CN113712121B - Application of niacinamide in improving milk components of dairy goats in lactation period - Google Patents
Application of niacinamide in improving milk components of dairy goats in lactation period Download PDFInfo
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Abstract
The invention relates to application of niacinamide in improving milk components of a milk goat in lactation period, and the system reveals the action mechanism of niacinamide in regulating and controlling the metabolism of mammary fatty acid of the milk goat from the whole, cell and molecular level. The invention discovers that the niacinamide has beneficial effect on the milk components of the dairy goats, and the niacinamide can be applied to daily feed of the dairy goats in the lactation period, so that the milk yield of the dairy goats can be improved, and the composition ratio of polyunsaturated fatty acids in the goat milk can be improved. The invention not only improves the milk yield of the milk goats, but also improves the quality of the goat milk and improves the economic benefit of the milk goat breeding. The invention clarifies the action mechanism of the niacinamide for regulating and controlling the milk goat mammary fatty acid metabolism, and provides theoretical and experimental basis for the application of the niacinamide in improving the milk quality of the milk goat and the deep research of the milk goat mammary fatty acid metabolism regulation network.
Description
Technical Field
The invention relates to a new application of niacinamide, in particular to an application of niacinamide in improving milk components of dairy goats in lactation.
Background
Nicotinamide (NAM) is a B-group vitamin with simple structure and stable physicochemical properties, is an amide form of vitamin B3, forms coenzyme I and coenzyme II with ribose, phosphoric acid and adenine in animal bodies and participates in metabolism of lipids, carbohydrates and proteins in the bodies. NAM naturally occurs in animal by-products and plant-derived feed materials. NAM is mainly absorbed through the gastrointestinal mucosa and enters the metabolism in the body. Sirtuin 2-related enzyme 1 (SIRT 1) is NAD + Dependent deacetylases, which are key upstream regulatory factors regulating lipid metabolism, modify the lysine residues of proteins by deacetylation to regulate physiological processes. SIRT1 activity can be inhibited by Niacinamide (NAM), which acts primarily by inhibiting SIRT1 deacetylase activity, and fat formation can be achieved not only by modulating expression of adipogenic transcription factors, but also by modulating gene expression during fatty acid oxidation.
Goat milk has rich nutritional value and has unique advantages in the aspect of milk fat. On one hand, the milk fat drops are smaller and are more easily digested and absorbed; on the other hand, the goat milk has higher content of short-medium chain fatty acid. Milk fat is taken as one of the main components of the goat milk, the fat content and the fatty acid composition of the milk are the main reasons for influencing the nutritional value and the flavor of the goat milk, the special flavor (namely the mutton smell) of the goat milk is a key constraint factor for developing the goat milk industry and expanding the consumer market, and if the special flavor can be changed, the taste of the goat milk is improved, and the development of the milk and goat industry is greatly promoted. Therefore, the influence of nutrient substances on the milk fat content and the fatty acid composition of the sheep and the milk fat metabolism regulation mechanism of the mammary gland of the milk goat are deeply studied, and the method has important theoretical and practical significance for further developing the goat milk consumer market and promoting the development of the milk goat industry.
The niacinamide is used as premix of ruminants or as feed additive, the nutrition requirements of ruminants in different physiological periods and different physiological states are different, and the purpose of nutrition regulation of the niacinamide is also different. Therefore, the control mechanism, effective control dosage, addition mode, action period and the like of the niacinamide are fully known, and the method is helpful for more reasonably utilizing the nutrition control means so as to achieve accurate nutrition control. In order to further improve the quality of the milk goat and improve the flavor of the goat milk, the invention tries to add the niacinamide into the basic ration of the milk goat so as to influence the milk yield, the milk components and other properties of the milk goat.
Disclosure of Invention
The invention aims to provide an application of niacinamide in improving milk components of a milk goat in lactation, and the system reveals the action mechanism of niacinamide in regulating and controlling the metabolism of mammary fatty acid of the milk goat from the whole, cell and molecular level.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
application of niacinamide in improving milk yield of milk goat in lactation period is provided.
The application of the niacinamide in improving the milk components of the dairy goats in the lactation period.
The milk components include milk fat and milk fat acid.
The milk fatty acids include C4:0, C16:0 and C18:1.
Application of the niacinamide in regulating transcription factor SREBP1 in FASN gene transcription.
The addition amount of the niacinamide in the basic ration of the milk goat is 5g/d.
The invention has the beneficial effects that:
the invention discovers that the niacinamide has beneficial effect on the milk components of the dairy goats, and the niacinamide can be applied to daily feed of the dairy goats in the lactation period, so that the milk yield of the dairy goats can be improved, and the composition ratio of polyunsaturated fatty acids in the goat milk can be improved.
The invention not only improves the milk yield of the milk goats, but also improves the quality of the goat milk and improves the economic benefit of the milk goat breeding.
The invention clarifies the action mechanism of the niacinamide for regulating and controlling the milk goat mammary fatty acid metabolism, and provides theoretical and experimental basis for the application of the niacinamide in improving the milk quality of the milk goat and the deep research of the milk goat mammary fatty acid metabolism regulation network.
Drawings
FIG. 1 is a graph showing the effect of varying concentrations of NAM on mRNA expression of an important signal molecule in the SIRT1/FOXO1 pathway.
FIG. 2 shows the effect of NAM at 300. Mu. Mol/L on milk fat synthesis-related gene expression.
Figure 3 is the effect of NAM on triglyceride content and lipid droplet aggregation in mammary epithelial cells of milk goats.
Figure 4 is the effect of NAM on fatty acid composition in mammary epithelial cells.
FIG. 5 is a schematic representation of potential transcription factor binding sites for the FASN gene promoter.
FIG. 6 is the effect of SIRT1 agonists and inhibitors on FASN promoter activity.
FIG. 7 is a schematic sequence alignment of SRE1 and SRE2 mutations.
FIG. 8 is a graph showing the effect of SRE1, SRE2 mutations on FASN promoter activity.
FIG. 9 is the effect of NAM on FASN wild-type, SRE mutant promoter activity.
Detailed Description
The following describes the embodiments of the present invention in further detail with reference to examples.
The embodiment of the invention processes data: test data are expressed as mean ± standard deviation, and the difference significance is analyzed by t-test by comparing the mean of two samples of SPSS software. P <0.05 indicates that the difference is significant, and P <0.01 indicates that the difference is extremely significant.
Example 1 design of experiments and feeding management of milk goats
40 milk goats with the same birth number (2 birth number), similar lactation days (30+/-5 d), the same or similar weight and good health condition are selected and randomly divided into two groups (a control group and a test group), 20 goats are fed with basic diet in each group, and 5g/d of Niacinamide (NAM) is added on the basis of the basic diet in the test group. The test diet design refers to NRC (2007) goat feeding standard, and the basic diet composition and the nutritional ingredients are shown in tables 1 and 2. The whole test period is 60 days, wherein the pre-test period is 10 days, and the positive test period is 50 days. The test was performed in a certain body milk goat farm in the western isthmus county of south yang city.
The test sheep were subjected to epidemic prevention, insect repellent, cleaning of the test shed and disinfection before the start of the test, after which 2 groups of test sheep were individually bred in 2 independent shed. Feeding twice a day. All sheep only drink water and move freely.
Table 1 basic diet composition (Dry matter basis)
| Raw materials% | Control group | Test group |
| Silage corn | 32 | 32 |
| Peanut seedling | 8 | 8 |
| Sweet potato seedling | 8 | 8 |
| Corn | 27 | 27 |
| Wheat bran | 6 | 6 |
| Bean pulp | 12 | 12 |
| Distiller's dried grain | 3 | 3 |
| Stone powder | 1 | 1 |
| Dibasic calcium phosphate | 1.13 | 1.13 |
| Salt | 0.87 | 0.87 |
| Premix compound ① | 1 | 1 |
| Totalizing | 100 | 100 |
Note that: (1) the premix can provide VA 20000IU and VB for each kg of daily ration 5 25.74mg，VD 30000IU，VE 5000IU，Fe 56mg，Mn 31mg，Zn 92.5mg，Cu 30mg，I 1.25mg，Se 1.00mg。
Table 2 nutrient levels (dry matter basis)
| Nutrient level ② | Control group | Test group |
| Digestion energy/(MJ/kg) | 12.24 | 12.46 |
| Crude protein% | 16.96 | 16.87 |
| Coarse ash% | 3.49 | 3.88 |
| Neutral washing fiber% | 37.32 | 37.47 |
| Acid washing fiber% | 22.68 | 22.44 |
| Calcium% | 1.26 | 1.26 |
| Phosphorus% | 0.54 | 0.54 |
Note that: (2) the digestion energy in each nutrient level is calculated, and the other nutrient levels are measured values.
EXAMPLE 2 Effect of NAM on milk yield, milk ingredient and milk fatty acid content of milk goats
(1) Milk yield
Milk production data were recorded after each milking, 2 daily milkings were made in the morning and evening during the test period.
The milk yield difference between the control group and the test group is not obvious at the beginning of the experiment, and the milk yield difference belongs to the difference of milk production traits among normal individuals. As the test proceeds, the milk goats enter the lactation period from the pre-lactation period, and the milk yield of the milk goats is increased at the end of the test compared with that of the beginning of the test. The results showed that the milk yield of the daily ration added NAM group of dairy goats was significantly higher than that of the control group (P <0.05, table 3). The method shows that the addition of the niacinamide in the daily ration can obviously improve the milk yield of the dairy goat and improve the economic benefit of the dairy goat.
TABLE 3 influence of NAM on milk yield of milk goats
| Project | Control group | Test group |
| Milk yield (kg/day 1) at the beginning of the test | 2.04±0.14 | 2.09±0.09 |
| Milk yield (kg/day 60) at the end of the test | 2.32±0.06 b | 2.48±0.07 a |
Note that: the same row of shoulders indicates that the difference is significant (P < 0.05), the different capital letters indicate that the difference is extremely significant (P < 0.01), and the unlabeled indicates that the difference is not significant (P > 0.05). The table below is the same.
(2) Milk component
And (3) collecting 1 time of milk samples from all the experimental sheep at the end of the experimental period, preparing 20mL of mixed samples in the morning and evening for detection, collecting 40 samples from all the experimental sheep, sending the 40 samples to a Henan province dairy cow production performance measuring center, and analyzing milk ingredient indexes such as lactose rate, milk fat rate, milk protein rate and the like.
As can be seen from table 4, the milk fat percentage in the goat milk component of the NAM group was significantly higher than that of the control group (P < 0.05); lactose, milk proteins and dry matter were not significantly changed (P > 0.05).
TABLE 4 influence of NAM on milk goat milk composition
| Project | Control group | Test group | P value |
| Milk fat/% | 3.48±0.08 b | 3.65±0.06 a | 0.045 |
| Milk protein/% | 3.22±0.09 | 3.14±0.10 | 0.347 |
| Lactose/% | 4.41±0.10 | 4.29±0.30 | 0.315 |
| Dry matter/% | 12.29±0.15 | 12.25±0.23 | 0.798 |
(3) Milk fatty acid
At the end of the test period, 1 milk sample was collected from all the experimental sheep, and 20mL of the milk samples were collected in the morning and evening to prepare a mixed sample for fatty acid extraction and measurement.
The method for extracting fatty acid from goat milk comprises the following steps: placing the collected milk sample into a water bath kettle at 30 ℃ for incubation for 20 minutes, centrifuging for 30 minutes at 20 ℃ under the centrifugal force of 17800g, transferring the upper fat into a 2mL centrifuge tube after centrifuging, and centrifuging for 20 minutes at 19300 g; taking a 10mL glass tube to absorb 80 mu L of upper oily liquid in a centrifuge tube, adding 5mL of normal hexane, shaking and mixing uniformly; then 0.2mL of methyl esterification reagent (11.2 g KOH is dissolved in 100mL of methanol) is added, and the mixture is sealed, vortex and shake for 1 minute and then stand for 30 minutes at room temperature; then 0.5g of anhydrous sodium sulfate is added, and 350g is centrifuged for 3 minutes; 1mL of the supernatant was aspirated into a 2mL chromatographic bottle for gas chromatography. Determining fatty acid components by gas chromatography, and calculating various fatty acid contents in goat milk by using an area normalization method.
As can be seen from Table 5, the amounts of fatty acids C4:0, C16:0 and C18:1 in the goat milk were significantly increased (P < 0.05) and the amounts of other fatty acids were not significantly changed (P > 0.05) in the test group compared to the control group. The result shows that NAM added into daily ration has the function of improving the composition of the goat milk fatty acid, and the composition of the goat milk easy to digest and absorb fatty acid and the content of beneficial fatty acid are improved.
TABLE 5 Effect of NAM addition of ration on goat milk fatty acid composition
| Fatty acid species | Control (%) | Test group (%) | P value |
| C4:0 | 1.54±0.23 b | 1.77±0.21 a | 0.009 |
| C6:0 | 1.81±0.23 | 1.89±0.19 | 0.274 |
| C8:0 | 2.33±0.41 | 2.32±0.32 | 0.988 |
| C10:0 | 9.01±1.51 | 8.87±1.22 | 0.794 |
| C12:0 | 4.99±0.79 | 4.70±1.00 | 0.402 |
| C14:0 | 10.63±1.50 | 10.87±1.17 | 0.649 |
| C16:0 | 26.20±2.11 b | 28.16±2.62 a | 0.041 |
| C16:1 | 1.18±0.29 | 1.11±0.38 | 0.589 |
| C18:0 | 8.20±1.75 | 8.05±2.16 | 0.849 |
| C18:1 | 24.99±1.51 b | 26.08±1.15 a | 0.039 |
| C18:2 | 2.47±0.47 | 2.50±0.34 | 0.850 |
EXAMPLE 3 Effect of NAM at different concentrations on SIRT1 and milk fat synthetic transcription factor expression
To study the mechanism of action of NAM on goat milk butter fat and the effect of NAM on lipid metabolism at the cellular level, milk goat mammary epithelial cells were treated with NAM (0,100,300,500 μmol/L) at different concentrations, respectively.
(1) Cell culture and treatment
Separating mammary epithelial cells from mammary tissue of milk goat by pancreatin digestion, subculturing by high density inoculation method, culturing with DMEM/F12 cell culture medium containing 10% FBS, 100U/mL penicillin, 100 μg/mL streptomycin, 10ng/mL epidermal growth factor, 5 μg/mL hydrocortisone and 5 μg/mL insulin at 37deg.C, 5% CO 2 Culturing in an incubator for 24 hours, replacing a fresh culture medium, and performing subculture when the confluence of cells is more than 90% under an inverted microscope. Cells are inoculated into a 12-hole culture plate, NAM induction culture medium containing 0 mu mol/L (control), 100 mu mol/L, 300 mu mol/L and 500 mu mol/L is respectively added when the cell confluency reaches 60% -70%, the cells are collected after 48h of culture, and the cells are used for detecting the expression of genes related to milk fat synthesis in subsequent experiments, so that the optimal treatment concentration is determined.
(2) Extraction of total RNA from cells and synthesis of cDNA
After NAM treatment for 48 hours, cells were collected, and total RNA of the cells was extracted using the Beijing-day-root Biochemical technology Co.Ltd cell/tissue total RNA extraction kit, and the procedure was performed according to the specification. The absorbance ratio value of the RNA sample A260 nm/A280 nm is measured by an ultraviolet spectrophotometer (NANODROP 2000), the sample is qualified when the detection concentration is more than 200 ng/mu L and the purity is 1.8-2.0, the extracted RNA is used as a template, and all the detected qualified RNA is reversely transcribed into cDNA according to the specification steps of reverse transcription kit of Takara company.
(3) Real-time fluorescent quantitative PCR
The real-time quantitative primers of the milk fat synthesis gene and the reference gene are shown in Table 6, specific real-time fluorescent quantitative primers are respectively designed by utilizing Primer Premier 5.0 and Oligo 6 software according to the published goat gene sequences of GenBank, the method of crossing introns is adopted, the upstream Primer and the downstream Primer of each pair of quantitative primers are distributed on different exons, the length of amplified products is about 200bp, and each Primer sequence is synthesized by Shanghai biological engineering Co.
The test uses GAPDH and UXT genes as internal reference for real-time fluorescence quantitative PCR reaction. PCR reaction system 20. Mu.L: 10.0. Mu.L of fluorescent quantitative PCR reaction mixture (2X SYBR Premix Ex Taq Mix), 1.0. Mu.L of cDNA template, 0.8. Mu.L of each of the upstream and downstream primers (10. Mu. Mol/L), and RNase-free H were added 2 O makes up the system. The reaction is carried out on an ABI 7500 fluorescence quantitative PCR instrument, and the reaction conditions are as follows: 95 ℃ for 30s,95 ℃ for 5s,60 ℃ for 30s,40 cycles; a melting curve is added. By 2 －△△Ct The data were analyzed by the method, wherein Δct=ct Target gene -Ct Internal reference ；△△Ct＝△Ct Test group -△Ct Control group . Each sample was set up with 3 replicates.
TABLE 6 real-time quantitative primer sequences
As a result, it was found that 300. Mu. Mol/L NAM down-regulated the mRNA expression of the sirtuin 2-related enzyme 1 (SIRT 1) and FoxO1, whereas 100. Mu. Mol/L NAM had no effect on SIRT1 and FOXO1 expression and 500. Mu. Mol/L NAM had no significant effect on FOXO1 expression (FIG. 1A); NAM at 100. Mu. Mol/L and 300. Mu. Mol/L significantly up-regulated the creamer key regulator PPARgamma (P < 0.05) (FIG. 1B), thus judging that 300. Mu. Mol/L is the optimal concentration. Cells were treated with 300. Mu. Mol/L NAM, and it was found that the expression levels of fatty acid synthase FASN and fatty acid desaturase SCD1 were significantly up-regulated (P < 0.05), and the expression of lipolytic gene ATGL was significantly down-regulated (P < 0.05) (FIG. 2).
Example 4 Effect of NAM on mammary cell triglyceride and fatty acid Synthesis
(1) Triglyceride (TAG) content determination
Treating milk goat mammary epithelial cells with NAM of 300 μmol/L for 48h, digesting with 0.25% trypsin/EDTA mixture, collecting cell precipitate, washing the precipitate with PBS for 2 times, transferring the cells into 1.5mL centrifuge tubes, adding 200 μL PBS into each tube, ultrasonically crushing the cells, and detecting intracellular triglyceride according to the operation method given by the cell triglyceride enzyme assay kitHeating appropriate amount of lysate in 70deg.C water bath for 10min, centrifuging at room temperature for 5min at 2000r/min, retaining supernatant for enzymatic determination, reacting 10 μl supernatant with 190 μl working solution at 37deg.C for 10min, and measuring absorbance (D) at 550nm wavelength with full-automatic enzyme marker 550nm )。
(2) Oil red O dyeing
After 48h of NAM treatment of the cells, the medium was discarded and washed twice with PBS; adding 10% neutral formaldehyde onto the culture plate, and fixing cells for 45min; adding 1mL of oil red O into each hole of cells for dyeing for 30min; finally, the sample is washed 3 times by PBS, and is observed and photographed under an inverted microscope.
(3) Determination of fatty acids in cells
Mammary gland epithelial cells are cultured in a 60mm cell culture dish, after NAM treatment for 48 hours, the culture medium is discarded, PBS is used for washing, then 0.25% trypsin/EDTA mixed solution is used for digestion, and cell sediment is collected in an 8mL glass tube, 2mL sulfuric acid/methanol solution with the volume ratio of 0.25% is added, after ultrasonic treatment for 10 minutes, incubation is carried out at 80 ℃ for 1 hour, after the temperature is cooled to room temperature, 2mL hydrochloric acid solution with the volume ratio of 0.1mol/L and 800 mu L of n-hexane are added, vortex shaking for 30s,900g is carried out for centrifugation for 5 minutes, supernatant is collected into a 2mL siliconized glass tube, 0.5g anhydrous sodium sulfate is added for absorbing redundant moisture, the mixture is placed for 12-14 hours, 13800g is centrifuged for 3 minutes after shaking, the supernatant is absorbed, and fatty acid components in the cells are analyzed by a GC-MS gas chromatography, and C19:0 fatty acid is used as a measurement standard. The relative proportions of the various fatty acids were calculated as the percentage of the area of the fatty acid peak, with 3 biological replicates per treatment set.
As a result, it was found that, after treatment of cells with niacinamide, the triglyceride content in the cells increased significantly (P < 0.05) (FIG. 3A), the oil red O staining showed increased accumulation of lipid droplets (FIG. 3B), the ratio of fatty acids C16:0 and C18:1 increased significantly (P <0.05, FIGS. 4B, 4D), while the ratio of C14:0 and C18:0 did not change significantly in the cells (FIGS. 4A, 4C). The results show that the niacinamide can promote the lipid synthesis of mammary epithelial cells of the dairy goat, change the content of beneficial fatty acids in the mammary cells, and provide basic data for further improving the fatty acid composition, the nutritional value and the flavor of the goat milk.
EXAMPLE 5 SIRT1-mediated NAM transcriptional control of FASN Gene
(1) Cloning and structural analysis of FASN gene promoter
Previous studies found that NAM changed milk fat synthesis and milk fat acid composition at cellular level, in order to intensively study the regulation mechanism of SIRT 1-mediated NAM on fatty acid composition in cells, we cloned the FASN promoter, a key enzyme for fatty acid synthesis, and studied the transcriptional regulation mechanism of NAM on FASN.
And obtaining the FASN promoter sequence of the milk goat by using a PCR method. The nucleotide sequence in NCBI is compared to find that the homology of FASN gene promoter sequence and cow, human and mouse reaches over 90%. The core region of the FASN promoter has a plurality of characteristic promoter elements and potential transcription factor binding sites, such as CAAT box, TATABOX, GC-box, E-box, transcription factors AP2, sp1, NF-Y and binding Sites (SRE) of two SREBPs 1, which are found by on-line software predictive analysis of transcription factors (FIG. 5). In conjunction with earlier studies, SREBP1 was found to regulate transcription of the FASN promoter.
(2) SIRT 1-mediated effect of NAM on FASN promoter Activity
Cells were treated with SIRT1 inhibitor (NAM) while the FASN promoter luciferase reporter vector was transfected and FASN promoter activity was detected using a dual luciferase system.
Luciferase activity assay: after cells are transfected with FASN promoter recombinant plasmid for 48 hours, the cells are collected, luciferase activity determination is carried out according to a Promega dual-reporter gene detection kit, culture solution is discarded, PBS is used for washing twice, 20 mu L of cell lysate is added into each hole, and the cells are incubated for 20 minutes at room temperature. Sucking 20 mu L of lysate into 96 Kong Baiban, adding 100 mu L of LAR II, rapidly mixing, and immediately detecting pGL3 luciferase activity (F value) on a multifunctional microplate luminometer; pRL-TK luciferase activity (R value) was detected by adding 100. Mu.L of 1 XStop & Glo solution. The calculated F/R value, i.e., relative luciferase activity, is used to represent the relative transcriptional activity of the promoter.
The experimental results show that: the FASN promoter activity was significantly increased (P < 0.05) after NAM treatment (FIG. 6).
(3) SREBP1 mediated effect of NAM on FASN promoter Activity
In order to study the effect of SREBP1 in SIRT1 mediated NAM regulation of FASN transcription, site-directed mutation is carried out on SRE1 and SRE2 sites on the FASN promoter respectively, recombinant vector transfected cells are constructed, and the change condition of FASN promoter activity before and after mutation is detected. Site-directed mutagenesis was performed by overlap extension PCR, and the site-directed mutagenesis primer information is shown in Table 7:
TABLE 7 site-directed mutagenesis primer
Note that: wherein italics are enzyme cleavage sites and bold are mutation sites.
The mutated fragment was ligated to the luciferase reporter vector and sequenced to confirm that the mutation was successful (fig. 7).
As a result, it was found that the FASN promoter activity was significantly decreased (P < 0.05) after SRE site mutation (FIG. 8). However, treatment of cells with NAM found that NAM significantly increased FASN wild-type promoter activity (P < 0.05) while there was no effect on SRE1 and SRE2 mutant promoter activity (FIG. 9).
The results can confirm that SIRT1 mediated NAM regulates the activity of a FASN gene promoter of the milk goat by regulating SREBP1, and provides a theoretical basis for improving the fatty acid composition and the flavor of the goat milk.
The foregoing description of the preferred embodiment of the invention is illustrative only, and various modifications and changes in the invention will be apparent to those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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<213> Artificial sequence ()
<400> 20
gatacaggct ccactttgat tgc 23
<210> 21
<211> 19
<212> DNA
<213> Artificial sequence ()
<400> 21
ccatcgcctg tggagtcac 19
<210> 22
<211> 22
<212> DNA
<213> Artificial sequence ()
<400> 22
gtcggataaa tctagcgtag ca 22
<210> 23
<211> 21
<212> DNA
<213> Artificial sequence ()
<400> 23
cagcatcttg cctgatttgt a 21
<210> 24
<211> 19
<212> DNA
<213> Artificial sequence ()
<400> 24
ctgggcatct aggacatcg 19
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 25
acgccatcga gaaacgctac 20
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 26
gtgcgcagac tcaggttctc 20
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 27
tgtggccctt ggatatggtt 20
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 28
ggttgtcgct gagctctgtg 20
<210> 29
<211> 29
<212> DNA
<213> Artificial sequence ()
<400> 29
attacgcgta agaggtgtcc gtgcatagg 29
<210> 30
<211> 24
<212> DNA
<213> Artificial sequence ()
<400> 30
cggcgcgccg catgacggca ctgg 24
<210> 31
<211> 24
<212> DNA
<213> Artificial sequence ()
<400> 31
cggcgcgccg cataacttca ctgg 24
<210> 32
<211> 30
<212> DNA
<213> Artificial sequence ()
<400> 32
cagccaagct gtcagcccat gtggcgtgtc 30
<210> 33
<211> 30
<212> DNA
<213> Artificial sequence ()
<400> 33
cagccaagct gtcagtttat gtggcgtgtc 30
<210> 34
<211> 28
<212> DNA
<213> Artificial sequence ()
<400> 34
ggaagatctg ggttcccgac tcacaact 28
Claims (4)
1. Application of niacinamide in improving milk yield of milk goat in lactation period is provided.
2. Use of niacinamide for improving the milk composition of a dairy goat during lactation, wherein the milk composition is milk fat and milk fatty acids, and the milk fatty acids are c4:0, c16:0 and c18:1.
3. Application of niacinamide to regulation of transcription factor SREBP1 in FASN gene transcription.
4. Use according to any one of claims 1-3, wherein niacinamide is added to the dairy goat base ration in an amount of 5g/d.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101061832A (en) * | 2006-04-27 | 2007-10-31 | 韩凌云 | Premix feed specially for high yield cow in perinatal period and the method of producing the same |
| CN101675165A (en) * | 2006-12-08 | 2010-03-17 | 奥斯瑞根公司 | The function of LET-7 Microrna and target |
| CN104026388A (en) * | 2014-06-03 | 2014-09-10 | 赵兰 | Female Huzhou sheep feed used in milk production period and preparation method of female Huzhou sheep feed |
| AU2013204272A1 (en) * | 2009-10-07 | 2014-10-30 | Ceva Sante Animale Sa | Veterinary Antiprolactinic Composition for Ruminants |
| WO2016083481A1 (en) * | 2014-11-26 | 2016-06-02 | Dupont Nutrition Biosciences Aps | Strains and methods for energy partitioning in ruminants |
| CN107006698A (en) * | 2017-04-18 | 2017-08-04 | 柳州市康小乐牛奶有限公司 | It is a kind of to improve TRM of the lactating cow output of milk and milk-quality and preparation method thereof suitable for southern |
| WO2018049243A1 (en) * | 2016-09-09 | 2018-03-15 | Omnigen Research, Llc | Feed additive comprising allicin |
| CN108719194A (en) * | 2018-06-07 | 2018-11-02 | 西北农林科技大学 | Enhance that lamb is anti-oxidant and product, the method and its application of immune function by adding niacinamide/or niacin to perinatal period milch goat |
| CN108813169A (en) * | 2018-05-09 | 2018-11-16 | 赤峰中正饲料有限公司 | Promote the beneficiated burden material complemental feed and preparation method thereof of Small-fat-tail sheep ewe lactation amount |
| CN111084283A (en) * | 2018-10-23 | 2020-05-01 | 孙亚妮 | Complete granulated feed for Guanzhong dairy goats in lactation period and preparation method thereof |
| CN113142398A (en) * | 2021-04-27 | 2021-07-23 | 张自力 | Functional feed additive for ruminants and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103732745A (en) * | 2011-06-15 | 2014-04-16 | Nse产品公司 | Identifying markers of caloric restriction and caloric restriction mimetics |
-
2021
- 2021-08-31 CN CN202111013715.7A patent/CN113712121B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101061832A (en) * | 2006-04-27 | 2007-10-31 | 韩凌云 | Premix feed specially for high yield cow in perinatal period and the method of producing the same |
| CN101675165A (en) * | 2006-12-08 | 2010-03-17 | 奥斯瑞根公司 | The function of LET-7 Microrna and target |
| AU2013204272A1 (en) * | 2009-10-07 | 2014-10-30 | Ceva Sante Animale Sa | Veterinary Antiprolactinic Composition for Ruminants |
| CN104026388A (en) * | 2014-06-03 | 2014-09-10 | 赵兰 | Female Huzhou sheep feed used in milk production period and preparation method of female Huzhou sheep feed |
| WO2016083481A1 (en) * | 2014-11-26 | 2016-06-02 | Dupont Nutrition Biosciences Aps | Strains and methods for energy partitioning in ruminants |
| WO2018049243A1 (en) * | 2016-09-09 | 2018-03-15 | Omnigen Research, Llc | Feed additive comprising allicin |
| CN107006698A (en) * | 2017-04-18 | 2017-08-04 | 柳州市康小乐牛奶有限公司 | It is a kind of to improve TRM of the lactating cow output of milk and milk-quality and preparation method thereof suitable for southern |
| CN108813169A (en) * | 2018-05-09 | 2018-11-16 | 赤峰中正饲料有限公司 | Promote the beneficiated burden material complemental feed and preparation method thereof of Small-fat-tail sheep ewe lactation amount |
| CN108719194A (en) * | 2018-06-07 | 2018-11-02 | 西北农林科技大学 | Enhance that lamb is anti-oxidant and product, the method and its application of immune function by adding niacinamide/or niacin to perinatal period milch goat |
| CN111084283A (en) * | 2018-10-23 | 2020-05-01 | 孙亚妮 | Complete granulated feed for Guanzhong dairy goats in lactation period and preparation method thereof |
| CN113142398A (en) * | 2021-04-27 | 2021-07-23 | 张自力 | Functional feed additive for ruminants and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 奶山羊围产期添加烟酰胺对羔羊腹脂脂质代谢的影响;赵会会等;农业生物技术学报;第26卷(第9期);1527-1534 * |
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