of C17:0 and C18:0 fatty acids.
- This sentence is equivalent with an increased content of at least one of C16:lcis9, C17:lcis9 and CLA and/or a decreased content of at least one of C17:0 and C18:0 fatty acids.
- the same meaning is given to the use of the expression "at least one" in combination with a group of features.
- a SCD 878V allele is associated with milk having a healthy milk- fat composition: a healthy fatty acid composition wherein a decreased content of saturated C17 and C18 fatty acids and an increased content of unsaturated C 16, C 17, and Cl 8 fatty acids is seen.
- An unsaturated Cl 8 fatty acid is preferably CLA.
- an increased desaturation index for C 16, C 17, Cl 8 or CLA means a desaturation index which is higher than the corresponding desaturation index for C 16, C 17, C18 or CLA in the milk-fat of the milk of a non- human mammal, preferably a bovine not having a SCD 878V allele.
- the increase is approximately of 2% or more. More preferably, the increase is approximately of 5% or more, or approximately of 7% or more, or approximately of 10% or more, or approximately of 15 % or more, or approximately of 20% or more, or approximately of 25% or more.
- a decreased content of C17:0 or C18:0 fatty acids means a content of C17:0 or C18:0 fatty acids which is lower than the content of C 17:0 or C 18:0 fatty acids present in the milk-fat of the milk of a non-human mammal, preferably a bovine not having a SCD 878V allele.
- the decrease is approximately of 4% or more, or approximately of 5% or more. More preferably, the decrease is approximately of 7% or more, or approximately of 10% or even more.
- an increased content of C16:lcis9, C17:lcis9 or CLA fatty acids means a content of C16:lcis9, C17:lcis9 or CLA fatty acids which is higher than the content of C16:lcis9, C17:lcis9 or CLA fatty acids present in the milk- fat of the milk of a non- human mammal, preferably a bovine not having a SCD 878V allele.
- the increase is approximately of 5% or more. More preferably, the increase is approximately of 7% or more, or approximately of 10% or more, or approximately of 15% or more, or approximately of 20% or more, or approximately of 25% or more.
- a DGATl 232 A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a different milk-fat composition than the one from a milk produced by a non- human mammal not having a DGATl 232 A allele.
- the presence of a DGATl 232 A allele is associated with a milk having at least one of a reduced content of saturated C16 fatty acids and an increased content of unsaturated Cl 8 fatty acids.
- the invention relates to the use of means that detect the presence of the DGATl 232A allele in a method for identifying a non- human mammal, preferably a bovine which produces milk having a milk-fat composition with at least one of a reduced content of saturated C 16 fatty acids and an increased content of at least one of C18 cis-isomers such as: C18:l cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid), conjugated linoleic acid (CLA) and C18:3 cis 9,12,15 (linolenic acid).
- the Cl 8 cis-isomers are selected from the group consisting of: C18:l cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid), CLA and C18:3 cis 9,12,15 (linolenic acid).
- the C18 cis-isomers are selected from the group consisting of oleic acid, linoleic acid, CLA and linolenic acid.
- the CLA is preferably selected from the following isomers consisting of: C18:2 cis 9, trans 11 and C18:2 translO, cis ⁇ 2.
- the invention relates to the use of means that detect the presence of the DGATl 232A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk-fat composition with at least one of a reduced content of saturated C16 fatty acids and an increased content of at least one of Cl 8 cis-isomers such as: C 18:1 cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid), and C18:3 cis 9,12,15 (linolenic acid).
- the C18 cis-isomers are selected from the group consisting of: C18:l cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid) and C18:3 cis 9,12,15 (linolenic acid).
- the Cl 8 cis-isomers are selected from the group consisting of oleic acid, linoleic acid, and linolenic acid.
- the DGATl 232A allele is associated with milk having a healthy milk- fat composition: a healthy fatty acid composition wherein a reduced content of saturated C16 fatty acids and an increased content of unsaturated Cl 8 fatty acids is seen. Furthermore, the DGATl 232A allele has unexpectedly been found to be associated with a relative small increase in saturated C 14 fatty acid. The total effect on fatty acid composition is positive for the human health because the effect of the DGATl 232A allele is much more pronounced on saturated C16 fatty acids, unsaturated C18 fatty acids than on saturated C14 fatty acids (see table 5).
- the invention relates to the use of means that detect the presence of the DGATl 232A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk- fat composition with at least one of a reduced content of saturated C16 fatty acids, an increased content of unsaturated C 18 fatty acids, and an increased content in saturated C 14 fatty acids. Furthermore, if one looks at the overall effect on fatty acid composition, it has been observed (table 6) that the DGATl K232A mutation has a statistically significant effect (reduction) on C5, C6, C7, C8, C9, CI l, C13, C15, C16 and C17 saturated fatty acids.
- the DGATl K232A mutation also has a statistically significant effect (increase) on C18 (C 18:1 cis9 (oleic acid), C 18:1 cisl l, C 18:2 cis 9,12 (linoleic acid), CLA, and C18:3 cis 9,12,15 (linolenic acid)) unsaturated fatty acids (table 7).
- the invention relates to the use of means that detect the presence of the DGATl 232A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk- fat composition with at least one of a reduced content of saturated C5, C6, C7, C8, C9, CI l, C13, C15, C16 and C17 fatty acids and an increased content of unsaturated on Cl 8 (Cl 8:1 cis9 (oleic acid), C 18:1 cisl l, C 18:2 cis 9,12 (linoleic acid), CLA, and C18:3 cis 9,12,15 (linoleic acid)) fatty acids.
- a reduced content of saturated C16 fatty acids means a content of saturated C16 fatty acids which is lower than the content of saturated C 16 fatty acids present in the milk-fat composition of the milk of a non-human mammal, preferably a bovine not having a DGATl 232 A allele.
- the reduction is approximately of 5% or more. More preferably, the reduction is approximately of 7% or more or approximately of 10% or even more.
- an increased content of unsaturated C 18 fatty acids means a content of unsaturated Cl 8 fatty acids which is higher than the content of unsaturated C18 fatty acids present in the milk-fat composition of the milk of a non- human mammal, preferably a bovine not having a DGATl 232 A allele.
- the increase is approximately of 5% or more. More preferably, the increase is approximately of 7% or more, or approximately of 10% or more.
- an increased content of saturated C 14 fatty acid means a content of saturated C 14 fatty acid which is higher than the content of saturated C 14 fatty acids present in the milk- fat composition of the milk of a non-human mammal, preferably a bovine not having a DGATl 232 A allele.
- the increase is approximately of 2% or more. More preferably, the increase is approximately of 5% or more or approximately of 7% or even more.
- a SCD 878V allele detects the presence of a SCD 878V allele and of additional means that detect the presence of a DGATl 232 A allele in a method for identifying a non- human mammal, preferably a bovine which produces milk having a different (more preferably healthier) milk-fat composition than the one from a milk produced by a non-human mammal not having a SCD 878V and/or DGATl 232 A allele.
- the invention relates to a use of means that detect the presence of a SCD 878V allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk-fat with an increased desaturation index for at least one of C 16, C 17, C18 and CLA fatty acids, wherein additional means are used to detect the presence of a DGATl 232 A allele.
- a SCD 575F aIIeIe and a DGATl 232 A allele explain a distinct part of the variation seen for the desaturation index of at least one of C 16, C18 and CLA fatty acids.
- the effect of the SCD A878V polymorphism and of the DGATl K232A polymorphism are mostly additive and in the same direction. Consequently, the combined homozygous genotypes have progressively decreasing effects on the ClO, C12 and C14 indexes, and progressively increasing effects on the Cl 8 and CLA indexes.
- the effect of the SCD A878V polymorphism and of the DGATl K232A polymorphism are mostly additive, but in opposite directions.
- the effect of the SCD A 878 V polymorphism is larger than the effect of the DGATl K232A polymorphism.
- the combination of homozygous SCD 878 A and homozygous DGATl 232 A has the most decreasing effect on the C16 index
- the combination of homozygous SCD 575 F and homozygous DGATl 232K has the most increasing effect on the C16 index.
- the combination of homozygous SCD 575 F and homozygous DGATl 232 A has an increasing effect on the C16 index also.
- the effect of the DGATl K232A polymorphism is not significant, therefore, does not add to the effect of the SCD A 878 V polymorphism.
- DGATl 232 A allele has unexpectedly been found to be associated with a relative small increase in saturated C 14 fatty acids.
- the total effect on fatty acid composition is positive for the human health because the effect of a DGATl 232 A allele is much more pronounced on saturated C16 fatty acids and unsaturated Cl 8 fatty acids than on saturated C 14 fatty acids.
- the invention relates to the use of means that detect the presence of a DGATl 232 A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk- fat composition with at least one of a reduced content of saturated C16 fatty acids, an increased content of unsaturated Cl 8 fatty acids, and an increased content in saturated C 14 fatty acids. Furthermore, if one looks at the overall effect on fatty acid composition, it has been observed that a DGATl K232A mutation has a statistically significant effect (decrease) on C5, C6, C7, C8, C9, CI l, C13, C15, C16 and C17 saturated fatty acids.
- DGATl K232A mutation also has a statistically significant effect (increase) on Cl 8 (C 18:1 cis9 (oleic acid), C 18:1 cisl l, C18:2 cis 9,12 (linoleic acid), CLA, and C18:3 cis 9,12,15 (linolenic acid)) unsaturated fatty acids.
- the invention relates to the use of means that detect the presence of a DGATl 232 A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk- fat composition with at least one of a reduced content of saturated C5, C6, C7, C8, C9, CI l, C13, C15, C16 and C17 fatty acids and an increased content of unsaturated C 18 (C 18 : 1 cis9 (oleic acid), C 18 : 1 cis 11 , C 18 :2 cis 9, 12 (linoleic acid), CLA, and C18:3 cis 9,12,15 (linoleic acid)) fatty acids.
- the milk has a milk-fat with an increased desaturation index for at least one of C 16, C 17, C18 and CLA fatty acids.
- the increased desaturation index is the CLA index.
- C 16, C 17, C18 or CLA means a desaturation index which is higher than the corresponding desaturation index for C 16, C 17, Cl 8 or CLA in the milk- fat of the milk of a non-human mammal, preferably a bovine not having a SCD 878V and a DGATl
- the increase is approximately of 5% or more. More preferably, the increase is approximately of 7% or more, or approximately of 10% or more, or approximately of 15% or more.
- the milk has a milk-fat composition with at least one of a decreased content of saturated C 16, C 17 and C 18 fatty acids and an increased content of unsaturated C 16, C17 and Cl 8 fatty acids.
- a decreased content of saturated C16, C17 or C18 fatty acids means a content of C16:0, C17:0 or C18:0 fatty acids which is lower than the content of C16:0, C17:0 or C18:0 fatty acids present in the milk-fat of the milk of a non- human mammal, preferably a bovine not having a SCD 878 V and a DGATl 232 A allele.
- the decrease is approximately of 4% or more, or approximately of 5% or more. More preferably, the decrease is approximately of 7% or more, or approximately of 10% or even more.
- an increased content of unsaturated C 16, C17 or C18 fatty acids means a content of C16:lcis9, C17:lcis9, C18:lcis9 or CLA fatty acids which is higher than the content of C16:lcis9, C17:lcis9, C18:lcis9 or CLA fatty acids present in the milk-fat of the milk of a non-human mammal, preferably a bovine not having a SCD 878V and a DGATl 232 A allele.
- the increase is approximately of 1% or more, or approximately of 5% or more. More preferably, the increase is approximately of 7% or more, or approximately of 10% or more, or approximately of 15% or more, or approximately of 20% or more.
- fatty acid content preferably means the amount of fatty acid in gram per 100 gram fatty acids in the milk-fat (w/w). This content is therefore preferably expressed by means of weight percentages.
- the milk-fat is preferably extracted from the milk as defined later herein.
- Fatty acid methyl esters are preferably prepared from fat fractions and analyzed as defined later herein.
- decrease and/or increase of fatty acid contents are preferably estimated by comparison with the corresponding control or average contents in the milk-fat of a control non-human mammal, preferably a bovine which is milked in the morning during winter (february-march) in its first lactation (between Day 63 and Day 282 in lactation).
- the bovine is preferably a Dutch Holstein Friesian cow that is milked twice a day, in which population the frequency of the SCD 878V allele is 0.27 and the frequency of the DGATl 232 A allele is 0.60.
- To have a representative average content at least 1000 cows are preferably used.
- the invention may be applied to any non-human mammal for obtaining milk having an improved milk-fat composition or an improved fatty acid composition.
- the non-human mammal is an ungulate and/or a ruminant.
- Preferred ungulates include a cow (or bovine), a horse, a sheep, a camel, a donkey, or a goat. More preferably, the non-human mammal is a bovine.
- Milk -fat is preferably extracted from the milk samples as follows. Milk -fat is extracted from the milk samples by adding 5 ml HCL (4M) to 100 ml of milk which is kept at 28°C and is shaken during 45 min. When butterfat is clearly visible, the water phase is poured off and the fat is rinsed with 200 ml cold water twice. Subsequently, the fat is melted at 70 0 C during 60 min, then transferred into a centrifuge tube and stored at -20 0 C until further processing. After defrosting, the tube is warmed to 40 0 C and centrifuged for 15 min at 1300g and 40 0 C.
- the fat phase is cleared at 40 0 C and used for the preparation of fatty acid methyl esters.
- Fatty acid methyl esters are prepared from fat fractions as described in ISO Standard 15884 (29). Fatty acid methyl esters are analyzed according to ISO Standard 15885 (3 0) on a Trace GC Ultra chromatograph (Thermo Electron Corporation, Waltham, MA, USA), using a Varian Fame Select column (100m x 0.25mm ID, Varian Inc. , Palo Alto, CA, USA). The initial temperature is held at 7O 0 C for 1 min, raised to 225 0 C at 3°C/min, and held at 225 0 C for 5 min. A volume of 1 ⁇ l is injected. Each peak is identified and quantified using pure methyl ester samples (Sigma-Aldrich, Zwijndrecht, the Netherlands; Larodan, Malmo, Sweden).
- a SCD 878V ox both a SCD 878V and a DGATl 232A allele are determined with respect to DNA, mRNA, and/or protein obtained from the non-human mammal by direct or indirect methods.
- the method used for detecting a SCD 878V or both a SCD 878V and a DGATl 232 A allele is not critical for the invention. Several methods were identified in WO 02/36824 using DNA, mRNA, and/or protein obtained from the bovine by direct or indirect methods.
- the presence of a SCD 878 V or both a SCD 878 V and a DGATl 232 A allele is directly assessed in the milk of the non-human mammal, preferably a bovine.
- somatic cells are first isolated from the milk. More preferably, in this preferred method, the Taqman assay or the SNaPshot assay is used as defined below.
- the type of means used in the method of the invention is also not critical for the invention as long as these means are able to identify a SCD 878V or both a SCD 878V and a DGATl 232 A allele.
- Preferred means include primers, nucleic acid probes or antibodies.
- a bovine coding nucleic acid sequence of the SCD 575FaIIeIe is given in SEQ ID NO:1.
- An amino acid sequence of the bovine SCD protein with the 878V allele encoded by SEQ ID NO:1 is SEQ ID NO:2.
- a bovine coding nucleic acid sequence of the SCD 878 A allele is given in SEQ ID NO:3.
- An amino acid sequence of the bovine SCD protein with the 878A allele encoded by SEQ ID NO:3 is SEQ ID NO:4.
- the nucleic acid coding sequence and amino acid sequence of the allele A are also available at GenBank data library under accession number AY241932.
- a bovine coding nucleic acid sequence of the DGATl 232 A allele is given in SEQ ID NO:5.
- An amino acid sequence encoded by SEQ ID NO:5 is SEQ ID NO:6.
- a bovine coding nucleic acid sequence of the DGATl 232K allele is given in SEQ ID NO:7.
- An amino acid sequence encoded by SEQ ID NO:7 is SEQ ID NO:8.
- PCR Polymerase Chain Reaction
- Taqman assay the SNaPshot single-base extension assay: PCR followed by allele-specific single-base extension as means, - detection of protein variants of SCD or DGATl using antibodies directed against these variants as means, hybridisation with probes that are complementary either for the SCD 878V allele or the DGATl 232 A allele (without amplification) as means and PCR with three primers as means, one specific for the locus of SCD or DGATl, one specific for the desired allele (SCD 878V or DGATl 232A) and one for the undesired allele (SCD 878A or DGATl 232K).
- the method used is the TaqMan assay: PCR using two primers and two probes as means.
- This preferred method is extensively presented below taken DGATl as example.
- This method includes a step in which ascertaining whether the K232A polymorphism is present in the sequence of DGATl DNA, includes amplifying the DNA in the presence of primers based on the nucleotide sequence of the DGATl gene and flanking sequence in the presence of allele-specific probes complementary to either the DGATl 232 A allele or the DGATl 232K allele.
- a primer of the present invention used in PCR for example, is a nucleic acid molecule sufficiently complementary to the sequence on which it is based and of sufficient length to selectively hybridise to the corresponding portion of a nucleic acid molecule intended to be amplified and to prime synthesis thereof under in vitro conditions commonly used in PCR.
- a probe of the present invention is a molecule, for example a nucleic acid molecule of sufficient length and sufficiently complementary to the nucleic acid molecule of interest, which selectively binds under high or low stringency conditions with the nucleic acid sequence of interest for detection thereof in the presence of nucleic acid molecules having differing sequences.
- Preferred primers for the detection of a DGATl 232A allele are the following: forward primer 5'- CGCTTGCTCGTAGCTTTGG -3' and reverse primer: 5'- CGCGGTAGGTCAGGTTGTC -3' (SEQ ID NO:9 and 10 respectively).
- Preferred probes for the detection of a DGATl allele 232A are the following: VIC MGB-probe: 5'- CGTTGGCC TTCTTAC -3' (detects K allele) and FAM MGB -probe: 5'-TTGGCCGCCTTAC-S' (detects A allele) (SEQ ID NO:11 and 12 respectively).
- FAM and VIC are fluoresent reporter dyes.
- the exication wavelength for FAM is 495 nm and the fluorescence has its emission peak at 520 nm
- the exication wavelength for VIC is 540 nm
- the fluorescence has its emmision peak at 555 nm.
- the use of fluorogenic probes in the 5 'nuclease assay combines PCR amplification and detection into a single step.
- an oligonucleotide probe is included in the PCR amplification reaction along with the forward and reverse primers. If the target sequence of the probe is amplified in the reaction, then the probe will hybridize to this target sequence during the annealing/extension step of PCR.
- the fluorogenic probe consists of an oligonucleotide labelled with both a fluorescent reporter dye and a quencher dye. In the intact probe, proximity of the quencher reduces the fluorescence signal observed from the reporter dye.
- Fluorogenic probes and the 5 'nuclease assay can be used for allelic discrimination.
- probes specific for each allele are included in the PCR assay.
- the probes can be distinguished because they are labelled with different fluorescent reporter dyes (e.g. FAM and VIC).
- FAM and VIC fluorescent reporter dyes
- a mismatch between probe and target greatly reduces the efficiency of probe hybridization and cleavage.
- substantial increase in FAM or VIC fluorescent signal indicates homozygosity for the FAM- or VIC-specific allele.
- An increase in both signals indicates heterozygosity (26).
- Genotyping with fluorogenic probes requires that fluorescence measurements be made after PCR is completed. This is conveniently done in a multicolour real-time PCR system.
- a multicolour real-time PCR system exicates fluorescent reporter dyes at their respective wavelength (e.g. 495nm for FAM and 540 nm for VIC) and subsequently measures the emitted fluorescence at the respective peak wavelengths (e.g. 520 nm for FAM and 555 nm for VIC).
- the power of discrimination between alleles is determined by the difference in melting temperature ( ⁇ T m ) between match and mismatch probe.
- the ⁇ T m is primarily dependent on the type of mutation, together with the length of the probe.
- oligonucleotides are designed that can anneal to their target at elevated (PCR) temperatures. This may result in a small ⁇ T m discrimination window and difficult allelic discrimination, especially when G to A or G to T mutations are present (27).
- Oligonucleotide probes conjugated with a minor groove binder (MGB) ligand have been developed.
- the MGB has a high affinity for the minor groove of double-stranded DNA and stabilizes the oligoprobe with the complementary single-strand DNA target.
- These MGB probes have a higher melting temperature (T m ) for a given length.
- T m melting temperature
- a 12-mer probe with MGB group had an identical T m as a 27-mer DNA probe without MGB group.
- Single base mismatches between target DNA and such short MGB probes significantly decrease the T m of the duplex resulting in a large ⁇ T m discrimination window. This means that shorter fluorogenic TaqMan probes can be used thus improving specificity and sensitivity.
- NFQ non- fluorescent quencher
- the method used is the SNaPshot single-base extension assay: PCR followed by allele-specific single-base extension as means.
- This preferred method is extensively presented below taken SCD as example. This method includes a step in which ascertaining whether the A878 V polymorphism is present in the sequence of SCD DNA, includes amplifying the DNA in the presence of primers based on the nucleotide sequence of the SCD gene and flanking sequence, followed by allele-specific single-base extension of either the SCD 878A allele or the SCD 878V allele.
- a primer of the present invention used in PCR for example, is a nucleic acid molecule sufficiently complementary to the sequence on which it is based and of sufficient length to selectively hybridise to the corresponding portion of a nucleic acid molecule intended to be amplified and to prime synthesis thereof under in vitro conditions commonly used in PCR.
- Preferred primers for the detection of a SCD 575F aIIeIe are the following: forward primer 5'- TCATTTAACCCCTCATTACCTCA -3' and reverse primer: 5'- TGTAAAATACTAGGCTTTCTGG -3' ( SEQ ID NO: 13 and 14 respectively).
- Preferred single-base extension primer for the detection of a SCD 878 V allele is the following: 5'- TGGTTTCCCTGGGAGCTG - 3' (SEQ ID NO: 15).
- the SNaPshot assay investigates SNP markers by employing PCR amplification followed by dideoxy single-base extension of an unlabeled primer.
- This unlabeled, single-base extension primer is designed to anneal to the sequence adjacent to the SNP site. Once the primer anneals, the single-base extension occurs by the addition of the complementary dye-labelled ddNTP (dye terminator) to the annealed primer.
- ddNTP dye terminator
- Each of the four ddNTPs is fluorescently labelled with a different colour dye. The result is marker fragments for the different SNP alleles that are all the same length, but vary by colour.
- Electrophoresis and multicolour fluorescence detection are preferably carried out with a DNA sequencer ( 48-capillary 3730 DNA analyser, Applied Biosystems), and GeneMapper software (Applied Biosystems) is preferably used to size and genotype the data.
- the SNaPshot multiplex assay can investigate up to ten SNP markers simultaneously by using single-base extension primers of different lengths for the different markers. It may be necessary to add a non-annealing tail to a primer to make its length sufficiently different from other primers. This prevents different SNP markers from overlapping.
- the invention relates to a method for selecting a non-human mammal, preferably a bovine which produces milk with an increased desaturation index for at least one of C 16, C 17, C 18, and CLA fatty acids and/or with a milk-fat composition with at least one of a reduced content of saturated C 16 fatty acids and an increased content of unsaturated Cl 8 fatty acids.
- the invention relates to a method for selecting a non- human mammal, preferably a bovine which produces milk with an increased desaturation index for at least one of C 16, C 17, C 18, and CLA fatty acids.
- the index is preferably the CLA index.
- the milk has a milk-fat composition with at least one of an increased content of C16:lcis9, C17:lcis9 and CLA and a decreased content of C17:0 and C18:0 fatty acids.
- the non-human mammal, preferably a bovine is obtained by applying the method of the invention wherein one screens for the presence of a SCD 878V allele. Therefore, the non- human mammal preferably possesses one allele SCD 878V, more preferably two.
- the invention relates to a method for selecting a non -human mammal, preferably a bovine which produces milk having a milk-fat composition with at least one of a reduced content of saturated C16 fatty acids and an increased content of unsaturated Cl 8 fatty acids.
- the milk- fat composition has a reduced content of saturated C16 fatty acids, an increased content of at least one of C18 cis-isomers such as C18:l cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9, ⁇ 2 (linoleic acid), CLA and C18:3 cis 9,12,15 (linolenic acid).
- the C18 cis- isomers are selected from the group consisting of: C 18:1 cis 9 (oleic acid), C 18:1 cis 11, C18:2 cis 9,12 (linoleic acid), CLA and C18:3 cis 9,12,15 (linolenic acid).
- the Cl 8 cis-isomers are selected from the group consisting of oleic acid, linolenic acid, CLA and linoleic acid.
- this non-human mammal preferably a bovine is obtained by applying the method of the invention wherein one screens for the presence of a DGATl 232 A allele. Therefore, the non- human mammal preferably possesses one allele DGATl 232 A, more preferably two.
- the invention relates to a further preferred embodiment, wherein the milk produced has an increased desaturation index for at least one of C 16, C 17, C18 and CLA fatty acids.
- the index is the CLA index.
- the milk produced has at least one of a decreased content of saturated C16, C17 and C18 fatty acids and an increased content of unsaturated C16, C17 and Cl 8 fatty acids.
- the non-human mammal preferably a bovine is obtained by applying the method of the invention wherein one screens for the presence of a SCD 878V allele in combination with a DGATl 232 A allele .
- the non- human mammal preferably possesses one allele SCD 878V and one allele DGATl 232 A, more preferably two alleles SCD 878 V and one allele DGATl 232 A, or one allele SCD 878V and two alleles DGATl 232 A and most preferably two alleles SCD 878V and two alleles DGATl 232 A.
- the method not only enables to identify a non-human mammal, preferably a bovine, producing milk with an improved (preferably healthier) fatty-acid composition.
- the invention provides a further third preferred embodiment wherein the method also enables to select a non- human mammal, preferably a bovine, as a parent of offspring producing milk with an improved fatty acid composition. Mating of such selected parents will result in an increase of the frequency of the alleles that are favorable for an improved fatty acid composition (such as SCD 878V and/or DGATl 232A) in the offspring, thus, result in offspring producing milk with an improved fatty acid composition compared to offspring of parents that were not selected with this method.
- an improved fatty acid composition such as SCD 878V and/or DGATl 232A
- the invention provides a further fourth preferred embodiment for the method of the invention wherein the non- human mammal, preferably a bovine producing such milk is obtained by further selecting and/or feeding a specific diet (12).
- This selection preferably includes the identification and selection of additional favorable alleles and/or further identification and selection of specific breeds.
- Identification and selection may encompass any method for ensuring that the non-human mammal produces milk with a further improved fatty acid composition (increased unsaturated fatty acid content and decreased saturated fatty acid content by comparison to a non-selected non-human mammal, preferably an increased desaturation index for at least one of C 16, C 17, Cl 8 and CLA fatty acids).
- a further improved fatty acid composition increased unsaturated fatty acid content and decreased saturated fatty acid content by comparison to a non-selected non-human mammal, preferably an increased desaturation index for at least one of C 16, C 17, Cl 8 and CLA fatty acids.
- a further improved fatty acid composition increased unsaturated fatty acid content and decreased saturated fatty acid content by comparison to a non-selected non-human mammal, preferably an increased desaturation index for at least one of C 16, C 17, Cl 8 and CLA fatty acids.
- the method combines second, third and/or fourth preferred embodiments for selecting a non-human mammal, preferably a bovine which produces milk having a milk-fat composition with a reduced content of saturated C16 fatty acids, an increased content of unsaturated C18 fatty acids and a control content of saturated C 14 fatty acid.
- a non-human mammal preferably a bovine which produces milk having a milk-fat composition with a reduced content of saturated C16 fatty acids, an increased content of unsaturated C18 fatty acids and a control content of saturated C 14 fatty acid.
- the method combines former first, second, third and/or fourth preferred embodiments for selecting a non-human mammal, preferably a bovine which produces milk having a milk-fat with an increased desaturation index for at least one of C 16, C 17, Cl 8 and CLA fatty acids.
- the invention relates to milk or milk-fat composition obtainable from the non- human mammal, preferably a bovine obtained by the method of the invention or by the use of the invention both as defined earlier herein.
- the milk of the invention is therefore much more healthy than the milk of control non-human mammal, preferably control bovine milk.
- the milk has an increased desaturation index for at least one of C 16, C 17,Cl 8 and CLA fatty acids.
- the index is preferably the CLA index.
- the milk has a milk-fat composition with at least one of an increased content of C16:lcis9, C17:lcis9 and CLA and a decreased content of C 17:0 and C 18:0 fatty acids.
- the milk has a milk- fat composition with at least one of a reduced content of saturated C16 fatty acids and an increased content of unsaturated C18 fatty acids.
- the milk obtained has a milk-fat composition with a reduced content of saturated C16 fatty acids, an increased content of at least one of Cl 8 cis-isomers such as C 18:1 cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid) and/or C18:3 cis 9,12,15 (linolenic acid) and/or an increased content of CLA.
- the C18 cis-isomers are selected from the group consisting of oleic acid, linolenic acid, CLA and linoleic acid.
- the milk has a milk-fat composition with at least one of a reduced content of saturated C 16 fatty acids, an increased content of unsaturated Cl 8 fatty acids and a control content of saturated C 14 fatty acid.
- the milk produced has further an increased desaturation index for at least one of C 16, C 17,Cl 8 and CLA fatty acids.
- the index is the CLA index.
- the milk produced has at least one of a decreased content of saturated C 16, C17 and Cl 8 fatty acids and an increased content of unsaturated C 16, C17 and Cl 8 fatty acids.
- Such products include all milk- based and/or milk-derived products such as yoghurt, butter or cheese.
- Such products further include food products that comprise a milk and/or milk-fat composition containing ingredient such as bakery products, chocolate or ice cream.
- the invention relates to a food product comprising a milk-fat composition derived from the milk of the invention.
- Figure 1 shows the relationship between available substrate (substrate yield) in one morning milk sample and desaturation index for C 14.
- the substrate yield is the amount of substrate that was available before desaturation took place. This is the amount of C14:0 plus the amount of C14:lcis9 that was measured in the morning milk sample (mean 13.47 kg milk per sample).
- Figure 1 shows that there is no effect of substrate yield on the desaturation index for C 14, thus, the desaturation index is not affected by the amount of substrate that is available. This holds also true for the desaturation indexes of ClO, C12, C16, C17, C18 and CLA.
- Figure 2 shows the relationship between the C 14 desaturation index and the C 16 desaturation index.
- Three groups can be discriminated.
- the phenotypes that were used to calculate the desaturation indexes were corrected for fixed effects, such as herd, days in milk, age at calving and season of calving. Therefore, the three groups might well demonstrate a genetic effect.
- the three groups overlap for 94% with the genotypes of the SCD A878V polymorphism. This indicates that the SCD A878V polymorphism underlies the three groups that can be discriminated in Figure 2.
- the fat composition of winter milk samples was measured in 1918 Dutch Holstein Friesian cows in their first lactation.
- the average milk-fat percentage is 4.36 (Table 1).
- the most abundant fatty acid is C 16:0, which accounts for about 33% of total fat (Table 2).
- Trans fatty acids contribute 1.54%.
- the ratio of saturated to unsaturated fatty acids (SFA/UFA) averages 2.80, meaning that saturated fatty acids account for about 70% of total fat.
- the coefficient of variation (CV) is highest (28%) for conjugated linoleic acid (CLA) and trans fatty acids, and lowest (about 9%) for C14:0, C16:0, and C4:0-C12:0.
- heritabilities to estimate what proportion of total phenotypic variation is additive genetic variation, i.e. heritable.
- Heritability for fat percentage is high (0.51), meaning that about half of the total variation is additive.
- Short- and medium-chain fatty acids (C4-C16) also have high heritabilities, ranging from 0.43 to 0.59.
- Saturated and unsaturated Cl 8 show lower heritabilities (around 0.25), as does the trans fatty acids (0.20).
- C 16:0 which is the most abundant and most unfavorable fatty acid from a nutritionist's point of view, shows a high negative genetic correlation with C14:0 (-0.84), CLA (-0.59), and unsaturared C18 (-0.53).
- the DGATl K232A mutation explains large proportions of the genetic variance: 50% for fat percentage, 53% for unsaturated C 18, 40% for C 16:0, and 36% for SF A/UFA. Effects of DGATl on fat composition are in line with expectations based on the effect of DGATl on fat percentage and the genetic correlations between fat percentage and fat composition (Table3).
- the effect of the DGATl K232A mutation on all saturated and respectively unsaturated fatty acid composition is given in Tables 6 and 7 respectively. It can be observed from table 6 that the DGATl K232A mutation has a statistically significant effect (reduction) on C5, C6, C7, C8, C9, CI l, C13, C15, C16 and C17 saturated fatty acids.
- the C18 fatty acids and, to a lesser extent, C 16:0 arise from the cow's plasma lipids and have a dietary origin (12); they are, therefore, likely to be under less genetic control.
- Heritability for trans fatty acids was also low (0.20). This low heritability may be because trans fatty acids are produced by microbial biohydrogenation in the rumen of the cow (33), which may be under less genetic control as well.
- Heritabilities for milk, protein and fat yield and protein and fat percentage are generally in line with previous studies (13-15, 34, 35). Genetic parameters for fat composition were reported only previously by Karijord et al. (20), who estimated lower heritabilities.
- the effect of the DGATl K232A mutation on fat composition may have different causes: a higher activity of DGATl and alteration of specificity of DGATl.
- the DGATl K allele has been shown to have a higher Vmax than the A allele in producing triglycerides, which is consistent with the in vivo effect of the K232A mutation (38).
- DGATl seems to have a preference for short-chain and unsaturated fatty acids, since the sn-3 position of the glycerol backbone is occupied predominantly by these fatty acids (39).
- Specificity has been shown for other acyltransferases (40, 41 ).
- the K232A mutation could alter the specificity of the DGATl enzyme, which may result in a change in fat composition.
- Genetic correlations show that an increase in fat percentage implies an increase in the fraction of C 16:0, while decreasing the fractions of unsaturated C 18, CLA, and C 14:0. It is likely that selection in the past decades in the Dutch dairy has resulted not only in increased fat percentage and fat yield, but also in a more saturated fat composition, with more C16:0, less unsaturated C18, less CLA, and less C14:0.
- Our results show that it is possible to change fat composition of milk-fat by selective breeding, and that efficiency of selective breeding can be improved using the K232A polymorphism in DGATl. From a public health point of view, increasing the frequency of the DGATl A allele is desirable because of its association with more unsaturated milk-fat, less C16:0, and more unsaturated C18.
- a 0.5 liter milk sample was collected from each cow at one morning milking between February and March 2005.
- Milk- fat composition was measured at the laboratory of COKZ (Leusden, the Netherlands).
- Milk-fat was extracted from the milk samples, and fatty acid methyl esters were prepared from fat fractions, as described in ISO Standard 15884 (29).
- Methyl esters were analyzed according to ISO Standard 15885 (30) on a Trace GC Ultra chromatograph (Thermo Electron Corporation, Waltham, MA, USA), using a Varian Fame Select column (100m x 0.25mm ID, Varian Inc. , Palo Alto, CA, USA).
- the initial temperature was held at 7O 0 C for 1 min, raised to 225 0 C at 3°C/min, and held at 225 0 C for 5 min.
- a volume of 1 ⁇ l was injected.
- Each peak was identified and quantified using pure methyl ester samples (Sigma-Aldrich, Zwijndrecht, the Netherlands; Larodan, Malmo, Sweden).
- the fatty acids included in this study were grouped according to their relevance to human nutrition and health. Fat and protein percentage were determined by infra red spectroscopy, using a MilkoScan FT6000 (Foss Electric, Hillerod, Denmark) at the Milk Control Station (Zutphen, the Netherlands). Fat and protein yields were calculated by multiplying each percentage by milk yield. Yield data were missing for 135 cows. Genotypes
- Genotyping of the DGATl K232A dinucleotide polymorphism was performed using a Taqman allelic discrimination method in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).
- the primers and labeled oligonucleotide probes for this reaction were: forward, 5'- CGCTTGCTCGTAGCTTTGG -3'; reverse, 5'- CGCGGTAGGTCAGGTTGTC -3'; VIC probe (detects ⁇ allele ), 5'- CGTTGGCCTTCTTAC -3'; FAM probe (detects A allele), 5'-TTGGCCGCCTTAC-S' (SEQ ID NO: 5, 6, 7 and 8 respectively).
- yykinm ⁇ + bi *dim 1 + b 2 *e ⁇ ° 05*dim +b 3 *afc, + b 4 *afc, 2 + season k + scodei + herd m + U n +
- Syklmn where y is the dependent variable, ⁇ is the general mean, dim is the covariate describing the effect of days in milk modelled with a Wilmink curve (32), afc is the covariate describing the effect of age at first calving, season is the fixed effect of the class of calving season (June -August 2004, September-November 2004, or December 2004-February 2005), scode is the fixed effect of the differences in genetic level between groups of proven bull daughters and young bull daughters, herd is the random effect of groups of animals sampled in the same herd, U n is the random additive genetic effect of animal n, and e is the random residual effect.
- Example 2 SCD 878V and optionally DGAT 232A Material and methods
- Phenotypes A 0.5 liter milk sample was collected from each cow at one morning milking between February and March 2005. Milk- fat composition was measured at the COKZ laboratory (Netherlands Controlling Authority for Milk and Milk Products, Leusden, the
- Fat and protein percentages were measured by infra red spectroscopy, using a MilkoScan FT6000 (Foss Electric, Hillerod, Denmark) at the Milk Control Station (Zutphen, the Netherlands). Fat and protein yields were calculated by multiplying each percentage by the milk yield. Yield data were missing for 145 cows.
- Genotypes for the SCD A 878 V polymorphism were assayed by SNaP shot single base primer extension method (Applied Biosystems, Foster City, CA, USA).
- the primer designs were based on the Genbank sequence (AY241932): forward, 5'- TCATTTAACCCCTCATTACCTCA -3'; reverse, 5'-
- 12 ⁇ l reactions were set up containing 20 ng of genomic DNA, 0.2 ⁇ M of each primer and 2X AccuPrime Supermix II (Invitrogen, Carlsbad, CA, USA). PCR cycling conditions were 94°C for 5 min, 36 cycles of 94°C for 30 s, 55°C for 45 s, 68°C for 90 s, followed by an extension cycle of 68°C for 10 min.
- PCR products were purified by incubation with shrimp alkaline phosphatase (SAP) (USB, Cleveland, OH, USA) and Exo I (USB) at 37°C for 1 h and 72°C for 15 min.
- Extension reactions using 3 ⁇ l of purified PCR product and 5 pmol of genotyping primer and SNaPshot multiplex Ready reaction mix (Applied Biosystems), were performed using 40 cycles of 96°C for 10 s, 50 0 C for 5 s, and 60 0 C for 30 s.
- the extension products were incubated with SAP at 37°C for 1 h and 72°C for 15 min.
- Genotyping of the DGATl K232A dinucleotide polymorphism was performed using a Taqman allelic discrimination method in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems).
- the primers and labeled oligonucleotide probes for this reaction were designed based on the DGATl sequence (Genbank accession no. AY065621): forward, 5'- CGCTTGCTCGTAGCTTTGG -3'; reverse, 5'- CGCGGTAGGTCAGGTTGTC -3'; VIC probe (detects K allele), 5'- CGTTGGCCTTCTTAC -3'; FAM probe (detects A allele), 5'-TTGGCCGCCTTAC-S'.
- PCR cycling conditions were 94°C for 5 min, 40 cycles of 92°C for 15 s and 60 0 C for 1 min. In total, 1779 animals were genotyped for the DGATl K232A polymorphism.
- Analyses were performed first using SAS 9.1 (2002) procedures to determine fixed effects.
- yi j ki ⁇ m ⁇ + bi *dim 1 + b 2 *e ⁇ ° 05*dim +b 3 *afc, + b 4 *afc, 2 + season k + scodei + herd m + U n +
- y was the dependent variable
- ⁇ was the general mean
- dim was the covariate describing the effect of days in milk modelled with a Wilmink curve (32)
- afc was the covariate describing the effect of age at first calving
- season was the fixed effect of the class of calving season (June -August 2004, September-November 2004, or December 2004-February 2005)
- scode was the fixed effect of the differences in genetic level between groups of proven bull daughters and young bull daughters
- herd was the random effect of groups of animals sampled in the same herd
- U n was the random additive genetic effect of animal n
- e was the random residual effect.
- Effects of the SCD A 878 V or the DGATl K232A mutation were estimated using the same model, but extended with effect g: the fixed effect of the SCD genotype (AA, AV, W) or the fixed effect of the DGATl genotype (KK, KA, AA). Ungenotyped individuals were included as a separate group, and appeared to be random.
- Var(U) A ⁇ u 2 , where A was a matrix of additive genetic relationships between individuals and ⁇ u 2 was the additive genetic variance.
- Heritabilities were estimated using univariate analyses, and phenotypic and genetic correlations were estimated using bivariate analyses. Summary:
- Figure 2 shows the C 14 and the C16 indexes (corrected phenotypes).
- SCD stearoyl- Co enzyme A desaturase
- This SNP is known to affect mono -unsaturated fatty acids (MUFA) percentage and melting point in intramuscular fat of Japanese black cattle (Taniguchi et al 2004, Mamm. Genome 15: 142-148).
- MUFA mono -unsaturated fatty acids
- melting point in intramuscular fat of Japanese black cattle
- V allele is associated with a lower index for ClO, C 12 and C 14, but is associated with a higher index for C 16, C 17, Cl 8 and CLA.
- the polymorphism in the gene DGATl known to have a clear influence on milk- fat composition, is also associated with desaturation indexes (Table 13). This indicates that not only the conversion of saturated to unsaturated fatty acids by SCD is determinative for saturation of milk- fat, but also the esterification of specific fatty acids to glycerol by DGATl.
- the percentages of the genetic variance explained by the SCD A 878 V and DGATl K232A polymorphisms are in Table 14. Correction for the SCD genotype doesn't notably change the effect (size) of the DGATl genotype, meaning that the two genes explain a different part of the variation.
- Table 15 provides the combined effects of the SCD A 878 V polymorphism and the DGATl K232A polymorphism on fatty acid composition and desaturation indexes.
- the effect of the SCD A 878 V polymorphism and of the DGATl K232A polymorphism are mostly additive and in the same direction. Consequently, the combined homozygous genotypes have progressively decreasing effects on the ClO, C 12 and C 14 indexes, and progressively increasing effects on the Cl 8 and CLA indexes.
- C16 the effect of the SCD A 878 V polymorphism and of the DGATl K232A polymorphism are mostly additive, but in opposite directions.
- the effect of the SCD A878V polymorphism is larger than the effect of the DGA Tl K232A polymorphism.
- the combination of homozygous SCD 878 A and homozygous DGATl 232 A has the most decreasing effect on the C16 index
- the combination of homozygous SCD 878 V and homozygous DGATl 232K has the most increasing effect on the C16 index.
- the combination of homozygous SCD 878 V and homozygous DGATl 232 A has an increasing effect on the C16 index also.
- the effect of the DGATl K232A polymorphism is not significant, therefore, does not add to the effect of the SCD A 878 V polymorphism.
- C4:0-C12:0 includes saturated fatty acids C4:0, C6:0, C8:0, C10:0 and C12:0.
- C18u includes unsaturated Cl 8 fatty acids: Cl 8:1 trans 6, Cl 8:1 trans 9, Cl 8:1 trans 11, Cl 8:1 cis 9, C18:l cis 11, C18:2 cis 9,12, C18:3 CM 9,12,15.
- trans includes C16:l trans 9, C18:l trans 4-8, C18:l trans 9, C18:l trans 10, C18:l trans 11, C18:l trans 12.
- SFA saturated fatty acids
- UFA unsaturated fatty acids
- Standard errors of phenotypic correlations were between 0.02 and 0.03.
- Standard errors of genetic correlations were between 0.08 and 0.23.
- KA contrast of KA -KK genotypes.
- AA contrast of AA -KK genotypes.
- P value statistical significance of the DGATl K232A effect.
- r 2 genetlc % percentage of the genetic variance explained by the DGATl K232A mutation.
- KA contrast of KA -KK genotypes.
- AA contrast of AA -KK genotypes.
- P value statistical significance of the DGATl K232A effect.
- KA contrast of KA -KK genotypes.
- AA contrast of AA -KK genotypes.
- P value statistical significance of the DGATl K232A effect.
- contrast of KA-KK genotypes " contrast of AA-KK genotypes.
- ISO-IDF 2002a Milkfat - Preparation of fatty acid methyl esters. ISO 15884-IDF 182; International Dairy Federation, Brussels, Belgium.
- ISO-IDF 2002b Milkfat - Determination of the fatty acid composition by gas- liquid chromatography.
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Abstract
The invention relates to the u se of means that detect the presence of a SC D 878V allele and /or a DGAT 1 232A allele in a method for identifying a non - human mammal, preferably a bovine which produces milk with having a n improved fatty acid composition.
Description
Method for selection of non- human mammal producing milk with improved fatty acid composition
Field of the invention The invention relates to the use of means that detect the presence of a SCD 878V and/or of additional means that detect the presence of a DGATl 232A allele in a method for identifying a non-human mammal which produces milk with an improved fatty acid composition.
Background of the invention
The impact of dietary fat on chronic disease, such as coronary heart disease, has been a topic of interest for decades (1). For almost 50 years, effects of fatty acid intake on serum lipids have been investigated. It is now clear that intake of saturated fatty acids increases total and low-density lipoprotein (LDL) cholesterol levels, whereas intake of polyunsaturated fatty acids decreases LDL cholesterol (2-4). Not all saturated fatty acids affect cholesterol concentrations to the same extent. Laurie acid (C 12:0), for example, reduces the ratio of total to high-density lipoprotein (total:HDL) cholesterol; reduction in this ratio is associated with reduction in the risk of coronary heart disease. Myristic acid (C14:0) and stearic acid (C18:0), furthermore, reduce total:HDL cholesterol less than C12:0, whereas palmitic acid (C16:0) has the unfavorable effect of increasing the ratio (4). More recently, trans fatty acids have been reported to increase total:HDL cholesterol, hence increasing the risk of coronary heart disease (5). A high intake of saturated and trans fatty acids has also been related to insulin resistance and, subsequently, to type 2 diabetes (6-7). It has been suggested, furthermore, that dietary fatty acids play a role in the development of obesity and cancer (8-10). These findings suggest that an alteration of the dietary fat composition could have a major impact on public health.
Hulshof et al. (11) showed that milk and milk-derived foods (including cheese and butter) were the main source of dietary saturated fatty acids across Europe, ranging between 27% and 58%. The contribution from milk and milk-derived foods to dietary trans fatty acids was between 17% and 72%. Milk- fat is relatively high in saturated fatty acids, especially C14:0 and C16:0, and low in polyunsaturated fatty acids.
Milk-fat composition can be altered through the nutrition of dairy cows (12), and possibly by selective breeding, although prospects for the latter have not been studied extensively. The major prerequisite for selective breeding is existence of genetic variation in milk-fat composition among cows. For milk-fat percentage, around half the observed variation is estimated to be due to genetic variation (13-16). Phenotypic variation in milk-fat composition has been reported as well, both between and within breeds, although the number of reports on genetic variation is limited and recent publications are lacking (17-21).
Recently, a quantitative trait locus (QTL) mapping study in cattle resulted in the identification of the K232A mutation in the gene coding for acyl CoA:diacylglycerol acyltransferase 1 (DGATl), which is a key enzyme in triglyceride synthesis (22) and has a strong effect on milk-fat percentage and other milk-production characteristics (23-24). WO02/36824 discloses that bovine carrying the DGATl 232A allele produce more milk, said milk containing more milk-protein and less milk-fat than milk from cows that do not carry the DGATl 232A allele. The effect of the DGATl 232A allele on milk fatty acid composition is not known.
There is still a need of finding genetic markers associated with a non-human mammal, preferably a bovine producing milk having attractive healthy properties for human beings such as its fatty acids composition.
Description of the invention
Surprisingly, the present invention establishes that the DGATl 232 A allele is further associated with at least one of: a reduced content of saturated C 16 fatty acids, and an increased content of unsaturated Cl 8 fatty acids in the milk- fat composition. Furthermore, another single nucleotide polymorphism (SNP) was identified in another gene which also influences the milk fatty acid composition. This gene codes for the Stearoyl-Coenzyme A desaturase (SCD). The mutation A878V in the SCD enzyme was found particularly attractive for conferring healthier fatty acid composition to the milk. Even more surprisingly, the inventors established that the presence of both a SCD 878 V and a DGATl 232 A allele is associated with a milk having an even more healthy fatty acid composition.
Use
Therefore in a first aspect, there is provided the use of means that detect the presence of a SCD 878 V allele and/or of additional means that detect the presence of a DGATl 232A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a different milk-fat composition than the one from a milk produced by a non-human mammal not having a SCD 878 V and/or DGATl 232 A allele.
In the context of the application, milk or milk- fat could be replaced by milk or milk- fat composition. A milk or milk-fat composition is preferably defined by a fatty acid composition.
In the context of the invention, "different" preferably means that using any of the means as described herein the milk-fat composition of a milk produced by a non-human mammal having a SCD 878V and/or a DGATl 232 A allele is analyzed as being different (i.e. distinct) from the milk-fat composition of a milk produced by a non- human mammal not having a SCD 878 V and/or DGATl 232 A allele. In this context, a "different" milk-fat composition more preferably means a healthier milk-fat composition. A "healthy" milk-fat composition is further herein defined by reference to its fatty acid composition.
SCD 878V allele
In a first preferred embodiment, there is provided the use of means that detect the presence of a SCD 878 V allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a different milk-fat composition than the one from a milk produced by a non- human mammal not having a SCD 575FaIIeIe. More preferably, in this embodiment, the milk has a milk-fat with an increased desaturation index for at least one of C16, C17,C18 and CLA fatty acids.
Fatty acid molecules are well known to the skilled person. These are carboxylic acids having generally a long unbranched aliphatic tail which is either saturated or unsaturated. Saturated fatty acids do not contain any double bonds or other functional groups along the chain. Saturated C 16 fatty acid is palmitic acid also named hexadecanoic acid.
Unsaturated fatty acids are fatty acids containing one or more alkenyl functional groups along the chain, with each alkene substituting a singly-bonded "-CH2-CH2-" part of the chain with a doubly-bonded "-CH=CH-". Due to the presence of this double bond, cis- and trans- isomeres may be formed. In the context of the invention, unsaturated Cl 8 fatty acids are preferably selected from the following group: C18:l cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid), CLA (conjugated linoleic acid) and C18:3 cis 9,12,15 (linolenic acid). All these unsaturated C18 cis-isomers C18: 1 cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid), CLA and C18:3 cis 9,12,15 (linolenic acid) are known to have a positive effect on human health. In the context of the invention, the CLA is selected from the following isomers consisting of: C 18:2 cis 9, trans 11 and C18:2 translO, cis 12. Even more preferably, the CLA is the C18:2 cis9, transl l isomer.
Within the context of the invention, a desaturation index is defined as being the ratio between the product and the substrate plus the product, said ratio being expressed as a percentage. Substrate and product mean substrate and product for a SCD enzyme. A SCD enzyme specifically introduces a cis double bond at the 9 position of a fatty acid. The substrate and product are expressed as content of substrate and product as later defined herein. For example, the C16 index is: (C16:lcis9 / (C16:0 + C16:lcis9))xl00. For example, the C17 index is: (C17:lcis9 / (C17:0 + C17:lcis9))xl00.
The C18 index is: ((C18:lcis9 + C18:ltransl2) / (C18:0 + C18:lcis9 + C18:ltransl2))xl00. Preferably, the Cl 8 index is the index of C18: lcis9. The index of C18:lcis9 is as follows: (C18:lcis9 / (C18:0 + C18:lcis9))xl00. The index of CLA is as follows: (CLA/ (C 18: 1 transl l + CLA))xlOO, which is (C 18:2cis9transl 1/(C 18:1 transl l + C18:2cis9transl l))xl00. Therefore, the C18 index does not include the CLA index. In the context of the invention, for calculating desaturation indexes, the CLA index is calculated as an index which is separate from the Cl 8 index.
In another preferred embodiment, the invention relates to a use of means, that detect the presence of a SCD 878V allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk-fat composition with at least
one of an increased content of C16:lcis9, C17:lcis9 and CLA and a decreased content of C17:0 and C18:0 fatty acids. This sentence is equivalent with an increased content of at least one of C16:lcis9, C17:lcis9 and CLA and/or a decreased content of at least one of C17:0 and C18:0 fatty acids. Throughout the invention the same meaning is given to the use of the expression "at least one" in combination with a group of features.
It has been found that a SCD 878V allele is associated with milk having a healthy milk- fat composition: a healthy fatty acid composition wherein a decreased content of saturated C17 and C18 fatty acids and an increased content of unsaturated C 16, C 17, and Cl 8 fatty acids is seen. An unsaturated Cl 8 fatty acid is preferably CLA.
In the context of this embodiment, an increased desaturation index for C 16, C 17, Cl 8 or CLA means a desaturation index which is higher than the corresponding desaturation index for C 16, C 17, C18 or CLA in the milk-fat of the milk of a non- human mammal, preferably a bovine not having a SCD 878V allele. Preferably, the increase is approximately of 2% or more. More preferably, the increase is approximately of 5% or more, or approximately of 7% or more, or approximately of 10% or more, or approximately of 15 % or more, or approximately of 20% or more, or approximately of 25% or more. In the context of this embodiment, a decreased content of C17:0 or C18:0 fatty acids means a content of C17:0 or C18:0 fatty acids which is lower than the content of C 17:0 or C 18:0 fatty acids present in the milk-fat of the milk of a non-human mammal, preferably a bovine not having a SCD 878V allele. Preferably, the decrease is approximately of 4% or more, or approximately of 5% or more. More preferably, the decrease is approximately of 7% or more, or approximately of 10% or even more. In the context of this embodiment, an increased content of C16:lcis9, C17:lcis9 or CLA fatty acids means a content of C16:lcis9, C17:lcis9 or CLA fatty acids which is higher than the content of C16:lcis9, C17:lcis9 or CLA fatty acids present in the milk- fat of the milk of a non- human mammal, preferably a bovine not having a SCD 878V allele. Preferably, the increase is approximately of 5% or more. More preferably, the increase is approximately of 7% or more, or approximately of 10% or more, or approximately of 15% or more, or approximately of 20% or more, or approximately of 25% or more.
DGA Tl 232 A allele
In another preferred embodiment, there is provided the use of means that detect the presence of a DGATl 232 A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a different milk-fat composition than the one from a milk produced by a non- human mammal not having a DGATl 232 A allele.
More preferably, in the embodiment, the presence of a DGATl 232 A allele is associated with a milk having at least one of a reduced content of saturated C16 fatty acids and an increased content of unsaturated Cl 8 fatty acids.
In a preferred embodiment, the invention relates to the use of means that detect the presence of the DGATl 232A allele in a method for identifying a non- human mammal, preferably a bovine which produces milk having a milk-fat composition with at least one of a reduced content of saturated C 16 fatty acids and an increased content of at least one of C18 cis-isomers such as: C18:l cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid), conjugated linoleic acid (CLA) and C18:3 cis 9,12,15 (linolenic acid). More preferably, the Cl 8 cis-isomers are selected from the group consisting of: C18:l cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid), CLA and C18:3 cis 9,12,15 (linolenic acid). Even more preferably, the C18 cis-isomers are selected from the group consisting of oleic acid, linoleic acid, CLA and linolenic acid. In the context of the invention, the CLA is preferably selected from the following isomers consisting of: C18:2 cis 9, trans 11 and C18:2 translO, cis\2. Alternatively, in another preferred embodiment, the invention relates to the use of means that detect the presence of the DGATl 232A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk-fat composition with at least one of a reduced content of saturated C16 fatty acids and an increased content of at least one of Cl 8 cis-isomers such as: C 18:1 cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid), and C18:3 cis 9,12,15 (linolenic acid). More preferably, the C18 cis-isomers are selected from the group consisting of: C18:l cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid) and C18:3 cis 9,12,15
(linolenic acid). Even more preferably, the Cl 8 cis-isomers are selected from the group consisting of oleic acid, linoleic acid, and linolenic acid.
It has been found that the DGATl 232A allele is associated with milk having a healthy milk- fat composition: a healthy fatty acid composition wherein a reduced content of saturated C16 fatty acids and an increased content of unsaturated Cl 8 fatty acids is seen. Furthermore, the DGATl 232A allele has unexpectedly been found to be associated with a relative small increase in saturated C 14 fatty acid. The total effect on fatty acid composition is positive for the human health because the effect of the DGATl 232A allele is much more pronounced on saturated C16 fatty acids, unsaturated C18 fatty acids than on saturated C14 fatty acids (see table 5). Therefore, in another preferred embodiment, the invention relates to the use of means that detect the presence of the DGATl 232A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk- fat composition with at least one of a reduced content of saturated C16 fatty acids, an increased content of unsaturated C 18 fatty acids, and an increased content in saturated C 14 fatty acids. Furthermore, if one looks at the overall effect on fatty acid composition, it has been observed (table 6) that the DGATl K232A mutation has a statistically significant effect (reduction) on C5, C6, C7, C8, C9, CI l, C13, C15, C16 and C17 saturated fatty acids. It has further been observed that the DGATl K232A mutation also has a statistically significant effect (increase) on C18 (C 18:1 cis9 (oleic acid), C 18:1 cisl l, C 18:2 cis 9,12 (linoleic acid), CLA, and C18:3 cis 9,12,15 (linolenic acid)) unsaturated fatty acids (table 7). Therefore, alternatively or in combination with earlier embodiment, the invention relates to the use of means that detect the presence of the DGATl 232A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk- fat composition with at least one of a reduced content of saturated C5, C6, C7, C8, C9, CI l, C13, C15, C16 and C17 fatty acids and an increased content of unsaturated on Cl 8 (Cl 8:1 cis9 (oleic acid), C 18:1 cisl l, C 18:2 cis 9,12 (linoleic acid), CLA, and C18:3 cis 9,12,15 (linoleic acid)) fatty acids.
In the context of the invention, a reduced content of saturated C16 fatty acids means a content of saturated C16 fatty acids which is lower than the content of saturated C 16 fatty acids present in the milk-fat composition of the milk of a non-human mammal, preferably a bovine not having a DGATl 232 A allele. Preferably, the reduction is approximately of 5% or more. More preferably, the reduction is approximately of 7% or more or approximately of 10% or even more.
In the context of the invention, an increased content of unsaturated C 18 fatty acids means a content of unsaturated Cl 8 fatty acids which is higher than the content of unsaturated C18 fatty acids present in the milk-fat composition of the milk of a non- human mammal, preferably a bovine not having a DGATl 232 A allele. Preferably, the increase is approximately of 5% or more. More preferably, the increase is approximately of 7% or more, or approximately of 10% or more. In the context of the invention, an increased content of saturated C 14 fatty acid means a content of saturated C 14 fatty acid which is higher than the content of saturated C 14 fatty acids present in the milk- fat composition of the milk of a non-human mammal, preferably a bovine not having a DGATl 232 A allele. Preferably, the increase is approximately of 2% or more. More preferably, the increase is approximately of 5% or more or approximately of 7% or even more.
SCD 878VaIMe and DGATl 232 A allele
In a more preferred embodiment, there is provided the use of means that detect the presence of a SCD 878V allele and of additional means that detect the presence of a DGATl 232 A allele in a method for identifying a non- human mammal, preferably a bovine which produces milk having a different (more preferably healthier) milk-fat composition than the one from a milk produced by a non-human mammal not having a SCD 878V and/or DGATl 232 A allele.
In this more preferred embodiment, the invention relates to a use of means that detect the presence of a SCD 878V allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk-fat with an increased desaturation index for at least one of C 16, C 17, C18 and CLA fatty acids, wherein additional means are used to detect the presence of a DGATl 232 A allele. Surprisingly,
the inventors found that a SCD 575F aIIeIe and a DGATl 232 A allele explain a distinct part of the variation seen for the desaturation index of at least one of C 16, C18 and CLA fatty acids. Therefore, by selecting for both a SCD 878V and a DGATl 232 A allele, one expects to take into account about 50% of the genetic variation seen for the desaturation index for at least one of C16, C17, C18 and CLA fatty acids (see table 14). Even more surprisingly, the inventors found that the effect of a SCD 575FaIIeIe and a DGATl 232 A allele are synergetic for the desaturation index of at least one of Cl 8 and CLA fatty acids. Table 15 provides the combined effects of the SCD A878V polymorphism and the DGATl K232A polymorphism on fatty acid composition and desaturation indexes. For all indexes except C16 and C17, the effect of the SCD A878V polymorphism and of the DGATl K232A polymorphism are mostly additive and in the same direction. Consequently, the combined homozygous genotypes have progressively decreasing effects on the ClO, C12 and C14 indexes, and progressively increasing effects on the Cl 8 and CLA indexes. For C 16 the effect of the SCD A878V polymorphism and of the DGATl K232A polymorphism are mostly additive, but in opposite directions. The effect of the SCD A 878 V polymorphism is larger than the effect of the DGATl K232A polymorphism. As a result, the combination of homozygous SCD 878 A and homozygous DGATl 232 A has the most decreasing effect on the C16 index, and the combination of homozygous SCD 575 F and homozygous DGATl 232K has the most increasing effect on the C16 index. The combination of homozygous SCD 575 F and homozygous DGATl 232 A has an increasing effect on the C16 index also. For C17 the effect of the DGATl K232A polymorphism is not significant, therefore, does not add to the effect of the SCD A 878 V polymorphism.
It has been found that a DGATl 232 A allele is associated with milk having a healthy milk-fat composition:
- a healthy fatty acid composition with an increased desaturation index for C18 and/or CLA fatty acids,
- a healthy fatty acid composition wherein a reduced content of saturated C16 fatty acids and an increased content of unsaturated C18 fatty acids is seen. Furthermore, a
DGATl 232 A allele has unexpectedly been found to be associated with a relative small increase in saturated C 14 fatty acids. The total effect on fatty acid composition is
positive for the human health because the effect of a DGATl 232 A allele is much more pronounced on saturated C16 fatty acids and unsaturated Cl 8 fatty acids than on saturated C 14 fatty acids. Therefore, in another preferred embodiment, the invention relates to the use of means that detect the presence of a DGATl 232 A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk- fat composition with at least one of a reduced content of saturated C16 fatty acids, an increased content of unsaturated Cl 8 fatty acids, and an increased content in saturated C 14 fatty acids. Furthermore, if one looks at the overall effect on fatty acid composition, it has been observed that a DGATl K232A mutation has a statistically significant effect (decrease) on C5, C6, C7, C8, C9, CI l, C13, C15, C16 and C17 saturated fatty acids. It has further been observed that a DGATl K232A mutation also has a statistically significant effect (increase) on Cl 8 (C 18:1 cis9 (oleic acid), C 18:1 cisl l, C18:2 cis 9,12 (linoleic acid), CLA, and C18:3 cis 9,12,15 (linolenic acid)) unsaturated fatty acids. Therefore, alternatively or in combination with earlier embodiment, the invention relates to the use of means that detect the presence of a DGATl 232 A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a milk- fat composition with at least one of a reduced content of saturated C5, C6, C7, C8, C9, CI l, C13, C15, C16 and C17 fatty acids and an increased content of unsaturated C 18 (C 18 : 1 cis9 (oleic acid), C 18 : 1 cis 11 , C 18 :2 cis 9, 12 (linoleic acid), CLA, and C18:3 cis 9,12,15 (linoleic acid)) fatty acids.
More preferably within this highly preferred embodiment, the milk has a milk-fat with an increased desaturation index for at least one of C 16, C 17, C18 and CLA fatty acids.
Even more preferably, the increased desaturation index is the CLA index.
In the context of this highly preferred embodiment, an increased desaturation index for
C 16, C 17, C18 or CLA means a desaturation index which is higher than the corresponding desaturation index for C 16, C 17, Cl 8 or CLA in the milk- fat of the milk of a non-human mammal, preferably a bovine not having a SCD 878V and a DGATl
232A allele. Preferably, the increase is approximately of 5% or more. More preferably,
the increase is approximately of 7% or more, or approximately of 10% or more, or approximately of 15% or more.
More preferably within this highly preferred embodiment, the milk has a milk-fat composition with at least one of a decreased content of saturated C 16, C 17 and C 18 fatty acids and an increased content of unsaturated C 16, C17 and Cl 8 fatty acids.
In the context of this embodiment, a decreased content of saturated C16, C17 or C18 fatty acids means a content of C16:0, C17:0 or C18:0 fatty acids which is lower than the content of C16:0, C17:0 or C18:0 fatty acids present in the milk-fat of the milk of a non- human mammal, preferably a bovine not having a SCD 878 V and a DGATl 232 A allele. Preferably, the decrease is approximately of 4% or more, or approximately of 5% or more. More preferably, the decrease is approximately of 7% or more, or approximately of 10% or even more.
In the context of this embodiment, an increased content of unsaturated C 16, C17 or C18 fatty acids means a content of C16:lcis9, C17:lcis9, C18:lcis9 or CLA fatty acids which is higher than the content of C16:lcis9, C17:lcis9, C18:lcis9 or CLA fatty acids present in the milk-fat of the milk of a non-human mammal, preferably a bovine not having a SCD 878V and a DGATl 232 A allele. Preferably, the increase is approximately of 1% or more, or approximately of 5% or more. More preferably, the increase is approximately of 7% or more, or approximately of 10% or more, or approximately of 15% or more, or approximately of 20% or more.
In the context of the invention, fatty acid content preferably means the amount of fatty acid in gram per 100 gram fatty acids in the milk-fat (w/w). This content is therefore preferably expressed by means of weight percentages. The milk-fat is preferably extracted from the milk as defined later herein. Fatty acid methyl esters are preferably prepared from fat fractions and analyzed as defined later herein.
In the context of the invention, decrease and/or increase of fatty acid contents are preferably estimated by comparison with the corresponding control or average contents in the milk-fat of a control non-human mammal, preferably a bovine which is milked in the morning during winter (february-march) in its first lactation (between Day 63 and Day 282 in lactation). The bovine is preferably a Dutch Holstein Friesian cow that is milked twice a day, in which population the frequency of the SCD 878V allele is 0.27
and the frequency of the DGATl 232 A allele is 0.60. To have a representative average content, at least 1000 cows are preferably used.
The invention may be applied to any non-human mammal for obtaining milk having an improved milk-fat composition or an improved fatty acid composition. In a preferred embodiment, the non-human mammal is an ungulate and/or a ruminant. Preferred ungulates include a cow (or bovine), a horse, a sheep, a camel, a donkey, or a goat. More preferably, the non-human mammal is a bovine.
The content of all fatty acids mentioned in this invention is preferably assessed as follows. Milk -fat is preferably extracted from the milk samples as follows. Milk -fat is extracted from the milk samples by adding 5 ml HCL (4M) to 100 ml of milk which is kept at 28°C and is shaken during 45 min. When butterfat is clearly visible, the water phase is poured off and the fat is rinsed with 200 ml cold water twice. Subsequently, the fat is melted at 700C during 60 min, then transferred into a centrifuge tube and stored at -200C until further processing. After defrosting, the tube is warmed to 400C and centrifuged for 15 min at 1300g and 400C. The fat phase is cleared at 400C and used for the preparation of fatty acid methyl esters. Fatty acid methyl esters are prepared from fat fractions as described in ISO Standard 15884 (29). Fatty acid methyl esters are analyzed according to ISO Standard 15885 (3 0) on a Trace GC Ultra chromatograph (Thermo Electron Corporation, Waltham, MA, USA), using a Varian Fame Select column (100m x 0.25mm ID, Varian Inc. , Palo Alto, CA, USA). The initial temperature is held at 7O0C for 1 min, raised to 2250C at 3°C/min, and held at 2250C for 5 min. A volume of 1 μl is injected. Each peak is identified and quantified using pure methyl ester samples (Sigma-Aldrich, Zwijndrecht, the Netherlands; Larodan, Malmo, Sweden).
In a further preferred embodiment, a SCD 878V ox both a SCD 878V and a DGATl 232A allele are determined with respect to DNA, mRNA, and/or protein obtained from the non-human mammal by direct or indirect methods. The method used for detecting a SCD 878V or both a SCD 878V and a DGATl 232 A allele is not critical for the invention. Several methods were identified in WO 02/36824 using DNA, mRNA, and/or protein obtained from the bovine by direct or indirect
methods. In a preferred method, the presence of a SCD 878 V or both a SCD 878 V and a DGATl 232 A allele is directly assessed in the milk of the non-human mammal, preferably a bovine. In this preferred method, somatic cells are first isolated from the milk. More preferably, in this preferred method, the Taqman assay or the SNaPshot assay is used as defined below.
The type of means used in the method of the invention is also not critical for the invention as long as these means are able to identify a SCD 878V or both a SCD 878V and a DGATl 232 A allele. Preferred means include primers, nucleic acid probes or antibodies.
A bovine coding nucleic acid sequence of the SCD 575FaIIeIe is given in SEQ ID NO:1. An amino acid sequence of the bovine SCD protein with the 878V allele encoded by SEQ ID NO:1 is SEQ ID NO:2. A bovine coding nucleic acid sequence of the SCD 878 A allele is given in SEQ ID NO:3. An amino acid sequence of the bovine SCD protein with the 878A allele encoded by SEQ ID NO:3 is SEQ ID NO:4. The nucleic acid coding sequence and amino acid sequence of the allele A are also available at GenBank data library under accession number AY241932. A bovine coding nucleic acid sequence of the DGATl 232 A allele is given in SEQ ID NO:5. An amino acid sequence encoded by SEQ ID NO:5 is SEQ ID NO:6. A bovine coding nucleic acid sequence of the DGATl 232K allele is given in SEQ ID NO:7. An amino acid sequence encoded by SEQ ID NO:7 is SEQ ID NO:8.
Five methods may be cited to identify a SCD 878 V allele or a DGATl 232 A allele:
Polymerase Chain Reaction (PCR) using two primers and two probes as means per allele to be detected (so-called Taqman assay), the SNaPshot single-base extension assay: PCR followed by allele-specific single-base extension as means, - detection of protein variants of SCD or DGATl using antibodies directed against these variants as means,
hybridisation with probes that are complementary either for the SCD 878V allele or the DGATl 232 A allele (without amplification) as means and PCR with three primers as means, one specific for the locus of SCD or DGATl, one specific for the desired allele (SCD 878V or DGATl 232A) and one for the undesired allele (SCD 878A or DGATl 232K).
All these methods have been extensively presented in WO 02/36824 and are well known to the skilled person. Other methods, based on e.g. hybridisation such as Southern blot, FISH (Fluorescent In Situ Hybridization), ASO (Allele-Specific Oligonucleotide hybridization), molecular beacons, array technology; mobility shift such as SSCA (Single-Strand Conformation Analysis), DGGE (Denaturing Gradient Gel Electrophoresis) DHPLC (Denaturing High-Performance Liquid Chromatography), melting curve; and enzymatic reactions such as cleavage (RFLP, Invader), ligation, nucleotide incorporation (sequencing, pyrosequencing, minisequencing) can also be used to identify a SCD 878V ox Ά DGATl 232 A allele. In a preferred embodiment, the method used is the TaqMan assay: PCR using two primers and two probes as means. This preferred method is extensively presented below taken DGATl as example. This method includes a step in which ascertaining whether the K232A polymorphism is present in the sequence of DGATl DNA, includes amplifying the DNA in the presence of primers based on the nucleotide sequence of the DGATl gene and flanking sequence in the presence of allele-specific probes complementary to either the DGATl 232 A allele or the DGATl 232K allele.
A primer of the present invention, used in PCR for example, is a nucleic acid molecule sufficiently complementary to the sequence on which it is based and of sufficient length to selectively hybridise to the corresponding portion of a nucleic acid molecule intended to be amplified and to prime synthesis thereof under in vitro conditions commonly used in PCR. Likewise, a probe of the present invention, is a molecule, for example a nucleic acid molecule of sufficient length and sufficiently complementary to the nucleic acid molecule of interest, which selectively binds under high or low stringency conditions with the nucleic acid sequence of interest for detection thereof in the presence of nucleic acid molecules having differing sequences. Preferred primers for the detection of a DGATl 232A allele are the following: forward
primer 5'- CGCTTGCTCGTAGCTTTGG -3' and reverse primer: 5'- CGCGGTAGGTCAGGTTGTC -3' (SEQ ID NO:9 and 10 respectively). Preferred probes for the detection of a DGATl allele 232A are the following: VIC MGB-probe: 5'- CGTTGGCC TTCTTAC -3' (detects K allele) and FAM MGB -probe: 5'-TTGGCCGCCTTAC-S' (detects A allele) (SEQ ID NO:11 and 12 respectively). FAM and VIC are fluoresent reporter dyes. The exication wavelength for FAM is 495 nm and the fluorescence has its emission peak at 520 nm, the exication wavelength for VIC is 540 nm and the fluorescence has its emmision peak at 555 nm. The use of fluorogenic probes in the 5 'nuclease assay combines PCR amplification and detection into a single step. In the 5 'nuclease assay, an oligonucleotide probe is included in the PCR amplification reaction along with the forward and reverse primers. If the target sequence of the probe is amplified in the reaction, then the probe will hybridize to this target sequence during the annealing/extension step of PCR. When the DNA polymerase with 5 'nuclease activity (e.g. Taq) encounters the hybridized probe, the probe is cleaved by the 5 'nuclease activity of the DNA polymerase. It is important to note that this cleavage occurs only if the probe is specifically hybridized to its target sequence. By using a fluorogenic probe, cleavage of the probe can be detected without post-PCR processing. The fluorogenic probe consists of an oligonucleotide labelled with both a fluorescent reporter dye and a quencher dye. In the intact probe, proximity of the quencher reduces the fluorescence signal observed from the reporter dye.
Cleavage of the fluorogenic probe during the PCR-assay liberates the reporter dye, causing an increase in its fluorescence intensity. Thus, an increase in reporter fluorescence indicates that the probe-specific target has been amplified (26). Fluorogenic probes and the 5 'nuclease assay can be used for allelic discrimination. For a bi-allelic system, probes specific for each allele are included in the PCR assay. The probes can be distinguished because they are labelled with different fluorescent reporter dyes (e.g. FAM and VIC). A mismatch between probe and target greatly reduces the efficiency of probe hybridization and cleavage. Thus, substantial increase in FAM or VIC fluorescent signal indicates homozygosity for the FAM- or VIC-specific allele. An increase in both signals indicates heterozygosity (26).
Genotyping with fluorogenic probes requires that fluorescence measurements be made after PCR is completed. This is conveniently done in a multicolour real-time PCR
system. Such a systems exicates fluorescent reporter dyes at their respective wavelength (e.g. 495nm for FAM and 540 nm for VIC) and subsequently measures the emitted fluorescence at the respective peak wavelengths (e.g. 520 nm for FAM and 555 nm for VIC). The power of discrimination between alleles is determined by the difference in melting temperature (ΔTm) between match and mismatch probe. The ΔTm is primarily dependent on the type of mutation, together with the length of the probe. Usually, relatively long oligonucleotides are designed that can anneal to their target at elevated (PCR) temperatures. This may result in a small ΔTm discrimination window and difficult allelic discrimination, especially when G to A or G to T mutations are present (27).
Oligonucleotide probes conjugated with a minor groove binder (MGB) ligand have been developed. The MGB has a high affinity for the minor groove of double-stranded DNA and stabilizes the oligoprobe with the complementary single-strand DNA target. These MGB probes have a higher melting temperature (Tm) for a given length. For instance, a 12-mer probe with MGB group had an identical Tm as a 27-mer DNA probe without MGB group. Single base mismatches between target DNA and such short MGB probes significantly decrease the Tm of the duplex resulting in a large ΔTm discrimination window. This means that shorter fluorogenic TaqMan probes can be used thus improving specificity and sensitivity. Furthermore, the use of a non- fluorescent quencher (NFQ) decreases the background fluorescence level (27, 28). In the TaqMan assay for allelic discrimination of a DGATl K232A polymorphism: 100% VIC-fluorescence indicates a homozygote KK, 100% FAM-fluorescence indicates a homozygote AA, and approximately -50% VIC-fluorescence and 50% FAM- fluorescence indicates a heterozygote KA.
In another preferred embodiment, the method used is the SNaPshot single-base extension assay: PCR followed by allele-specific single-base extension as means. This preferred method is extensively presented below taken SCD as example. This method includes a step in which ascertaining whether the A878 V polymorphism is present in the sequence of SCD DNA, includes amplifying the DNA in the presence of primers based on the nucleotide sequence of the SCD gene and flanking sequence, followed by
allele-specific single-base extension of either the SCD 878A allele or the SCD 878V allele.
A primer of the present invention, used in PCR for example, is a nucleic acid molecule sufficiently complementary to the sequence on which it is based and of sufficient length to selectively hybridise to the corresponding portion of a nucleic acid molecule intended to be amplified and to prime synthesis thereof under in vitro conditions commonly used in PCR. Preferred primers for the detection of a SCD 575F aIIeIe are the following: forward primer 5'- TCATTTAACCCCTCATTACCTCA -3' and reverse primer: 5'- TGTAAAATACTAGGCTTTCTGG -3' ( SEQ ID NO: 13 and 14 respectively). Preferred single-base extension primer for the detection of a SCD 878 V allele is the following: 5'- TGGTTTCCCTGGGAGCTG - 3' (SEQ ID NO: 15).
The SNaPshot assay investigates SNP markers by employing PCR amplification followed by dideoxy single-base extension of an unlabeled primer. This unlabeled, single-base extension primer is designed to anneal to the sequence adjacent to the SNP site. Once the primer anneals, the single-base extension occurs by the addition of the complementary dye-labelled ddNTP (dye terminator) to the annealed primer. Each of the four ddNTPs is fluorescently labelled with a different colour dye. The result is marker fragments for the different SNP alleles that are all the same length, but vary by colour. After electrophoresis and fluorescence detection, the alleles of a single marker appear as different colour peaks at roughly the same size in the electropherogram plot. The size of the different allele peaks will vary slightly due to differences in molecular weight of the dyes. Electrophoresis and multicolour fluorescence detection are preferably carried out with a DNA sequencer ( 48-capillary 3730 DNA analyser, Applied Biosystems), and GeneMapper software (Applied Biosystems) is preferably used to size and genotype the data.
The SNaPshot multiplex assay can investigate up to ten SNP markers simultaneously by using single-base extension primers of different lengths for the different markers. It may be necessary to add a non-annealing tail to a primer to make its length sufficiently different from other primers. This prevents different SNP markers from overlapping.
Method
In a further aspect, the invention relates to a method for selecting a non-human mammal, preferably a bovine which produces milk with an increased desaturation index for at least one of C 16, C 17, C 18, and CLA fatty acids and/or with a milk-fat composition with at least one of a reduced content of saturated C 16 fatty acids and an increased content of unsaturated Cl 8 fatty acids.
In a first preferred embodiment, the invention relates to a method for selecting a non- human mammal, preferably a bovine which produces milk with an increased desaturation index for at least one of C 16, C 17, C 18, and CLA fatty acids. In this first preferred embodiment, the index is preferably the CLA index. In a preferred embodiment, the milk has a milk-fat composition with at least one of an increased content of C16:lcis9, C17:lcis9 and CLA and a decreased content of C17:0 and C18:0 fatty acids. In this embodiment, the non-human mammal, preferably a bovine is obtained by applying the method of the invention wherein one screens for the presence of a SCD 878V allele. Therefore, the non- human mammal preferably possesses one allele SCD 878V, more preferably two.
Alternatively to a first embodiment as described above, in a second preferred embodiment, the invention relates to a method for selecting a non -human mammal, preferably a bovine which produces milk having a milk-fat composition with at least one of a reduced content of saturated C16 fatty acids and an increased content of unsaturated Cl 8 fatty acids. Preferably, in this method, the milk- fat composition has a reduced content of saturated C16 fatty acids, an increased content of at least one of C18 cis-isomers such as C18:l cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,\2 (linoleic acid), CLA and C18:3 cis 9,12,15 (linolenic acid). More preferably, the C18 cis- isomers are selected from the group consisting of: C 18:1 cis 9 (oleic acid), C 18:1 cis 11, C18:2 cis 9,12 (linoleic acid), CLA and C18:3 cis 9,12,15 (linolenic acid). Even more preferably, the Cl 8 cis-isomers are selected from the group consisting of oleic acid, linolenic acid, CLA and linoleic acid. In a more preferred embodiment of the method, this non-human mammal, preferably a bovine is obtained by applying the method of the invention wherein one screens for the
presence of a DGATl 232 A allele. Therefore, the non- human mammal preferably possesses one allele DGATl 232 A, more preferably two.
In combination with the first and/or second embodiment as described above, the invention relates to a further preferred embodiment, wherein the milk produced has an increased desaturation index for at least one of C 16, C 17, C18 and CLA fatty acids. In a more preferred embodiment, the index is the CLA index. In an even more preferred embodiment, the milk produced has at least one of a decreased content of saturated C16, C17 and C18 fatty acids and an increased content of unsaturated C16, C17 and Cl 8 fatty acids. In this embodiment, the non-human mammal, preferably a bovine is obtained by applying the method of the invention wherein one screens for the presence of a SCD 878V allele in combination with a DGATl 232 A allele . Therefore, the non- human mammal preferably possesses one allele SCD 878V and one allele DGATl 232 A, more preferably two alleles SCD 878 V and one allele DGATl 232 A, or one allele SCD 878V and two alleles DGATl 232 A and most preferably two alleles SCD 878V and two alleles DGATl 232 A.
All features of this method have already been defined under the section entitled "use".
The method not only enables to identify a non-human mammal, preferably a bovine, producing milk with an improved (preferably healthier) fatty-acid composition. Alternatively or in combination with previous embodiments, the invention provides a further third preferred embodiment wherein the method also enables to select a non- human mammal, preferably a bovine, as a parent of offspring producing milk with an improved fatty acid composition. Mating of such selected parents will result in an increase of the frequency of the alleles that are favorable for an improved fatty acid composition (such as SCD 878V and/or DGATl 232A) in the offspring, thus, result in offspring producing milk with an improved fatty acid composition compared to offspring of parents that were not selected with this method. Selection of parents and mating may include animals of the same breed but also animals of different breeds.
Alternatively or in combination with previous embodiments, the invention provides a further fourth preferred embodiment for the method of the invention wherein the non- human mammal, preferably a bovine producing such milk is obtained by further selecting and/or feeding a specific diet (12). This selection preferably includes the identification and selection of additional favorable alleles and/or further identification and selection of specific breeds. Identification and selection may encompass any method for ensuring that the non-human mammal produces milk with a further improved fatty acid composition (increased unsaturated fatty acid content and decreased saturated fatty acid content by comparison to a non-selected non-human mammal, preferably an increased desaturation index for at least one of C 16, C 17, Cl 8 and CLA fatty acids). For example, one may identify and select within or outside the breed for the presence of additional advantageous alleles for the desired phenotype and mate selected parents to obtain a further improved non-human mammal. In addition, one may also follow a specific feeding method and/or schedule in order to improve the non- human mammal any further.
In an even more preferred embodiment, the method combines second, third and/or fourth preferred embodiments for selecting a non-human mammal, preferably a bovine which produces milk having a milk-fat composition with a reduced content of saturated C16 fatty acids, an increased content of unsaturated C18 fatty acids and a control content of saturated C 14 fatty acid.
In another even more preferred embodiment, the method combines former first, second, third and/or fourth preferred embodiments for selecting a non-human mammal, preferably a bovine which produces milk having a milk-fat with an increased desaturation index for at least one of C 16, C 17, Cl 8 and CLA fatty acids.
Milk
In a further aspect, the invention relates to milk or milk-fat composition obtainable from the non- human mammal, preferably a bovine obtained by the method of the invention or by the use of the invention both as defined earlier herein. The milk of the
invention is therefore much more healthy than the milk of control non-human mammal, preferably control bovine milk.
In a first preferred embodiment (presence of a SCD 575FaIIeIe) , the milk has an increased desaturation index for at least one of C 16, C 17,Cl 8 and CLA fatty acids. In this first preferred embodiment, the index is preferably the CLA index. In a more preferred embodiment, the milk has a milk-fat composition with at least one of an increased content of C16:lcis9, C17:lcis9 and CLA and a decreased content of C 17:0 and C 18:0 fatty acids. In a second preferred embodiment (presence of a DGATl 232 A allele), the milk has a milk- fat composition with at least one of a reduced content of saturated C16 fatty acids and an increased content of unsaturated C18 fatty acids. Preferably, the milk obtained has a milk-fat composition with a reduced content of saturated C16 fatty acids, an increased content of at least one of Cl 8 cis-isomers such as C 18:1 cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid) and/or C18:3 cis 9,12,15 (linolenic acid) and/or an increased content of CLA. Even more preferably, the C18 cis-isomers are selected from the group consisting of oleic acid, linolenic acid, CLA and linoleic acid. Even more preferably, the milk has a milk-fat composition with at least one of a reduced content of saturated C 16 fatty acids, an increased content of unsaturated Cl 8 fatty acids and a control content of saturated C 14 fatty acid.
In a third preferred embodiment (combination of the first and second preferred embodiments: presence of a SCD 878V and a DGATl 232A allele), the milk produced has further an increased desaturation index for at least one of C 16, C 17,Cl 8 and CLA fatty acids. In a more preferred embodiment, the index is the CLA index. In an even more preferred embodiment, the milk produced has at least one of a decreased content of saturated C 16, C17 and Cl 8 fatty acids and an increased content of unsaturated C 16, C17 and Cl 8 fatty acids.
Product The skilled person will understand that all products prepared using this milk and/or milk-fat composition and/or products derived from this milk and/or milk-fat composition and/or comprising this milk and/or milk-fat composition are also
encompassed by the scope of the present invention. Such products include all milk- based and/or milk-derived products such as yoghurt, butter or cheese. Such products further include food products that comprise a milk and/or milk-fat composition containing ingredient such as bakery products, chocolate or ice cream. In a preferred embodiment, the invention relates to a food product comprising a milk-fat composition derived from the milk of the invention.
In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".
The invention is further decribed by the following examples which should not be construed as limiting the scope of the invention.
Brief description of the figures
Figure 1 shows the relationship between available substrate (substrate yield) in one morning milk sample and desaturation index for C 14. The substrate yield is the amount of substrate that was available before desaturation took place. This is the amount of C14:0 plus the amount of C14:lcis9 that was measured in the morning milk sample (mean 13.47 kg milk per sample). Figure 1 shows that there is no effect of substrate yield on the desaturation index for C 14, thus, the desaturation index is not affected by the amount of substrate that is available. This holds also true for the desaturation indexes of ClO, C12, C16, C17, C18 and CLA.
Figure 2 shows the relationship between the C 14 desaturation index and the C 16 desaturation index. Three groups can be discriminated. The phenotypes that were used to calculate the desaturation indexes were corrected for fixed effects, such as herd, days in milk, age at calving and season of calving. Therefore, the three groups might well
demonstrate a genetic effect. The three groups overlap for 94% with the genotypes of the SCD A878V polymorphism. This indicates that the SCD A878V polymorphism underlies the three groups that can be discriminated in Figure 2.
Examples
Example 1: DGA T 232 A
The fat composition of winter milk samples was measured in 1918 Dutch Holstein Friesian cows in their first lactation. The average milk-fat percentage is 4.36 (Table 1). The most abundant fatty acid is C 16:0, which accounts for about 33% of total fat (Table 2). The unsaturated C18 fatty acids (C18u), of which oleic acid (C 18:1 cis 9) is the principal one, account for more than 21% of total fat. Trans fatty acids contribute 1.54%. The ratio of saturated to unsaturated fatty acids (SFA/UFA) averages 2.80, meaning that saturated fatty acids account for about 70% of total fat. The coefficient of variation (CV) is highest (28%) for conjugated linoleic acid (CLA) and trans fatty acids, and lowest (about 9%) for C14:0, C16:0, and C4:0-C12:0.
We computed heritabilities to estimate what proportion of total phenotypic variation is additive genetic variation, i.e. heritable. Heritability for fat percentage is high (0.51), meaning that about half of the total variation is additive. Short- and medium-chain fatty acids (C4-C16) also have high heritabilities, ranging from 0.43 to 0.59. Saturated and unsaturated Cl 8 show lower heritabilities (around 0.25), as does the trans fatty acids (0.20).
Heritabilities indicate that genetic variation for milk-fat composition is present and, therefore, that selective breeding to alter composition is possible. We computed genetic correlations to estimate to what extent different traits are influenced by the same genes (table 3). Fat percentage shows a positive genetic correlation with C 16:0 (0.65) and a negative genetic correlation with unsaturated C18 (-0.72), CLA (-0.58), and C14:0 (-0.43). These results imply that selection for an increasd milk-fat percentage will lead to a correlated response in fat composition; increased fat percentage will lead to a correlated increase in the fraction of C 16:0 and a correlated decrease in the fractions of unsaturated C 18, CLA and C 14:0. Genetic correlations for fat yield show similar directions as genetic correlations for fat percentage. C 16:0,
which is the most abundant and most unfavorable fatty acid from a nutritionist's point of view, shows a high negative genetic correlation with C14:0 (-0.84), CLA (-0.59), and unsaturared C18 (-0.53).
To study the effect of the DGATl K232A mutation, a total of 1762 cows was genotyped for this polymorphism. The frequency of the K allele was 0.40. The estimated effects of this polymorphism on milk-production traits are in table 4. The K allele is associated with increased fat percentage, protein percentage and fat yield, whereas it is associated with decreased milk yield and protein yield. Interestingly, the K allele leads to an increase in the fraction of C 16:0 and the ratio SF A/UFA, whereas it leads to a decrease in the fractions of C14:0, unsaturated C18 and CLA (Table 5). The DGATl K232A mutation explains large proportions of the genetic variance: 50% for fat percentage, 53% for unsaturated C 18, 40% for C 16:0, and 36% for SF A/UFA. Effects of DGATl on fat composition are in line with expectations based on the effect of DGATl on fat percentage and the genetic correlations between fat percentage and fat composition (Table3). The effect of the DGATl K232A mutation on all saturated and respectively unsaturated fatty acid composition is given in Tables 6 and 7 respectively. It can be observed from table 6 that the DGATl K232A mutation has a statistically significant effect (reduction) on C5, C6, C7, C8, C9, CI l, C13, C15, C16 and C17 saturated fatty acids. In parallel, from table 7, it can be observed that the DGATl K232A mutation has a statistically significant effect (increase) on C12:l, C18 (C18:l cis9, C 18:1 cisM, C 18:1 transβ, C 18:1 trans9, C 18:1 transl l, linoleic acid, CLA, and linolenic acid) unsaturated fatty acids.
DISCUSSION We have demonstrated the existence of substantial genetic variation in milk-fat composition within a dairy cattle population. Heritabilities were high for the short- chain saturated fatty acids C4:0-C12:0 (0.59), and for the medium-chain fatty acids C14:0 (0.59) and C16:0 (0.43). Heritabilities for the long-chain C18 fatty acids were lower, around 0.25. This contrast may be explained by the dual origins of fatty acids in the milk. Short-chain and medium-chain fatty acids, C4:0 to C14:0 and also some C 16:0, are synthesized de novo in the mammary gland and may be influenced by genetics. The C18 fatty acids and, to a lesser extent, C 16:0 arise from the cow's plasma
lipids and have a dietary origin (12); they are, therefore, likely to be under less genetic control. Heritability for trans fatty acids was also low (0.20). This low heritability may be because trans fatty acids are produced by microbial biohydrogenation in the rumen of the cow (33), which may be under less genetic control as well. Heritabilities for milk, protein and fat yield and protein and fat percentage are generally in line with previous studies (13-15, 34, 35). Genetic parameters for fat composition were reported only previously by Karijord et al. (20), who estimated lower heritabilities.
Estimated effects of the DGATl K232A mutation on fat percentage, protein percentage, and yield traits are consistent with previous studies (23, 36, 37). The K allele increases fat percentage, protein percentage, and fat yield, whereas it decreases milk yield and protein yield. Ours is the first report on the effects of the DGATl K232A mutation on milk-fat composition. We show that the K allele is associated with a larger fraction of C16:0; smaller fractions of C14:0, unsaturated C18, and CLA; and a higher ratio SF A/UFA. DGATl catalyses the last step in triglyceride synthesis: the esterification of a fatty acyl-CoA to the sn-3 position of a diacylglycerol. The effect of the DGATl K232A mutation on fat composition may have different causes: a higher activity of DGATl and alteration of specificity of DGATl. For the first, using a baculovirus expression system, the DGATl K allele has been shown to have a higher Vmax than the A allele in producing triglycerides, which is consistent with the in vivo effect of the K232A mutation (38). For the second, DGATl seems to have a preference for short-chain and unsaturated fatty acids, since the sn-3 position of the glycerol backbone is occupied predominantly by these fatty acids (39). Specificity has been shown for other acyltransferases (40, 41 ). The K232A mutation could alter the specificity of the DGATl enzyme, which may result in a change in fat composition. Genetic correlations show that an increase in fat percentage implies an increase in the fraction of C 16:0, while decreasing the fractions of unsaturated C 18, CLA, and C 14:0. It is likely that selection in the past decades in the Dutch dairy has resulted not only in increased fat percentage and fat yield, but also in a more saturated fat composition, with more C16:0, less unsaturated C18, less CLA, and less C14:0. Our results show that it is possible to change fat composition of milk-fat by selective breeding, and that efficiency of selective breeding can be improved using the K232A polymorphism in DGATl. From a public health point of view, increasing the frequency
of the DGATl A allele is desirable because of its association with more unsaturated milk-fat, less C16:0, and more unsaturated C18.
MATERIALS AND METHODS Animals
This study is part of the Milk Genomics Initiative, which focuses on the genetic background of detailed milk composition. As part of this study, morning milk samples and blood samples were collected from 1918 first lactation cows on 398 commercial herds in The Netherlands. At least three cows per herd were sampled; cows were milked twice a day. Cows descended from one of fifty young bulls (843 cows), from one of five proven bulls (888 cows), or from other proven bulls (187 cows). The NRS (Arnhem, the Netherlands) provided the pedigree of the cows. Each cow was over 87.5 percent Holstein-Friesian, and was in lactation between Day 63 and Day 263.
Phenotypes
A 0.5 liter milk sample was collected from each cow at one morning milking between February and March 2005. Milk- fat composition was measured at the laboratory of COKZ (Leusden, the Netherlands). Milk-fat was extracted from the milk samples, and fatty acid methyl esters were prepared from fat fractions, as described in ISO Standard 15884 (29). Methyl esters were analyzed according to ISO Standard 15885 (30) on a Trace GC Ultra chromatograph (Thermo Electron Corporation, Waltham, MA, USA), using a Varian Fame Select column (100m x 0.25mm ID, Varian Inc. , Palo Alto, CA, USA). The initial temperature was held at 7O0C for 1 min, raised to 2250C at 3°C/min, and held at 2250C for 5 min. A volume of 1 μl was injected. Each peak was identified and quantified using pure methyl ester samples (Sigma-Aldrich, Zwijndrecht, the Netherlands; Larodan, Malmo, Sweden). The fatty acids included in this study were grouped according to their relevance to human nutrition and health. Fat and protein percentage were determined by infra red spectroscopy, using a MilkoScan FT6000 (Foss Electric, Hillerod, Denmark) at the Milk Control Station (Zutphen, the Netherlands). Fat and protein yields were calculated by multiplying each percentage by milk yield. Yield data were missing for 135 cows.
Genotypes
Blood samples for DNA isolation were collected between April and June 2005. Genotyping of the DGATl K232A dinucleotide polymorphism was performed using a Taqman allelic discrimination method in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The primers and labeled oligonucleotide probes for this reaction were: forward, 5'- CGCTTGCTCGTAGCTTTGG -3'; reverse, 5'- CGCGGTAGGTCAGGTTGTC -3'; VIC probe (detects ^ allele ), 5'- CGTTGGCCTTCTTAC -3'; FAM probe (detects A allele), 5'-TTGGCCGCCTTAC-S' (SEQ ID NO: 5, 6, 7 and 8 respectively). PCR cycling conditions were 94°C for 5 min, 40 cycles of 92°C for 15 s and 600C for 1 min. About 7% of samples were genotyped in duplicate and repeatability was 100%. In total, 1762 animals were genotyped. Genotypes were missing for 156 animals, because either no DNA sample was available (n=144) or the sample could not be genotyped unambiguously (n=12).
Statistical analysis
Variance components and genetic parameters were estimated using an Animal Model in
ASReml (31):
yykinm = μ + bi *dim1 + b2*e~° 05*dim +b3*afc, + b4*afc,2 + seasonk + scodei + herdm+ Un +
Syklmn where y is the dependent variable, μ is the general mean, dim is the covariate describing the effect of days in milk modelled with a Wilmink curve (32), afc is the covariate describing the effect of age at first calving, season is the fixed effect of the class of calving season (June -August 2004, September-November 2004, or December 2004-February 2005), scode is the fixed effect of the differences in genetic level between groups of proven bull daughters and young bull daughters, herd is the random effect of groups of animals sampled in the same herd, Un is the random additive genetic effect of animal n, and e is the random residual effect. Effects of the DGATl K232A mutation were estimated using the same model, but extended with g: the fixed effect of the DGATl genotype (KK, KA, AA). Ungeno typed individuals were included as a separate group, and appeared to be random.
The variance-covariance structure of the additive genetic effects is Var(U)=Aσu 2, where A is a matrix of additive genetic relationships between individuals and σu 2 is the additive genetic variance. Heritabilities were estimated using univariate analyses, and phenotypic and genetic correlations were estimated using bivariate analyses.
Example 2: SCD 878V and optionally DGAT 232A Material and methods
Animals
This study is part of the Dutch Milk Genomics Initiative, which focuses on the genetic background of detailed milk composition. As part of this study, morning milk samples and blood samples were collected from 1,933 first-lactation cows on 398 commercial herds in the Netherlands. At least three cows per herd were sampled and cows were milked twice a day. Cows descended from one of fifty young bulls (845 cows), from one of five proven bulls (897 cows), or from other proven bulls (191 cows). The NRS (Arnhem, the Netherlands) provided pedigrees of the cows and the milk yield records. Each cow was over 87.5 percent Holstein-Friesian and was in lactation between Day 63 and Day 282.
Phenotypes A 0.5 liter milk sample was collected from each cow at one morning milking between February and March 2005. Milk- fat composition was measured at the COKZ laboratory (Netherlands Controlling Authority for Milk and Milk Products, Leusden, the
Netherlands) as described by Schennink et al (25). Milk-fat was extracted from the milk samples, and fatty acid methyl esters were prepared from fat fractions, as described in ISO Standard 15884 (29). Methyl esters were analyzed according to ISO Standard 15885 (30) on a Trace GC Ultra chromatograph (Thermo Electron
Corporation, Waltham, MA, USA), using a Varian Fame Select column (100m x 0.25mm ID, Varian Inc. , Palo Alto, CA, USA). The initial temperature was held at 7O0C for 1 min, raised to 2250C at 3°C/min, and held at 2250C for 5 min. A volume of 1 μl was injected. Each peak was identified and quantified using pure methyl ester samples (Sigma-Aldrich, Zwijndrecht, the Netherlands; Larodan, Malmo, Sweden). The fatty acids included in this study were grouped according to their relevance to human nutrition and health. Desaturation indexes were defined as: desaturation index = (unsaturated product / (substrate for desaturation + unsaturated product))* 100 . Fat and protein percentage were determined by infra red spectroscopy, using a MilkoScan FT6000 (Foss Electric, Hillerod, Denmark) at the Milk Control Station (Zutphen, the Netherlands). Fat and protein percentages were measured by infra red spectroscopy, using a MilkoScan FT6000 (Foss Electric, Hillerod, Denmark) at the Milk Control Station (Zutphen, the Netherlands). Fat and protein yields were calculated by multiplying each percentage by the milk yield. Yield data were missing for 145 cows.
Genotypes
Genotypes for the SCD A 878 V polymorphism were assayed by SNaP shot single base primer extension method (Applied Biosystems, Foster City, CA, USA). The primer designs were based on the Genbank sequence (AY241932): forward, 5'- TCATTTAACCCCTCATTACCTCA -3'; reverse, 5'-
TGTAAAATACTAGGCTTTCTGG -3'; genotyping primer, 5'- TGGTTTCCCTGGGAGCTG - 3'. TO amplify the SCD fragment, 12 μl reactions were set up containing 20 ng of genomic DNA, 0.2 μM of each primer and 2X AccuPrime Supermix II (Invitrogen, Carlsbad, CA, USA). PCR cycling conditions were 94°C for
5 min, 36 cycles of 94°C for 30 s, 55°C for 45 s, 68°C for 90 s, followed by an extension cycle of 68°C for 10 min. PCR products were purified by incubation with shrimp alkaline phosphatase (SAP) (USB, Cleveland, OH, USA) and Exo I (USB) at 37°C for 1 h and 72°C for 15 min. Extension reactions, using 3 μl of purified PCR product and 5 pmol of genotyping primer and SNaPshot multiplex Ready reaction mix (Applied Biosystems), were performed using 40 cycles of 96°C for 10 s, 500C for 5 s, and 600C for 30 s. The extension products were incubated with SAP at 37°C for 1 h and 72°C for 15 min. Two microliters of extension product were added to 8 μl of Hi-Di formamide and electrophoresed on an ABI 3730 DNA analyzer. Results were analyzed using the GeneMapper Software v4.0 (Applied Biosystems). In total, 1,725 animals were genotyped for the SCD A 878 V polymorphism.
Genotyping of the DGATl K232A dinucleotide polymorphism was performed using a Taqman allelic discrimination method in an Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems). The primers and labeled oligonucleotide probes for this reaction were designed based on the DGATl sequence (Genbank accession no. AY065621): forward, 5'- CGCTTGCTCGTAGCTTTGG -3'; reverse, 5'- CGCGGTAGGTCAGGTTGTC -3'; VIC probe (detects K allele), 5'- CGTTGGCCTTCTTAC -3'; FAM probe (detects A allele), 5'-TTGGCCGCCTTAC-S'. PCR cycling conditions were 94°C for 5 min, 40 cycles of 92°C for 15 s and 600C for 1 min. In total, 1779 animals were genotyped for the DGATl K232A polymorphism.
Statistical analysis
Analyses were performed first using SAS 9.1 (2002) procedures to determine fixed effects. The model should include stage of lactation (days in milk = time between
calving and date of sample), age at first calving, season of calving, and an effect of the differences in genetic level between groups of proven bull daughters and young bull daughters. Variance components and genetic parameters were estimated using an Animal Model in ASReml (31).
yijkiπm = μ + bi *dim1 + b2*e~° 05*dim +b3*afc, + b4*afc,2 + seasonk + scodei + herdm+ Un +
Syklmn
where y was the dependent variable, μ was the general mean, dim was the covariate describing the effect of days in milk modelled with a Wilmink curve (32), afc was the covariate describing the effect of age at first calving, season was the fixed effect of the class of calving season (June -August 2004, September-November 2004, or December 2004-February 2005), scode was the fixed effect of the differences in genetic level between groups of proven bull daughters and young bull daughters, herd was the random effect of groups of animals sampled in the same herd, Un was the random additive genetic effect of animal n, and e was the random residual effect. Effects of the SCD A 878 V or the DGATl K232A mutation were estimated using the same model, but extended with effect g: the fixed effect of the SCD genotype (AA, AV, W) or the fixed effect of the DGATl genotype (KK, KA, AA). Ungenotyped individuals were included as a separate group, and appeared to be random.
The variance-covariance structure of the additive genetic effects was Var(U)=Aσu 2, where A was a matrix of additive genetic relationships between individuals and σu 2 was the additive genetic variance. Heritabilities were estimated using univariate analyses, and phenotypic and genetic correlations were estimated using bivariate analyses.
Summary:
This study aims to identify genetic variation in desaturation of cow's milk. Milk- fat composition was measured on milk samples from 1933 Dutch Holstein Friesian cows using gas chromatography on fatty acid methyl esters (GC-FAME). Desaturation indexes were defined as:
desaturation index = (unsaturated product / (substrate for desaturation + unsaturated product)) * 100.
Means and coefficients of variation (CV) of cis9 desaturation indexes are in Table 8. Different fatty acids have different levels of desaturation: Cl 8 is desaturated the most (67.6%) and C12 the least (2.7%). The CV vary from 6% for C18 index to 20% for C12 index. Figure 1 shows that there is no substrate effect on the rate of desaturation of C14. This holds also true for the other indexes.
To visualize the relationship between the different indexes, they can be plotted in a graph. Figure 2 shows the C 14 and the C16 indexes (corrected phenotypes). Remarkably, we can discriminate three groups. Since we corrected phenotypes for herd and other effects, like DIM, age of calving, season of calving, the three groups might well represent a genetic effect.
Heritabilities were moderate to high for desaturation indexes, whereas herd effects were generally small (Table 9). The ratio of genetic variance to herd variance showed that for all traits genetic effects were much larger than herd effects.
Phenotypic and genetic correlations between desaturation indexes are in Table 10. Genetic correlations were high and positive between the medium chain ClO, C 12, and C14 indexes and between the long chain C17, C18 and CLA indexes. Correlations between medium chain and long chain indexes were much lower.
A candidate gene for desaturation was identified as stearoyl- Co enzyme A desaturase (SCD), which is an enzyme responsible for the conversion of saturated fatty acids to unsaturated fatty acids by introducing a cis double bond at the 9 position. We genotyped a SNP in exon 5 of SCD, causing a VaI to Ala amino acid substitution. This SNP is known to affect mono -unsaturated fatty acids (MUFA) percentage and melting point in intramuscular fat of Japanese black cattle (Taniguchi et al 2004, Mamm. Genome 15: 142-148). When we compare these genotypings with the three groups found as a result of Figure 2, they overlap for 94%. This high resemblance indicates that the genotyped mutation in the SCD gene (or one in LD with this mutation) underlies the three groups seen in Figure 2. In other words: based on the pheno types genotypes can be predicted highly reliable.
Effects of the SCD A 878 V polymorphism on milk production traits and desaturation indexes are shown in Table 11 and 12. The polymorphism had no effect on fat percentage or fat yield, nor on the overall ratio saturated to unsaturated fatty acids
(SFA/UFA). However, it has significant effects on all separate indexes. The V allele is associated with a lower index for ClO, C 12 and C 14, but is associated with a higher index for C 16, C 17, Cl 8 and CLA.
The polymorphism in the gene DGATl, known to have a clear influence on milk- fat composition, is also associated with desaturation indexes (Table 13). This indicates that not only the conversion of saturated to unsaturated fatty acids by SCD is determinative for saturation of milk- fat, but also the esterification of specific fatty acids to glycerol by DGATl. The percentages of the genetic variance explained by the SCD A 878 V and DGATl K232A polymorphisms are in Table 14. Correction for the SCD genotype doesn't notably change the effect (size) of the DGATl genotype, meaning that the two genes explain a different part of the variation.
Table 15 provides the combined effects of the SCD A 878 V polymorphism and the DGATl K232A polymorphism on fatty acid composition and desaturation indexes. For all indexes except C16 and C 17, the effect of the SCD A 878 V polymorphism and of the DGATl K232A polymorphism are mostly additive and in the same direction.
Consequently, the combined homozygous genotypes have progressively decreasing effects on the ClO, C 12 and C 14 indexes, and progressively increasing effects on the Cl 8 and CLA indexes. For C16 the effect of the SCD A 878 V polymorphism and of the DGATl K232A polymorphism are mostly additive, but in opposite directions. The effect of the SCD A878V polymorphism is larger than the effect of the DGA Tl K232A polymorphism. As a result, the combination of homozygous SCD 878 A and homozygous DGATl 232 A has the most decreasing effect on the C16 index, and the combination of homozygous SCD 878 V and homozygous DGATl 232K has the most increasing effect on the C16 index. The combination of homozygous SCD 878 V and homozygous DGATl 232 A has an increasing effect on the C16 index also. For C17 the effect of the DGATl K232A polymorphism is not significant, therefore, does not add to the effect of the SCD A 878 V polymorphism.
TABLES Table 1. Mean, coefficient of variation (CV), heritability (h ) and additive genetic standard deviation
(σu) of milk-production traits, measured on 1 morning milk sample of 1,918 first- lactation Dutch Holstein Friesian cows
Table 2. Mean, coefficient of variation (CV), heritability (h ) and additive genetic standard deviation (σu) of groups of fatty acids, measured on 1 morning milk sample of 1,918 first-lactation Dutch Holstein Friesian cows
Trait mean (% w/w) CV (%) (se) σu
C4:0-C12:0a 14.24 9 O.59(oii) 0.83
C14:0 11.62 8 O.59(oii) 0.66
C16:0 32.61 9 O.43(oii) 1.57
C18:0 8.73 16 O.23(oo7) 0.61
C18ub 21.58 11 0.26 (oo9) 1.02
CLAC 0.39 28 O.42(oo9) 0.05 transd 1.54 28 0.20 (oo8) 0.15
SFA/UFAe 2.80 13 0.28 (oo9) 0.16
a) C4:0-C12:0 includes saturated fatty acids C4:0, C6:0, C8:0, C10:0 and C12:0. b) C18u includes unsaturated Cl 8 fatty acids: Cl 8:1 trans 6, Cl 8:1 trans 9, Cl 8:1 trans 11, Cl 8:1 cis 9, C18:l cis 11, C18:2 cis 9,12, C18:3 CM 9,12,15. c) CLA: Cl 8:2 cis 9, trans 11. d) trans includes C16:l trans 9, C18:l trans 4-8, C18:l trans 9, C18:l trans 10, C18:l trans 11, C18:l trans 12. e) SFA (saturated fatty acids): C4:0, C5:0, C6:0, C7:0, C8:0, C9:0, C10:0, Cl 1:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, C18:0; UFA (unsaturated fatty acids): C10:l, C12:l, C14:l, C16:l, C18u, CLA.
Table 3. Phenotypic (below diagonal) and genetic (above diagonal) correlations'1 between groups of fatty acids, fat percentage, and fat yield
Trait C4:0- C14:0 C16:0 C18:0 C18u CLA Fat % Fat yield
C12:0
C4:0- 0.63 -0.48 -0.22 -0.37 0.07 0.20 0.35
C12:0
C14:0 0.66 -0.84 -0.34 0.23 0.33 -0.43 -0.11
C16:0 -0.22 -0.35 0.27 -0.53 -0.59 0.65 0.18
C18:0 -0.08 -0.22 -0.28 -0.41 -0.58 0.01 0.18
C18u -0.45 -0.23 -0.66 0.06 0.71 -0.72 -0.35
CLA -0.22 -0.02 -0.34 -0.35 0.58 -0.58 -0.30
Fat % 0.1 -0.27 0.43 0.08 -0.42 -0.32 0.51
Fat yield 0.23 0.01 0.22 -0.05 -0.29 -0.22 0.45
a) Standard errors of phenotypic correlations were between 0.02 and 0.03. Standard errors of genetic correlations were between 0.08 and 0.23.
Table 4. Effect of the DGATl K232A mutation on milk production traits
KK KA (se) AA (se)
Trait (n=289) (n=829) (n=644) P value" r genetic%
Milk yield
0 0.84 (o i6) 1.46 (oi8) <0.001 22 (kg)
Fat yield (kg)
0 -O.O2(ooi) -O.O7(ooi) <0.001 22
Protein yield
0 O.O2(ooi) O.O2(ooi) <0.001 14 (kg)
Fat (%)
0 -O.45(oo4) -O.98(oo4) <0.001 50
Protein (%)
0 -0.10(002) -O.25(oo2) <0.001 22
a) KA: contrast of KA -KK genotypes. b) AA: contrast of AA -KK genotypes. c) P value: statistical significance of the DGATl K232A effect. d) r2 genetlc%: percentage of the genetic variance explained by the DGATl K232A mutation.
KK KA" (ge) AA (se)
Trait (n=289) (n=829) (n=644) P value" r genetic%
C4:0-C12:0 0 0-16 (oo7) 0.03 (oo8) 0.05 1
C14:0 0 O.43(oo6) O.79(oo6) <0.001 23
C16:0 0 -1.02 (oi6) -2.52 (o i7) <0.001 40
C18:0 0 -0.18 (oo9) -O.lO(oio) 0.18 1
C18u 0 0.80 (o i4) 2.12 (o is) <0.001 53
CLA 0 O.O2(ooi) O.O5(ooi) <0.001 16
Trans 0 -0.01 (oo2) 0.04(003) 0.03 2
SFAAJFA 0 -0.11 (002) -O.27(oo2) <0.001 36
Table 5. Effect of the DGATl K232A mutation on fatty acid composition a) KA: contrast of KA -KK genotypes. b) AA: contrast of AA -KK genotypes. c) P value: statistical significance of the DGATl K232A effect. d) r2 genetlc%: percentage of the genetic variance explained by the DGATl K232A mutation.
Table 6. Effect of the DGATl K232A mutation on fatty acid composition (saturated fatty acids)
Mean KK KA" (ge) AAC (se)
Trait % (w/w) (n=289) (n=829) (n=644) P value"
C4:0 3.502 0 -0.018(0018) O.OO5(ooi9) 0.221
C5:0 0.029 0 -O.OO5(oooi) -O.OO9(oooi) <0.001
C6:0 2.225 0 -O.O18(ooio) -O.O62(ooii) <0.001
C7:0 0.031 0 -O.OO4(oooi) -O.O12(oooi) <0.001
C8:0 1.367 0 O.OO4(ooo9) -O.O28(ooo9) <0.001
C9:0 0.045 0 -O.OO6(oooi) -O.O16(oooi) <0.001
C10:0 3.032 0 0.065(0026) 0.014(0028) 0.016
Cl l :0 0.079 0 -O.OO8(ooo2) -O.O22(ooo2) <0.001
C12:0 4.113 0 0.129(0037) 0.097(0040) 0.003
C13:0 0.112 0 -O.OO7(ooo3) -O.O19(ooo3) <0.001
C14:0 11.620 0 0.427(0O55) 0.788(0060) <0.001
C15:0 1.176 0 -O.O65(ooi2) -O.118(ooi3) <0.001
C16:0 32.610 0 -l.O21(o 160) -2.524(o 173) <0.001
C17:0 0.455 0 -O.O14(ooo3) -O.OO8(ooo4) <0.001
C18:0 8.726 0 -U.I /Omnon -U.1U-Zm ΠQSΛ 0.176
a) KA: contrast of KA -KK genotypes. b) AA: contrast of AA -KK genotypes. c) P value: statistical significance of the DGATl K232A effect.
Table 7. Effect of the DGATl K232A mutation on fatty acid composition (unsaturated fatty acids)
Mean KK KA (se) AA (se)
Trait % (w/w) (n=289) (n=829) (n=644) P value"
C10:l 0.367 0 -O.OOl(ooo4) -O.O17(ooo5) <0.001
C12:l 0.115 0 O.OOl(ooo2) -O.OO5(ooo2) <0.001
C14:l 1.346 0 -O.OO9(ooi7) -O.O49(ooi8) 0.009
C16:l 1.443 0 -O.136(oo2o) -O.317(oo22) <0.001
C17:l 0.180 0 -O.OO3(ooo2) -O.OO2(ooo2) 0.374
C18:lcis9 (oleic acid) 18.020 0 O.687(oi2i) 1.804(0I3O) <0.001
C18:lcisll 0.413 0 O.O19(ooo6) O.O39(ooo6) <0.001
C18:ltrans6 0.214 0 O.OO6(ooo3) O.O18(ooo3) <0.001
C18:ltrans9 0.149 0 O.OO4(ooo2) O.Oll(ooo2) <0.001
C18:ltransll 0.773 0 -O.OO6(ooio) O.O27(ooii) <0.001
C18:2cis9,12 1.200 0 O.O65(ooi4) O.133(ooi6) <0.001 (linoleic acid) C18:2cis9transll (CLA) 0.393 0 O.O17(ooo6) O.O49(ooo6) <0.001
C18:3cis9,12,15 0.414 0 O.O15(ooo5) O.O43(ooo5) <0.001 (linolenic acid)
a) KA: contrast of KA -KK genotypes. b) AA: contrast of AA -KK genotypes. c) P value: statistical significance of the DGATl K232A effect.
Table 8. Mean and coefficient of variation (CV) for fatty acid desaturation indexes, measured on one morning milk sample of 1933 first-lactation Dutch Holstein Friesian cows
Trait Mean CV (%)
ClO index ΪO9 Ϊ7
C12 index 2.7 20
C14 index 10.5 17
C16 index 4.2 19
C17 index 28.3 11
Cl 8 index 67.6 6
CLA1 index 33.7 12
1 CLA: C18:2 cώ9, transW
Table 9. Heritability (h2), herd effect (hherd), and ratio between additive genetic variance and herd variance (σV σ2herd) for fatty acid desaturation indexes, measured on one morning milk sample of 1933 first-lactation Dutch Holstein Friesian cows
Trait h2 hherd σ A/ σ herd
ClO index 0.37 ± 0.09 0.06 ± 0.02 5.5
C 12 index 0.37 ± 0.09 0.06 ± 0.02 5.6
C 14 index 0.45 ± 0.09 0.06 ± 0.02 6.6
C16 index 0.46 ± 0.09 0.07 ± 0.02 6.2
C17 index 0.46 ± 0.10 0.19 ± 0.02 1.9
Cl 8 index 0.33 ± 0.08 0.06 ± 0.02 5.1
CLA1 index 0.23 ± 0.07 0.09 ± 0.02 2.5
CLA: C\8:2 cis9, trans W
Table 10. Phenotypic (below diagonal) and genetic (above diagonal) correlations1 between fatty acid desaturation indexes
errors of genetic correlations were between 0.02 and 0.20. 2 CLA: C\8:2 cis9, transW
Table 11. Effect of the SCD A 878 V polymorphism on milk production traits
AA VA1 W2
Trait Mean (n=919) (n=689) (n=117) P value3
Fat (%) 4.36 0 0.02 ± 0.04 0.05 ± 0.07 0.36
Protein (%) 3.51 0 0.00 ±0.01 -0.03 ± 0.03 0.22
Fat yield (kg) 0.58 0 0.00 ±0.01 0.00 ±0.01 <0.01
Protein yield 0.47 0 0.00 ±0.00 -0.01 ±0.01 0.02
(kg)
Milk yield 13.46 0 0.02 ±0.12 -0.20 ± 0.24 0.03
(kg)
Fat (%) 305d 4.32 0 0.01 ±0.02 0.04 ±0.05 0.63
Protein (%) 3.46 0 -0.01 ±0.01 -0.01 ±0.02 0.85
305d
Fat yield (kg) 326.00 0 3.33 ±2.12 3.34 ±4.16 0.04
305d
Protein yield 262.00 0 1.44 ±1.67 -2.14 ±3.27 0.11
(kg) 305d
Milk yield 7602.00 0 58.02 ±54.07 -55.79 ±106.00 0.29
(kg) 305d contrast of VA-AA genotypes, contrast of W-AA genotypes, statistical significance of the SCD A 878 V polymorphism.
Table 12. Effect of the SCD A878V polymorphism on fatty acid composition and desaturation indexes
AA VA1 VV2
Trait Mean (n=919) (n=689) (V1=Ul) P value3
C4:0 3.50 0 -0.02 ±0.01 0.01 ±0.03 0.02
C6:0 2.22 0 0.01 ±0.01 0.02 ± 0.02 0.27
C8:0 1.37 0 0.01 ±0.01 0.02 ±0.01 0.16
C10:0 3.03 0 0.10 ±0.02 0.16 ±0.04 <0.001
C10:l 0.37 0 -0.03 ± 0.00 -0.06 ±0.01 <0.001
C12:0 4.11 0 0.09 ±0.03 0.15 ±0.06 <0.01
C12:l 0.12 0 -0.01 ± 0.00 -0.02 ±0.00 <0.001
C14:0 11.61 0 0.22 ± 0.04 0.42 ± 0.09 <0.001
C14:lc9 1.36 0 -0.17 ±0.01 -0.33 ± 0.02 <0.001
C16:0 32.59 0 -0.12 ±0.13 -0.25 ± 0.25 0.58
C16:lc9 1.44 0 0.17 ±0.02 0.34 ±0.03 <0.001
C17:0 0.45 0 -0.01 ±0.00 -0.02 ±0.00 <0.001
C17:lc9 0.18 0 0.01 ±0.00 0.02 ± 0.00 <0.001
C18:0 8.73 0 -0.30 ±0.07 -0.43 ±0.13 <0.001
C18:lc9 18.04 0 O.IO±O.IO 0.19±0.19 0.44
CLA4 0.39 0 0.01 ± 0.00 0.02 ±0.01 <0.01
SFAAJFA5 2.79 0 -0.01 ± 0.02 -0.01 ± 0.03 0.97
ClOindex 10.89 0 -1.18 ±0.09 -2.16 ±0.17 <0.001
C12index 2.74 0 -0.29 ± 0.02 -0.56 ±0.05 <0.001
C14index 10.50 0 -1.34 ±0.08 -2.61 ±0.16 <0.001
Clόindex 4.24 0 0.48 ± 0.04 0.98 ±0.08 <0.001
C17index 28.28 0 1.62 ±0.14 3.27 ±0.27 <0.001
C18index 67.59 0 0.87 ±0.19 1.43 ±0.37 <0.001
CLA4index 33.71 0 1.32 ±0.20 2.39 ±0.40 <0.001
1 contrast of VA-AA genotypes, contrast of VV-AA genotypes.
3 statistical significance of the SCD JS7SFpolymorphism. 4CLA:C18:2cis9,transll
5 SFA (Saturated Fatty Acids): C4:0, C5:0, C6:0, C7:0, C8:0, C9:0, C10:0, Cl 1:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, Cl 8:0; UFA (Unsaturated Fatty Acids): C10:l, C12:l, C14:l,C16:l,C18u, CLA.
contrast of KA-KK genotypes. " contrast of AA-KK genotypes.
5 statistical significance of the DGATl K232A polymorphism. 'CLA: C18:2 cis9, trans 11
Table 14. Percentage of the genetic variance explained by the SCD A878V and DGATl K232A polymorphism
Trait SCD Λ878V DGATl K232A
ClO index 48 3
C12 index 41 6
C14 index 58 11
C16 index 48 16
C17 index 47 0
C18 index 9 19
CLA1 index 28 16 1 CLA: C18:2 cis9, transl l
Table 15. Combined effect of the SCD A878V polymorphism and the DGATl K232A polymorphism on fatty acid composition and desaturation indexes
AAsa/KK AAscr/AA DGAT1 VVscτ/KK o GA Ti VVSCD/AA
Trait Mean DGATl (n=316) (n=16) 3 DGATl P value4
(n=159) (n=41)
C4:0 3.50 0 0.00 ±0.03 0.00 ±0.07 0.02 ±0.05 0.19
C6:0 2.22 0 -0.07 ± 0.02 0.01 ±0.04 -0.03 ± 0.03 <0.001
C8:0 1.37 0 -0.04 ±0.01 0.02 ± 0.03 0.00 ± 0.02 <0.01
C10:0 3.03 0 -0.01 ± 0.04 0.16 ±0.10 0.17 ±0.07 <0.001
C10:l 0.37 0 -0.01 ±0.01 -0.05 ± 0.02 -0.07 ±0.01 <0.001
C12:0 4.11 0 0.07 ± 0.06 0.25 ±0.14 0.20 ±0.10 <0.01
C12:l 0.12 0 0.00 ± 0.00 -0.01 ±0.01 -0.02 ± 0.00 <0.001
C14:0 11.61 0 0.79 ±0.08 0.68 ±0.21 1.17 ± 0.14 <0.001
C14:lc9 1.36 0 -0.01 ± 0.02 -0.26 ± 0.06 -0.34 ±0.04 <0.001
C16:0 32.59 0 -2.29 ± 0.24 0.01 ±0.61 -2.70 ±0.41 <0.001
C16:lc9 1.44 0 0.00 ± 0.00 0.00 ± 0.00 0.00 ±0.00 <0.001
C17:0 0.45 0 -0.01 ±0.00 -0.05 ±0.01 -0.02 ±0.01 <0.001
C17:lc9 0.18 0 0.00 ± 0.00 0.01 ±0.01 0.02 ±0.01 <0.001
C18:0 8.73 0 -0.22 ±0.13 -0.80 ±0.34 -0.60 ±0.23 <0.001
C18:lc9 18.04 0 1.68 ±0.17 0.12 ±0.45 1.93 ±0.30 <0.001
CLA5 0.39 0 0.05 ±0.01 0.03 ± 0.02 0.07 ±0.01 <0.001
SFAAJFA6 2.79 0 -0.25 ± 0.03 -0.02 ±0.08 -0.26 ±0.05 <0.001
ClOindex 10.89 0 -0.28 ±0.17 -1.72 ±0.44 -2.42 ±0.30 <0.001
C12index 2.74 0 -0.12 ±0.05 -0.44 ±0.12 -0.67 ±0.09 <0.001
C14index 10.50 0 -0.79 ±0.15 -2.38 ±0.39 -3.34 ±0.27 <0.001
Clόindex 4.24 0 -0.49 ±0.07 1.08 ±0.19 0.41 ±0.13 <0.001
C17index 28.28 0 0.18 ±0.27 3.48 ±0.70 3.02 ±0.48 <0.001
C18index 67.59 0 2.54 ± 0.36 2.37 ± 0.93 4.05 ± 0.63 <0.001
CLA^ index 33.71 0 2.25 ± 0.39 3.31 ± 1.01 4.62 ± 0.69 <0.001
' contrast of SCD/DGAT1 AA/ AA-AAfKK genotypes.
2 contrast of SCD/DGAT1 VV/KK-AA/KK genotypes.
3 contrast of SCD/DGAT1 VV/KK-AA/KK genotypes.
4 statistical significance of the combination of the SCD A878V and the DGATl K232A polymorphism.
5 CLA: C18:2 cis9, transl l
6 SFA (Saturated Fatty Acids): C4:0, C5:0, C6:0, C7:0, C8:0, C9:0, C10:0, Cl 1:0, C12:0, C13:0, C14:0, C15:0, C16:0, C17:0, Cl 8:0; UFA (Unsaturated Fatty Acids): C10:l, C12:l, C14:l, C16:l, C18u, CLA.
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39. Parodi, P. W. (1982) J. Dairy Res. 49, 73-80. 40. Kinsella, J. E. (1916) Lipids W, 680-684.
41. Morand, L. Z., Morand, J. N., Matson, R. & German, J. B. (1998) J. Dairy ScL 81, 100-6.
Claims
1. Use of means that detect the presence of a SCD 878V allele and/or of additional means that detect the presence of a DGATl 232 A allele in a method for identifying a non-human mammal, preferably a bovine which produces milk having a different milk- fat composition than the one from a milk produced by a non- human mammal not having a SCD 878 V and/or DGATl 232 A allele.
2. Use of means according to claim 1, wherein the presence of a SCD 878V allele is associated with a milk having a milk-fat with an increased desaturation index for at least one of C16, C17,C18 and CLA fatty acids.
3. Use of means according to claim 2, wherein the milk has a milk- fat composition with at least one of an increased content of C16:lcis9, C17:lcis9 and CLA and a decreased content of C17:0 and C18:0 fatty acids.
4. Use of means according to any one of claims 1 to 3, wherein the presence of a DGATl 232 A allele is associated with a milk having a milk-fat with at least one of a reduced content of saturated C 16 fatty acids and an increased content of unsaturated Cl 8 fatty acids.
5. Use of means according to claim 4, wherein the unsaturated C18 fatty acid is selected from the following group: C18:l cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid), CLA and C18:3 cis 9,12,15 (linolenic acid).
6. Use according to any one of the preceding claims, wherein the presence of a SCD 878V and DGATl 232A allele is associated with a milk having a milk-fat with an increased desaturation index for at least one of C 16, C 17,Cl 8 and CLA fatty acids.
7. Use according to claim 6, wherein the milk has a milk-fat composition with at least one of a decreased content of saturated C 16, C17 and C 18 fatty acids and an increased content of unsaturated C 16, C17 and Cl 8 fatty acids.
8. Use according to claim 7, wherein the unsaturated C 16, C17 and Cl 8 fatty acid is selected from the following list: C16:l cis 9, C17:l cis 9, C18:l cis 9 (oleic acid) and CLA.
9. Use according to claim 7 or 8, wherein the unsaturated Cl 8 fatty acid is a CLA, preferably the C 18:2 cis 9, trans 11 isomer.
10. Use according to any one of claims 1 to 9, wherein a SCD 878V allele is determined with respect to DNA, mRNA, and/or protein obtained from the non- human mammal by direct or indirect methods.
11. Use according to any one of claims 1 to 9, wherein a DGATl 232 A allele is determined with respect to DNA, mRNA, and/or protein obtained from the non- human mammal by direct or indirect methods.
12. Method for selecting a non- human mammal, preferably a bovine which produces milk with:
- an increased desaturation index for at least one of C 16, C 17,Cl 8 and CLA fatty acids and/or
- a milk -fat composition with at least one of a reduced content of saturated C 16 fatty acids and an increased content of unsaturated Cl 8 fatty acids.
13. A method according to claim 12, wherein the unsaturated Cl 8 fatty acid is selected from the following group: C18:l cis 9 (oleic acid), C18:l cis 11, C18:2 cis 9,12 (linoleic acid), CLA and C18:3 cis 9,12,15 (linolenic acid).
14. A method according to claim 12 or 13, wherein the non-human mammal, preferably a bovine possesses a SCD 878 V and/or a DGATl 232 A allele.
15. A method according to any one of claims 12 to 14, wherein the non- human mammal, preferably a bovine is obtained by further selecting and/or feeding a specific diet.
16. Milk or milk -fat composition obtainable from the non- human mammal, preferably a bovine produced by the method of any one of claims 12 to 15 or by the use of any one of claims 1 to 11.
17. Food product derived from and/or comprising the milk or milk- fat composition of claim 16.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP08712611A EP2121976A2 (en) | 2007-02-15 | 2008-02-15 | Method for selection of non-human mammal producing milk with improved fatty acid composition |
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP07102480 | 2007-02-15 | ||
| EP07113639 | 2007-08-01 | ||
| EP08712611A EP2121976A2 (en) | 2007-02-15 | 2008-02-15 | Method for selection of non-human mammal producing milk with improved fatty acid composition |
| PCT/NL2008/050085 WO2008100145A2 (en) | 2007-02-15 | 2008-02-15 | Method for selection of bovines producing milk with improved fatty acid composition |
Publications (1)
| Publication Number | Publication Date |
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| EP2121976A2 true EP2121976A2 (en) | 2009-11-25 |
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| WO (1) | WO2008100145A2 (en) |
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| CA2748228A1 (en) * | 2008-12-24 | 2010-08-05 | Fonterra Co-Operative Group Limited | Selection of animals for desired milk and/or tissue profile |
| NL2005543C2 (en) * | 2010-10-18 | 2012-04-19 | Friesland Brands Bv | Method to produce milk with increased unsaturated fatty acids content. |
| NL2006696C2 (en) * | 2011-04-29 | 2012-10-30 | Friesland Brands Bv | Milk composition comprising vitamins and minerals. |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002036824A1 (en) * | 2000-10-31 | 2002-05-10 | Michel Alphonse Julien Georges | Marker assisted selection of bovine for improved milk production using diacylglycerol acyltransferase gene dgat1 |
| WO2004004450A1 (en) * | 2002-07-03 | 2004-01-15 | A2 Corporation Limited | Method for altering fatty acid composition of milk |
| US20040234986A1 (en) * | 2001-07-06 | 2004-11-25 | Hans-Rudolf Fries | Method of testing a mammal for its predisposition for fat content of milk and/ or its predisposition for meat marbling |
| US20040254104A1 (en) * | 2002-06-05 | 2004-12-16 | Sarah Blott | Marker assisted selection of bovine for improved milk composition |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3619833B2 (en) * | 2003-02-20 | 2005-02-16 | 財団法人新産業創造研究機構 | Method for determining beef flavor and texture based on the genotype of stearoyl-CoA desaturase |
-
2008
- 2008-02-15 EP EP08712611A patent/EP2121976A2/en not_active Withdrawn
- 2008-02-15 WO PCT/NL2008/050085 patent/WO2008100145A2/en active Application Filing
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002036824A1 (en) * | 2000-10-31 | 2002-05-10 | Michel Alphonse Julien Georges | Marker assisted selection of bovine for improved milk production using diacylglycerol acyltransferase gene dgat1 |
| US20040234986A1 (en) * | 2001-07-06 | 2004-11-25 | Hans-Rudolf Fries | Method of testing a mammal for its predisposition for fat content of milk and/ or its predisposition for meat marbling |
| US20040254104A1 (en) * | 2002-06-05 | 2004-12-16 | Sarah Blott | Marker assisted selection of bovine for improved milk composition |
| WO2004004450A1 (en) * | 2002-07-03 | 2004-01-15 | A2 Corporation Limited | Method for altering fatty acid composition of milk |
Non-Patent Citations (4)
| Title |
|---|
| GRISART B ET AL: "Genetic and functional confirmation of the causality of the DGAT1 K232A quantitative trait nucleotide in affecting milk yield and composition", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC; US, vol. 101, no. 8, 24 February 2004 (2004-02-24), pages 2398 - 2403, XP002326522, ISSN: 0027-8424, DOI: 10.1073/PNAS.0308518100 * |
| GRISART B ET AL: "Positional candidate cloning of a QTL in dairy cattle: Identification of a missense mutation in the bovine DGAT1 gene with major effect on milk yield and composition", GENOME RESEARCH, COLD SPRING HARBOR LABORATORY PRESS, WOODBURY, NY, US, vol. 12, no. 2, 1 February 2002 (2002-02-01), pages 222 - 231, XP002227895, ISSN: 1088-9051, DOI: 10.1101/GR.224202 * |
| LACORTE G A ET AL: "DGAT1 K232A polymorphism in Brazilian cattle breeds.", GENETICS AND MOLECULAR RESEARCH : GMR 2006, vol. 5, no. 3, 2006, pages 475 - 482, ISSN: 1676-5680 * |
| SPELMAN R J ET AL: "Characterization of the DGAT1 gene in the New Zealand dairy population", JOURNAL OF DAIRY SCIENCE, AMERICAN DAIRY SCIENCE ASSOCIATION, US, vol. 85, no. 12, 1 December 2002 (2002-12-01), pages 3514 - 3517, XP002436970, ISSN: 0022-0302, DOI: 10.3168/JDS.S0022-0302(02)74440-8 * |
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| Publication number | Publication date |
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| WO2008100145A2 (en) | 2008-08-21 |
| WO2008100145A3 (en) | 2008-10-09 |
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