CN110537631A - Method for promoting growth of mammalian offspring by adding plant sterol ester from mother source - Google Patents
Method for promoting growth of mammalian offspring by adding plant sterol ester from mother source Download PDFInfo
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- CN110537631A CN110537631A CN201910764922.2A CN201910764922A CN110537631A CN 110537631 A CN110537631 A CN 110537631A CN 201910764922 A CN201910764922 A CN 201910764922A CN 110537631 A CN110537631 A CN 110537631A
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- phytosterol
- feed
- ester
- bile acid
- phytosterol esters
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/158—Fatty acids; Fats; Products containing oils or fats
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Food Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Physical Education & Sports Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Neurology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Animal Husbandry (AREA)
- Epidemiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an application of phytosterol ester in preparation of a feed additive for mammals and a method for promoting growth of progeny of mammals by adding the phytosterol ester in a maternal source. The phytosterol ester is added into the grease-free daily ration feed of mammals instead of grease, so that the bile acid level in serum and amniotic fluid, the expression level of a placental bile acid transporter and the expression level of an embryonic hind limb bud bile acid receptor can be reduced, and the weight, the muscle content, the gripping force, the number of soleus muscle fibers, the cross-sectional area of gastrocnemius muscle fibers and the expression level of myosin heavy chain mRNA can be improved; in addition, after entering the body of a mammal, the phytosterol ester is converted into phytosterol and fatty acid through the digestion effect of the small intestine, and no toxic or side effect is generated, so that the phytosterol ester has wide popularization and application values in the aspect of preparing a feed additive for the mammal.
Description
Technical Field
The invention belongs to the technical field of food and feed. More particularly, it relates to a method for promoting growth of mammalian offspring by adding plant sterol ester from mother source.
Background
In recent years, a great deal of research has proved that the maternal nutrition level during gestation period has an important role in the aspects of growth, development, immunity, metabolism and the like of organism tissues and organs of fetuses and newborns, which marks that the theory of the programmed regulation of the maternal fetus growth and development makes an important progress. Therefore, both nutritional deficiencies and overnutrition of pregnant and parturient women may have adverse effects on the developing fetus.
Bile acids are important regulators of lipid metabolism in the animal body. Primary bile acids are synthesized from cholesterol in the liver, bind to glycine or taurine, increase their solubility, are secreted into the bile, and are converted by intestinal microorganisms in the colon into secondary bile acids, which are reabsorbed back into the liver in the ileum and colon. Recent studies have shown that circulating total bile acids are associated with skeletal muscle volume, 12 α -hydroxysteroid bile acids (including deoxycholic acid) are associated with decreased skeletal muscle volume, and non-12 α -hydroxysteroid bile acids (including chenodeoxycholic acid) are associated with increased skeletal muscle volume. However, it is not yet clear how maternal bile acid levels affect the growth and differentiation of progeny muscle fibers.
Phytosterol esters are the product of esterification of phytosterols with fatty acids and are structurally very similar to cholesterol. In the prior art, studies have shown that phytosterol esters are healthy and safe food ingredients and play an important role in lowering blood cholesterol levels and preventing cardiovascular disease. Phytosterol esters can alleviate cognitive deficits caused by high cholesterol diet and aging, can prevent non-alcoholic fatty liver disease caused by high fat diet, and can regulate the intestinal environment of rats by reducing the content of short chain fatty acids in the colon contents of rats on high fat diet. Thus, phytosterol esters play an important role in the breakdown and synthesis of cholesterol and are of great importance to the health and metabolism of the body. In addition, the prior patent CN 107048067a discloses an application of plant sterol ester in preparing chicken feed additive and a method for promoting the growth of offspring chicken by adding plant sterol ester from motherwort, which shows that plant sterol ester has good application value in being used as chicken feed additive. However, there has not been any report on the use of phytosterol esters as a raw material for the preparation of a feed supplement for mammals to promote growth and muscle development in offspring.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the blank of the existing plant sterol ester in preparing the feed additive for the mammals and provides a method for promoting the growth of the filial generation of the mammals by adding the plant sterol ester in a maternal source manner. The phytosterol ester is added into the grease-free daily ration feed of mammals instead of grease, and can regulate and control the metabolism of bile acid between mothers and fetuses and obviously promote the growth of offspring and the development of muscles, so the phytosterol ester has wide application value in preparing feed additives for the mammals.
The invention aims to provide application of phytosterol ester in preparation of a feed additive or feed for mammals.
it is another object of the present invention to provide a feed for mammals.
It is still another object of the present invention to provide a method for increasing growth of progeny of a mammal by maternal addition of phytosterol esters.
The above purpose of the invention is realized by the following technical scheme:
The research of the invention finds that the phytosterol ester can be added into the grease-free daily ration feed of mammals instead of grease, can reduce the bile acid level in serum and amniotic fluid, the expression level of a placental bile acid transporter and the expression level of a post-embryonic limb-bud bile acid receptor, can improve the weight, the muscle content, the gripping force, the number of soleus muscle fibers, the cross-sectional area of gastrocnemius muscle fibers and the expression level of myosin heavy chain mRNA, and can be used for preparing feed additives of the mammals.
Therefore, the following applications should be within the scope of the present invention:
Use of phytosterol esters in the preparation of a feed additive or feed for mammals.
Preferably, the application refers to the application of the phytosterol ester in preparing the feed additive capable of regulating and controlling the metabolism of bile acid between mothers and fetuses.
More preferably, the use refers to the use of phytosterol esters in the preparation of feed additives capable of reducing the levels of bile acids in serum and amniotic fluid, the expression level of placental bile acid transporters and the expression level of post-embryonic limb bud bile acid receptors.
Still further preferably, the placental bile acid transporter is organic anion transport polypeptide 2a1 and/or organic anion transport polypeptide 4a 1.
Still further preferably, the embryonic hind limb bud bile acid receptor is a farnesoid X receptor and/or a G protein-coupled bile acid receptor.
Preferably, the application refers to the application of the phytosterol ester in preparing the feed additive capable of promoting the growth and muscle development of offspring.
more preferably, the use refers to the use of phytosterol esters in the preparation of a feed additive capable of increasing body weight, muscle content, grip strength, number of soleus muscle fibers, cross-sectional area of gastrocnemius muscle fibers and myosin heavy chain mRNA expression level.
Still more preferably, the myosin heavy chain is any one or more of type 1, type 2a, type 2b or type 2x myosin heavy chain.
Preferably, the mammal is a pregnant mammal.
Preferably, the feed is a non-greasy feed.
Preferably, the phytosterol ester is any one or more of beta-sitosterol ester, stigmasterol ester or campesterol ester.
The invention also provides a mammal feed which comprises an effective amount of phytosterol ester and is free of grease.
In addition, the invention also provides a method for promoting the growth of the offspring of the mammals by adding the plant sterol ester from the motherland source instead of the grease, and the plant sterol ester is added into the daily ration feed of the mammals.
Preferably, the mass percentage of the phytosterol ester is 1-10%.
More preferably, the mass percentage of the phytosterol ester is 1-5%.
Even more preferably, the phytosterol ester is present in an amount of 3.33% by weight.
The invention has the following beneficial effects:
The invention provides a method for promoting growth of mammalian offspring by maternal addition of phytosterol ester. The research of the invention finds that the phytosterol ester can be added into the grease-free daily ration feed of mammals instead of grease, can reduce the bile acid level in serum and amniotic fluid, the expression level of a placental bile acid transporter and the expression level of a post-embryonic limb bud bile acid receptor, and can improve the weight, the muscle content, the gripping power, the number of soleus muscle fibers, the cross-sectional area of gastrocnemius muscle fibers and the expression level of myosin heavy chain mRNA.
The complete feed obtained by adding the phytosterol ester into the grease-free daily ration feed of the mammals is a safe feed, the phytosterol ester is converted into the phytosterol and the fatty acid through the digestion of the small intestine after entering the bodies of the mammals, no toxic or side effect is generated, and compared with a common feed additive, the complete feed can regulate and control the metabolism of bile acid between mothers and fetuses and obviously promote the growth and muscle development of filial generations; in addition, the method for adding the phytosterol ester into the grease-free daily feed of the mammals is simple and convenient, and has no requirement on special equipment, so the phytosterol ester has wide popularization and application values in the aspect of preparing the feed additive of the mammals.
Drawings
FIG. 1 shows the effect of phytosterol esters on serum bile acid levels in pregnant females; wherein PE represents a phytosterol ester.
FIG. 2 is a graph showing the effect of phytosterol esters on the levels of amniotic fluid bile acid in pregnant females; wherein PE represents a phytosterol ester.
Figure 3 is the results of the effect of phytosterol esters on placental bile acid transporters; wherein, OATP2a1 represents organic anion transport polypeptide 2a 1; OATP4A1 represents organic anion transport polypeptide 4A 1.
FIG. 4 shows the effect of phytosterol esters on post-embryonic limb bud bile acid receptors; wherein FXR represents the farnesoid X receptor; TGR5 represents a G protein-coupled bile acid receptor; tubulin stands for Tubulin.
Fig. 5 shows the results of the effect of phytosterol esters on the body weight of offspring mice (male and female).
Figure 6 is the results of the effect of phytosterol esters on muscle and fat content in offspring mice (male and female).
Fig. 7 is the results of the effect of phytosterol esters on the gripping power of offspring mice (male and female).
FIG. 8 is the results of the effect of phytosterol esters on the muscle tissue ratio of offspring mice (male and female); wherein GAS represents gastrocnemius, SOL represents soleus, TA represents tibialis anterior, EDL represents extensor digitorum longus, BAT represents brown fat, and iWAT represents subcutaneous fat.
Figure 9 is the results of the effect of phytosterol esters on the number of soleus muscle fibers in progeny mice.
Fig. 10 shows the effect of phytosterol esters on the cross-sectional area of gastrocnemius muscle fibers in offspring mice (male and female).
FIG. 11 is the results of the effect of phytosterol esters on the expression levels of myosin heavy chain mRNA in progeny mice (male and female); wherein, MHC 1, MHC 2a, MHC 2b and MHC 2x represent myosin heavy chains of 1, 2a, 2b and 2x types respectively.
FIG. 12 shows the effect of phytosterol esters on the ratio of muscle fibers in offspring mice (male and female); wherein, MHC 1, MHC 2a and MHC 2b represent myosin heavy chains of types 1, 2a and 2b, respectively.
Detailed Description
The present invention is further illustrated by the following specific examples, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 Effect of phytosterol esters on intercarrier bile acid metabolism
1. Preparation of test diets
(1) Weighing phytosterol ester (40% of beta-sitosterol ester, 30% of stigmasterol ester and 10% of campesterol ester) to prepare a non-fat daily ration;
(2) And mixing the phytosterol ester with the grease-free daily ration to prepare the complete feed, wherein the mass fraction of the phytosterol ester is 3.33 percent of the complete feed.
The formula, the nutrition level and the preparation method of the grease-free daily ration refer to the conventional method, and equipment for mixing the phytosterol ester and the grease-free daily ration is conventional equipment.
2. Experimental mouse grouping and handling
12C 57BL female mice with good body condition at 8-10 weeks are selected for the test, randomly divided into 2 groups of 6 mice, and fed with common daily ration before pregnancy. Combining the male mouse and the female mouse for hybridization at 20 days, taking out the male mouse and detecting the vaginal suppository of the female mouse at 8 morning the next day, recording as 0.5 th day of pregnancy if the vaginal suppository is found, and replacing the test daily ration; if no vaginal suppository is found, continue to feed normal ration and repeat the operation. The control group adopts grease-free daily ration and 1.97% oleic acid; the phytosterol ester group replaced oleic acid with a phytosterol ester equivalent value on a control group ration basis. In the test, single-cage breeding is adopted except for cage combination, and the mice can eat and drink water freely in the test period. The test set-up groups and the treatment diet composition are shown in table 1.
Table 1 test set-up group and treatment feed composition
3. Tissue sampling
Serum, embryonic hind limb buds, amniotic fluid and placenta of the female mouse are collected respectively on day 12.5 of gestation, day 17.5 of gestation and day of delivery. Blood was collected by the eyedrop method, and blood was collected by a 1.5mL centrifuge tube, left to stand until blood coagulated, and then centrifuged at 3000rpm at 4 ℃ for 15 minutes to collect serum, and stored in a refrigerator at-20 ℃ for further use. And (3) respectively putting the rest samples into a centrifuge tube and a container filled with liquid nitrogen, transferring the samples into a refrigerator at the temperature of-80 ℃ after sampling is finished, and storing for later use.
After blood collection of 3-week-old and 8-week-old offspring mice, cervical dislocation and sacrifice are completed, and muscle tissues of the offspring mice are collected, and the method comprises the following steps: gastrocnemius (GAS), Soleus (SOL), Tibialis Anterior (TA), Extensor Digitorum Longus (EDL), brown fat (BAT) and subcutaneous fat (iWAT), weighing, recording, placing in a centrifuge tube, sticking the left leg gastrocnemius on a hard paper sheet, keeping the original shape of the muscle as much as possible, completely placing in the centrifuge tube with an opening on the tube wall, quickly freezing with liquid nitrogen, transferring to a refrigerator at-80 deg.C, and storing for later use.
4. Test method
The detection of the serum bile acid and the amniotic fluid bile acid levels of the female mouse is carried out by a bile acid kit of Nanjing Biotechnology Ltd. The effect of phytosterol esters on placental bile acid transporters (organic anion transport polypeptide 2a1, organic anion transport polypeptide 4a1) was investigated by performing PCR experiments on placenta. The effect of phytosterol esters on postembryonic limb bud bile acid receptors (farnesoid X receptors, G protein-coupled bile acid receptors) was investigated by performing Western Blot experiments on postembryonic limb buds.
5. Test results
The effect of phytosterol esters on serum bile acid levels in pregnant dams the results are shown in figure 1, and it can be seen that phytosterol esters significantly reduced bile acid levels in serum of dams at day 17.5 of pregnancy. The effect of phytosterol esters on amniotic fluid bile acid levels in pregnant females is shown in figure 2, and it can be seen that phytosterol esters significantly reduced bile acid levels in amniotic fluid in females at day 17.5 of pregnancy. The results of the effect of phytosterol esters on placental bile acid transporters are shown in fig. 3, and it can be seen that phytosterol esters reduced the expression levels of organic anion transport polypeptide 2a1 and organic anion transport polypeptide 4a 1. The effect of phytosterol ester on the post-embryonic limb bud bile acid receptor results are shown in fig. 4, and it can be seen that phytosterol ester significantly reduced the expression level of farnesoid X receptor, G protein-coupled bile acid receptor.
The above results show that: phytosterol esters are capable of regulating bile acid metabolism between mothers and fetuses.
Example 2 Effect of phytosterol esters on growth in progeny
1. Test method
After birth, the weight of the offspring mice was measured to 8 weeks of age every week. After weaning for 3 weeks, feeding the male and female animals in cages and feeding the animals with common daily ration.
Body composition was determined after progeny mice grew to 8 weeks of age. The offspring mice were housed, their tails were stimulated to urinate and defecate, then the weights were measured and recorded, the offspring mice were gently fixed and placed in a nuclear magnetic resonance imager (shanghai niumei science and technology ltd., MesoQMR 23-060H), and the muscle and fat contents of the offspring mice were measured.
After the body composition of the 8-week-old offspring mouse is determined, the grip strength is determined after the offspring mouse recovers for three days. The offspring mice were placed on a paw grab meter (shenzhen ruivorad technologies ltd, model BIO-GS 3), the offspring mice were pulled parallel to the touchnet, the machine-displayed measurements were read and recorded, 10 measurements were made per offspring mouse, and the average was taken for subsequent statistics. After the grip strength measurement was completed, the offspring mice were sampled after three days of recovery.
The tissue ratios of GAS, SOL, TA, EDL, BAT and iWAT were determined for the offspring mice, respectively.
2. Test results
The results of the effect of phytosterol ester on the body weight of offspring mice (male and female) are shown in fig. 5, and it can be seen that phytosterol ester significantly increased the body weight of 0-6 weeks old offspring mice compared to the control group. The results of the effect of phytosterol ester on the muscle and fat content of offspring mice (male and female) are shown in fig. 6, and it can be seen that phytosterol ester can significantly improve the muscle content of 8-week-old offspring mice and female mice and reduce the fat content of offspring mice. The results of the effect of phytosterol ester on the gripping ability of offspring mice (male and female) are shown in fig. 7, and it can be seen that phytosterol ester can significantly improve the gripping ability of 8-week-old offspring mice and male. The results of the effect of phytosterol esters on the muscle tissue ratio of offspring mice (male and female) are shown in fig. 8, and it can be seen that phytosterol esters can significantly increase the GAS, TA, BAT ratio of offspring mice and significantly decrease iWAT ratio; phytosterol esters significantly increased the rate of TA in offspring mother rats.
The above results show that: phytosterol esters can significantly promote the growth of progeny mice.
Example 3 Effect of phytosterol esters on progeny muscle development
1. Test method
preparing slices of hind limb buds of a 1-day-old offspring mouse and gastrocnemius muscles of a 3-week-old offspring mouse, fixing the slices in 4% paraformaldehyde for 10-20 min, washing for 1-2 s, dyeing in hematoxylin for 20-30 s, washing for 5-10 s with running water, placing in 1% hydrochloric acid ethanol for 1-3 s, washing for 15-30 s with running water, placing in 1% eosin for dyeing for 30-60 s, washing for 1-2 s, 80% ethanol for 1-2 s, 95% ethanol for 1-2 s, anhydrous ethanol for 1-2 s, xylene (I) for 2-3 s, xylene (II) for 2-3 s, and sealing with neutral gum. After the H & E stained sections were photographed under a microscope, the number of Soleus muscle fibers (Soleus) and the cross-sectional area of gastrocnemius muscle fibers of offspring mice were counted using Image J software for subsequent data analysis.
The effect of phytosterol esters on the expression levels of their myosin heavy chains (type 1, type 2a, type 2b, type 2x myosin heavy chains) was investigated by performing PCR assays on gastrocnemius muscle of 3-week-old offspring mice.
Making sections of gastrocnemius of 8-week-old offspring mice, placing the sections in an acidic pre-incubation solution for incubation at room temperature for 10min, washing the sections twice with Tris-CaCl2 for 1min each time, placing the sections in an SDH incubation solution for incubation at 37 ℃ for 45min, washing the sections twice with double distilled water for 30S each time, placing the sections in an ATP incubation solution for incubation at 37 ℃ for 30min, washing the sections twice with double distilled water for 1min each time, incubating the sections for 5min with 2% CoCl2, washing the sections twice with double distilled water for 3min each time, placing the sections in 2% (NH4)2S for 30S each time, washing the sections twice with double distilled water for 5min each time, staining hematoxylin a dark place for 3min, washing the sections twice with double distilled water for 2min each time, decolorizing with gradient alcohol (50%, 75%, 100%, each 2min), soaking the sections in xylene for 2min, air-drying the. Stained sections were photographed under a microscope and the number of different types of muscle fibers was counted and the ratio was calculated using Image J software.
2. Test results
The effect of phytosterol ester on the number of soleus muscle fibers in offspring mice is shown in fig. 9, and it can be seen that phytosterol ester can significantly increase the number of soleus muscle fibers in 1-day-old offspring mice. The results of the effect of phytosterol ester on the cross-sectional area of the gastrocnemius fibers of the offspring mice (male and female mice) are shown in fig. 10, and it can be seen that phytosterol ester can increase the cross-sectional area of the gastrocnemius fibers of 3-day-old offspring mice.
The results of the effect of phytosterol ester on myosin heavy chain mRNA expression levels in progeny mice (male and female) are shown in fig. 11, and it can be seen that phytosterol ester significantly increases the mRNA expression levels of MHC 2a and MHC 2x in 3-week-old progeny male mice and MHC 2a in 3-week-old progeny female mice.
The results of the effect of phytosterol ester on the muscle fiber ratio of offspring mice (male mice and female mice) are shown in fig. 12, and it can be seen that phytosterol ester can significantly reduce the ratio of MHC type 1 and MHC type 2a muscle fibers of 8-week-old offspring mice, and significantly increase the ratio of MHC type 2b muscle fibers of 8-week-old offspring mice; meanwhile, the phytosterol ester can obviously reduce the MHC 1 muscle fiber ratio of 8-week-old filial generation female rats and obviously improve the MHC 2b muscle fiber ratio of 8-week-old filial generation female rats.
The above results show that: the phytosterol ester can remarkably promote the development of muscles of offspring mice.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. Use of phytosterol esters in the preparation of a feed additive or feed for mammals.
2. The use according to claim 1, wherein the use is the use of phytosterol esters in the preparation of a feed supplement capable of modulating the metabolism of bile acids between mothers and fetuses.
3. The use according to claim 1, wherein the use is the use of phytosterol esters in the manufacture of a feed supplement for the promotion of progeny growth and muscle development.
4. The use according to claim 2, wherein the use is the use of phytosterol esters in the preparation of a feed supplement capable of reducing the levels of bile acids in serum and amniotic fluid, the expression level of placental bile acid transporters and the expression level of post-embryonic limb bud bile acid receptors.
5. The use according to claim 3, wherein the use is of phytosterol esters in the preparation of a feed supplement for increasing body weight, muscle content, grip strength, number of soleus muscle fibers, cross-sectional area of gastrocnemius muscle fibers and myosin heavy chain mRNA expression level.
6. The use of claim 1, wherein the mammal is a pregnant mammal.
7. Use according to claim 1, wherein the feed is a fat-free feed.
8. The use according to any one of claims 1 to 5, wherein the phytosterol ester is any one or more of beta-sitosterol ester, stigmasterol ester or campesterol ester.
9. A mammalian feed comprising an effective amount of a phytosterol ester and being free of fats and oils.
10. A method for promoting growth of mammalian offspring by maternal addition of phytosterol esters is characterized in that the phytosterol esters are added to the ration feed of the mammal instead of oil.
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