CN114431186A - Feeding method for increasing selenium content in milk - Google Patents
Feeding method for increasing selenium content in milk Download PDFInfo
- Publication number
- CN114431186A CN114431186A CN202011197978.3A CN202011197978A CN114431186A CN 114431186 A CN114431186 A CN 114431186A CN 202011197978 A CN202011197978 A CN 202011197978A CN 114431186 A CN114431186 A CN 114431186A
- Authority
- CN
- China
- Prior art keywords
- selenium
- rich
- milk
- corn
- silage
- 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.)
- Pending
Links
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 title claims abstract description 247
- 239000011669 selenium Substances 0.000 title claims abstract description 247
- 229910052711 selenium Inorganic materials 0.000 title claims abstract description 247
- 235000013336 milk Nutrition 0.000 title claims abstract description 75
- 239000008267 milk Substances 0.000 title claims abstract description 75
- 210000004080 milk Anatomy 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 21
- 230000001965 increasing effect Effects 0.000 title description 8
- 240000008042 Zea mays Species 0.000 claims abstract description 89
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims abstract description 89
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims abstract description 88
- 235000005822 corn Nutrition 0.000 claims abstract description 88
- 241000283690 Bos taurus Species 0.000 claims abstract description 54
- 239000004460 silage Substances 0.000 claims abstract description 52
- 235000016709 nutrition Nutrition 0.000 claims abstract description 46
- 239000003337 fertilizer Substances 0.000 claims abstract description 28
- 208000003643 Callosities Diseases 0.000 claims abstract description 24
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 24
- 238000003306 harvesting Methods 0.000 claims abstract description 15
- 238000005507 spraying Methods 0.000 claims abstract description 14
- 238000000855 fermentation Methods 0.000 claims abstract description 11
- 230000004151 fermentation Effects 0.000 claims abstract description 11
- 235000013365 dairy product Nutrition 0.000 claims abstract description 9
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 claims description 30
- 230000035764 nutrition Effects 0.000 claims description 28
- 241000196324 Embryophyta Species 0.000 claims description 15
- 229930003427 Vitamin E Natural products 0.000 claims description 15
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 claims description 15
- 235000019165 vitamin E Nutrition 0.000 claims description 15
- 229940046009 vitamin E Drugs 0.000 claims description 15
- 239000011709 vitamin E Substances 0.000 claims description 15
- 238000002360 preparation method Methods 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 7
- 238000009331 sowing Methods 0.000 claims description 7
- 230000007226 seed germination Effects 0.000 claims description 5
- 241001057636 Dracaena deremensis Species 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 235000015872 dietary supplement Nutrition 0.000 claims description 3
- 235000020185 raw untreated milk Nutrition 0.000 abstract description 8
- 230000036541 health Effects 0.000 abstract description 5
- 229940091258 selenium supplement Drugs 0.000 description 199
- 239000008280 blood Substances 0.000 description 19
- 210000004369 blood Anatomy 0.000 description 19
- 230000002354 daily effect Effects 0.000 description 14
- 238000001514 detection method Methods 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 12
- 238000005516 engineering process Methods 0.000 description 10
- 239000003963 antioxidant agent Substances 0.000 description 9
- 230000003078 antioxidant effect Effects 0.000 description 9
- 235000006708 antioxidants Nutrition 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 229940118019 malondialdehyde Drugs 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000005070 sampling Methods 0.000 description 5
- 235000013305 food Nutrition 0.000 description 4
- 238000003825 pressing Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 206010039921 Selenium deficiency Diseases 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 238000007726 management method Methods 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000003674 animal food additive Substances 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 2
- 235000021056 liquid food Nutrition 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 201000008482 osteoarthritis Diseases 0.000 description 2
- 235000021485 packed food Nutrition 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- -1 10 parts by weight Chemical compound 0.000 description 1
- 108010027612 Batroxobin Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 208000019926 Keshan disease Diseases 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 102000014171 Milk Proteins Human genes 0.000 description 1
- 108010011756 Milk Proteins Proteins 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 206010057178 Osteoarthropathies Diseases 0.000 description 1
- 238000001069 Raman spectroscopy Methods 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 229960002210 batroxobin Drugs 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 230000036983 biotransformation Effects 0.000 description 1
- 230000014461 bone development Effects 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 235000020247 cow milk Nutrition 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000002795 fluorescence method Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 235000020604 functional milk Nutrition 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 229960001008 heparin sodium Drugs 0.000 description 1
- 244000144980 herd Species 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000006651 lactation Effects 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 229940127554 medical product Drugs 0.000 description 1
- 235000021243 milk fat Nutrition 0.000 description 1
- 235000021239 milk protein Nutrition 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 238000009928 pasteurization Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- 235000015921 sodium selenite Nutrition 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
- A01K67/02—Breeding vertebrates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
-
- 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
- A23K20/174—Vitamins
-
- 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
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/30—Oligoelements
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
Abstract
The invention provides a feeding method for improving selenium content in milk, which is characterized in that selenium-rich silage corns are fed to a lactating cow, wherein the selenium-rich silage corns are obtained by spraying an organic selenium-rich foliar fertilizer on the corns in the jointing stage to the milk stage of the corns, and performing sealed fermentation after harvesting; and/or feeding the dairy cattle with daily ration added with a selenium-rich functional nutritional bag, wherein the selenium-rich functional nutritional bag comprises inorganic selenium and organic selenium. According to the invention, the selenium-rich milk can be produced, the selenium content and structure enriched in the raw milk are stable, the product stability is good, the health of the cow organism is not affected, and the practicability of the invention is high.
Description
Technical Field
The invention relates to the field of milk production, in particular to a method for improving selenium content in milk by feeding selenium-rich feed for lactating cows.
Background
Selenium is a trace element essential to human body. The Chinese nutritional society also ranks selenium as one of the 15 nutrients essential to human body, and a large number of clinical experiments at home and abroad show that the selenium deficiency of human body can cause dysfunction of some important organs, thus causing a plurality of serious diseases.
Selenium deficiency is a significant cause of keshan disease, and is also considered to be a significant cause of Kaschin-Beck disease. Kaschin-Beck disease is an endemic, multiple and deformed osteoarthropathy. It mainly occurs in teenagers and seriously affects bone development and the ability to work and live in the future. Research shows that the people with low selenium or selenium deficiency can prevent tumor, liver disease, etc. by supplementing selenium with proper amount, improve immunity, maintain normal functions of heart, liver, lung, stomach and other important organs, and prevent senile cardiovascular and cerebrovascular diseases.
The selenium storage capacity in human body is very weak, and the selenium-containing food needs to be eaten frequently to obtain enough selenium, so that the development of the selenium-rich food has important significance.
Milk is one of the oldest natural beverages, contains rich nutrient substances and is known as 'white blood'. The selenium can be accumulated by feeding the lactating cows with the selenium-rich feed, and the selenium-rich feed is eaten by people and has the advantages of economy, convenience and high efficiency.
Disclosure of Invention
Technical problem to be solved
The invention aims to provide a method for producing selenium-rich milk, which is characterized in that high-quality selenium-rich milk is produced for people to eat by feeding lactating cows with selenium-rich feed.
The invention also aims to provide the selenium-rich feed for feeding the lactating cows, which comprises the selenium-rich silage corns and the selenium-rich functional nutrition bags.
Technical scheme for solving problems
The invention provides three technologies for improving the selenium content in raw milk, wherein one technology is that the most common corn silage with the largest use amount in daily ration of a milk cow is selected to prepare organic selenium-rich corn silage, and the selenium content in the raw milk is improved after the biological conversion of the milk cow. And secondly, a selenium-rich functional nutrition bag is researched and developed, and the selenium content in the milk is improved by enhancing the biological conversion of the milk cow organism through daily ration feeding of the milk cow. Thirdly, on the basis of feeding selenium-rich corn silage, a selenium-rich functional nutrition bag is added to strengthen the selenium content in TMR (Total Mixed Rations), and after the cow eats the feed, the cow carries out biotransformation through the cow organism to improve the selenium content in the milk.
Specifically, the present invention provides the following technical solutions.
According to one aspect of the invention, the feeding method for improving the selenium content in milk is characterized in that selenium-rich silage corns are fed to a lactating cow, the selenium-rich silage corns are obtained by spraying an organic selenium-rich foliar fertilizer on the corns in the corn jointing stage to the large horn mouth stage, and the selenium-rich silage corns are subjected to sealed fermentation after being harvested.
According to a further aspect of the invention, a feeding method for increasing the selenium content in milk is provided, characterized in that dairy cows are fed a daily ration supplemented with a selenium-rich functional nutritional package comprising inorganic selenium and organic selenium.
In the present invention, the above two feeding methods are preferably employed simultaneously.
The invention also provides a preparation method of the selenium-rich silage corn, and the selenium-rich silage corn is prepared by spraying a selenium-rich organic foliar fertilizer on the corn through mechanical equipment such as a plant protection operation machine or a plant protection unmanned aerial vehicle, harvesting through large machinery at the later stage, and pressing a cellar for fermentation. The organic selenium content concentration of the organic selenium foliar fertilizer is 4-10 g/L, such as 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L and 10 g/L. By controlling the content within the range, the toxic action of the inorganic selenium fertilizer on the corn and the risk brought to the health of the dairy cows by later feeding can be avoided, the loss in the process of inorganic selenium absorption effect and conversion into organic selenium is avoided, and the planting cost of the silage corn is considered at the same time.
The preparation method of the selenium-rich silage corn comprises the steps of spraying an organic selenium-rich foliar fertilizer on the corn in the jointing stage to the milk stage and 7-14 leaves of the corn, and performing sealed fermentation for 60-180 days after harvesting, wherein the organic selenium content concentration of the organic selenium-rich foliar fertilizer is preferably 4-10 g/L, and the spraying dosage is 200 ml/mu each time.
The preparation method of the selenium-rich silage corn comprises the following steps:
sowing: selecting corn varieties with the seed germination rate of more than 95 percent and the growth period of 95-110 days, and sowing the corn varieties according to the density of 4000-5000 plants/mu;
spraying: and (3) taking 200ml of organic selenium leaf fertilizer with the organic selenium concentration of 4-10 g/L and adding 10-20 kg of water when 7-10 leaves exist in the corn jointing stage-large horn mouth stage, and uniformly spraying the organic selenium leaf fertilizer on corn plants with the land amount of one mu by using a plant protection operation machine.
In the male-drawing period to the milk-ripe period of the corn and 10-14 leaves, 200ml of organic selenium leaf fertilizer with the concentration of 4-10 g/L is taken, 1-2 kg of water is added, and the organic selenium leaf fertilizer is sprayed on the corn with the amount of one mu of land by using a plant protection unmanned aerial vehicle.
The above-mentioned two spraying operations are preferably carried out in succession.
The selenium-rich silage corns obtained by the preparation method of the selenium-rich silage corns are used for feeding the lactating cows, and the feeding amount is 20-25 kg/head/day.
Still another aspect of the present invention provides a selenium-rich functional nutritional supplement including inorganic selenium and organic selenium, wherein 10 to 50 parts by weight of the inorganic selenium and 20 to 90 parts by weight of the organic selenium are included, for example, 10 parts by weight, 20 parts by weight, 30 parts by weight, 40 parts by weight, 50 parts by weight of the inorganic selenium and 20 parts by weight, 30 parts by weight, 40 parts by weight, 50 parts by weight, 60 parts by weight, 70 parts by weight, 80 parts by weight, 90 parts by weight of the organic selenium are included, based on 100 parts by weight of the total weight of the selenium-rich functional nutritional supplement,
preferably, the vitamin E further comprises 10-30 parts by weight of vitamin E, such as 10 parts by weight, 20 parts by weight and 30 parts by weight.
The selenium-rich functional nutritional bag is used for feeding lactating cows, and the addition amount of the selenium-rich functional nutritional bag is 1 g-5 g/head/day, such as 1 g/head/day, 2 g/head/day, 3 g/head/day, 4 g/head/day and 5 g/head/day.
Preferably, the selenium-rich functional nutrition package is additionally supplemented with yeast cultures, and the addition amount of the yeast cultures is 100 g-150 g/head/day, such as 100 g/head/day, 110 g/head/day, 120 g/head/day, 130 g/head/day, 140 g/head/day and 150 g/head/day.
Advantageous effects
According to the preparation technology of the selenium-rich corn silage, the intake of selenium in the whole corn silage, which is a main feed raw material of the dairy cow, can be improved, and the absorption rate of the dairy cow to the selenium in daily ration can be improved by feeding the selenium-rich silage corn. The selenium element has the effects of regulating the metabolism of the cow organism, improving the immunity of the cow, relieving the stress of the herd, strengthening the selenium nutrition of the cow and improving the content of the selenium in the raw milk.
According to the technical scheme of the selenium-rich functional nutrition bag, the invention provides a feed formula technology for improving the selenium content of milk, and the selenium content in the milk is improved by enhancing the biological conversion of a milk cow organism through daily ration feeding of the milk cow.
According to the feeding technology for improving the selenium content of the milk, the selenium-rich functional nutrition bag is added on the basis of feeding the selenium-rich corn silage, so that the selenium content in the TMR daily ration is enhanced, and the milk is subjected to biological conversion through the milk cow organism after the milk cow eats the feed, so that the selenium content in the milk is improved.
The raw milk rich in selenium and vitamin E obtained by the technical scheme is formed by metabolism of a milk cow organism, is easier to be absorbed by a human body compared with an exogenously added product, has higher contents of endogenous selenium and vitamin E compared with common milk, and is primary selenium-rich functional milk.
Detailed Description
Hereinafter, the present invention will be described in more detail with reference to specific embodiments for further illustrating technical features, objects and advantages of the present invention, but the scope of the present invention is not limited thereto.
The raw materials and equipment used in the invention are all the raw materials and equipment which are commonly used in the field if not specifically stated, and the method used in the invention is all the conventional method in the field if not specifically stated.
The organic selenium-rich foliar fertilizer adopts green vitamin-rich organic selenium-rich fertilizer produced by Shaanxi Yanling Australian Pont bioscience GmbH, the specification is 200 ml/bottle, and the total selenium content is more than or equal to 6 g/L.
The inorganic selenium is inorganic selenium feed additive produced by Hengyang Huadi feed additive limited company, and the content of sodium selenite (calculated by selenium) is more than or equal to 1 percent.
The organic selenium is obtained from Raman France, and the selenium content in yeast is not less than 2000 ppm.
Vitamin E is a Zhejiang medical product, and the vitamin E is more than or equal to 50 percent.
The yeast culture adopts Yikang XP produced by Danongwei biological fermentation engineering technology (Shenzhen) Limited.
EXAMPLE 1 preparation of selenium-containing silage maize with different fertilisation treatments
1) After harvesting the previous crops, deeply ploughing and harrowing the land, selecting corn varieties with the seed germination rate of more than 95 percent and the growth period of 95-110 days, and sowing the corn varieties according to the density of 4000-5000 plants/mu.
2) When 8-10 leaves of corn are planted, 200ml of organic selenium-rich foliar fertilizer with the organic selenium concentration of 4g/L is taken and diluted by adding 15kg of water, and the organic selenium-rich foliar fertilizer is uniformly sprayed on corn plants with the land size of 1 mu by using a plant protection operation machine.
3) During harvesting, a large-scale ensiling harvesting machine and a cellar pressing and sealing device are used for operation, sealed fermentation is carried out for 150 days, 5 samples are detected by opening the cellar, and the selenium content in the obtained selenium-rich ensiling corns is between 0.41 and 0.69 mg/kg.
EXAMPLE 2 preparation of selenium-containing silage corn with different fertilisation treatments
1) After harvesting the previous crops, deeply ploughing and harrowing the land, selecting corn varieties with the seed germination rate of more than 95 percent and the growth period of 95-110 days, and sowing the corn varieties according to the density of 4000-5000 plants/mu.
2) When 12-14 leaves of corn are planted, 200ml of organic selenium leaf fertilizer with the organic selenium concentration of 4g/L is taken, 1.5kg of water is added, and the organic selenium leaf fertilizer is sprayed on corn with the amount of 1 mu of land by using a plant protection unmanned aerial vehicle.
3) During harvesting, a large ensiling harvesting machine and a cellar pressing and sealing device are used for operation, sealed fermentation is carried out for 150 days, 5 samples are detected by opening the cellar, and the selenium content of the obtained selenium-enriched ensiling corn is about 0.25-0.43 mg/kg.
Example 3 preparation of selenium-containing silage corn with different fertilisation treatments
1) After harvesting the previous crops, deeply ploughing and harrowing the land, selecting corn varieties with the seed germination rate of more than 95 percent and the growth period of 95-110 days, and sowing the corn varieties according to the density of 4000-5000 plants/mu.
2) When 8-10 leaves of corn are planted, 200ml of organic selenium-rich foliar fertilizer with the organic selenium concentration of 4g/L is taken and diluted by adding 15kg of water, and the organic selenium-rich foliar fertilizer is uniformly sprayed on corn plants with the land size of 1 mu by using a plant protection operation machine.
3) When 12-14 leaves of corn are planted, 200ml of organic selenium leaf fertilizer with the organic selenium concentration of 4g/L is taken, 1.5kg of water is added, and the organic selenium leaf fertilizer is sprayed on the corn with the amount of 1 mu of land again by using a plant protection unmanned aerial vehicle.
4) During harvesting, a large ensiling harvesting machine and a cellar pressing and sealing device are used for operation, sealed fermentation is carried out for 150 days, 5 samples are detected by opening the cellar, and the content of the obtained selenium-rich ensiling corn is about 0.85-1.23 mg/kg.
Experimental example 1 research experiment on preparation technology of selenium-enriched corn silage
The selenium-rich corn prepared in the above examples 1 to 3 is stored in a cellar, and then starch, crude protein, neutral detergent fiber, acidic detergent fiber and pH value are measured, and the detection methods refer to national standards. The selenium detection method refers to the determination method of selenium in feed of national standard GB/T13883-2008.
From the measurement results, the content of the selenium in the corn silage prepared in the embodiment 3 is obviously higher than that in the embodiments 1-2, the selenium-rich silage corns prepared in the embodiments 1, 2 and 3 meet the silage quality standard, and the nutrition indexes are not obviously different. See table 1 for details.
TABLE 1 measurement results of physicochemical indexes of silage of selenium-enriched silage corn with different concentrations
Note: the shoulder marks containing different letters indicate significant statistical differences, and the same letters indicate no significant statistical differences, as follows.
Experimental example 2 silage feeding experiment for selenium-enriched corn with different concentrations
In order to further screen and research the feeding concentration and content of the selenium-rich corn silage, 22kg of the selenium-rich silage corn prepared in the embodiment 1-3 is selected to be mixed with other feed components according to a formula (see a basic daily ration formula table in a table 4), a high-yield Holstein lactating cow is fed for 120 days in total, milk samples are collected in 30 days before an experiment and in an experimental period in a tracking way, a vat mixed milk sample is collected once every 30 days, and the selenium content and the milk components in the milk of the cow are compared through different treatments; simultaneously randomly selecting 20 cows, tracking and collecting blood samples 1 month before and during the experiment, collecting 1 time every 30 days, and detecting the selenium content in the blood and the antioxidant index in the blood, wherein the conventional milk index detection method is DHI conventional detection, and the selenium content in the milk is GB 5009.93-2017 first method; the components and proportion of the daily grain, the sampling method, the detection index and the detection method are all carried out according to the method of the experimental example 3.
From the experimental results of the example groups, compared with the blank control group, the selenium content in milk in the example groups 1, 2 and 3 is respectively increased from 1.68 mu g/100g to 4.21 mu g/100g, 3.43 mu g/100g and 5.67 mu g/100g, and the selenium content in the milk fed to the lactating cow in the example group 3 is the highest, and can reach the selenium content specified in the "pre-packaged food nutrition labeling convention" (GB28050-2011), which is specifically shown in Table 2.
TABLE 2 feeding selenium content variation trends in corn silage cows with different concentrations of selenium
Each milk index and each blood antioxidant capacity index are average values of sampling detection within 120 days, wherein the content difference of the milk indexes is not obvious, the milk quality is not influenced, the average blood selenium content and the total antioxidant capacity (T-AOC) in each embodiment group are positively correlated with the selenium content in milk, and the Malondialdehyde (MDA) content is negatively correlated with the selenium content in milk, which is specifically shown in Table 3.
Table 3 influence of feeding corn silage rich in selenium at different concentrations on various milk and blood indices
Example 4 feeding selenium-enriched functional nutritional bag I
The selenium-rich functional nutritional package in this embodiment comprises: 50 parts of inorganic selenium, 20 parts of organic selenium and 30 parts of vitamin E, and 1g of vitamin E is added into the daily ration per head per day.
Example 5 feeding of selenium-enriched functional nutritional bag II
The selenium-rich functional nutritional package in this embodiment comprises: 20 parts of inorganic selenium, 50 parts of organic selenium and 30 parts of vitamin E, and 3g of vitamin E is added into the daily ration.
Example 6 feeding of selenium-enriched functional nutritional formula III
The selenium-rich functional nutritional package in this embodiment comprises: 20 parts of inorganic selenium, 50 parts of organic selenium and 30 parts of vitamin E, and 5g of the vitamin E is added into daily ration. The yeast culture was supplemented additionally 120 g/head/day.
Experimental example 3 feeding experiment of different selenium-rich functional nutrition packages
1. Experimental methods
In order to research and screen the optimal component combination of the selenium-rich functional nutrition package, the selenium-rich functional nutrition package composed of different components prepared in the embodiments 4-6 is selected and added into a basic daily ration formula to feed high-yield Hostan lactating cows. The experiment was divided into four groups, example 4, example 5 and example 6, and a blank control group, i.e. high yield holstein lactating cows fed with the basic ration formula, was also set.
1.1 daily ration formula
The composition and ratio of the ration are shown in table 4.
TABLE 4 basic daily ration prescription Table
Note: the high yield 50 concentrate is a trade name and is purchased from inner Mongolia Muquangxing fodder GmbH.
1.2 Experimental animals and raising management
The experimental animals are healthy Holstein lactating cows with the lactation period of 90-120 days, are raised in a scattered hurdle bed-lying mode, and are milked in parallel 3 times each day. TMR is fed for three times every day, and water is freely drunk; specially-assigned people are responsible for feeding and milking, and other management is executed according to a cattle farm management method.
1.3 sample Collection and detection
Collecting large jar milk sample 1 time every 30 days, freezing and storing for inspection. 1.0g of milk sample is digested by 10ml of mixed solution of nitric acid and perchloric acid, 2ml of hydrochloric acid is added, and then the selenium content in milk is determined by adopting inductively coupled plasma mass spectrometry (ICP-MS/MS).
20 cows were randomly selected and blood samples were collected 1 month and 1 time every 30 days, following the test period. The tail vein was used to collect 5mL of blood sample in a vacuum tube coated with heparin sodium, and then centrifuged at 3000 g for 10min using a centrifuge to obtain plasma, which was stored at-20 ℃. The kit is used for measuring the content of Malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in serum, and the atomic fluorescence method is used for measuring the content of blood selenium.
1.4 data collation analysis
The experimental data are sorted by Excel 2003 software, and the data are subjected to one-factor variance analysis by using a GLM program of SPSS 20.0, wherein the significant difference is that P is less than 0.05, and the difference trend is that P is more than or equal to 0.05 and less than 0.10.
2. Results of the experiment
Comparing the influence of different treatments on the selenium content in milk, milk components, the selenium content in blood and antioxidant indexes in blood of the dairy cows after 120 days of feeding.
From the results, the milk fat, milk protein, lactose and total solid content in the milk of each experimental group have no significant difference, but compared with a blank control group, the selenium content in the milk of the groups 4, 5 and 6 is respectively increased to 3.54 mug/100 g, 4.73 mug/100 g and 5.82 mug/100 g compared with the control group of 2.08 mug/100 g, which shows that the selenium content in the milk can be increased by adding the functional bag of each component, and the absorption effect of the organic selenium is better than that of the inorganic selenium; the milk selenium content of the group 6 is the highest and can reach the selenium-containing level specified in the general rule of prepackaged food nutrition labeling, which shows that the functional nutrition packet of the group has good effect when being matched with yeast cultures. The specific results are shown in Table 5.
TABLE 5 selenium content variation trend in cow milk fed by selenium-rich functional nutritional bag with different components
All milk indexes and blood antioxidant capacity indexes are average values of sampling detection within 120 days, and the results are similar to those of experimental example 2 and are shown in table 6.
Table 6 influence of feeding of selenium-rich functional nutrition bags with different components on various milk indexes and blood indexes
Experimental example 4 selenium-enriched corn silage + functional nutrition bag feeding experiment
Randomly selecting 20 lactating cows as an experimental group, simultaneously adding the selenium-rich silage corns prepared in the example 3 and the selenium-rich functional nutrition bag prepared in the example 6 into the experimental group, feeding the normal cows as a control group, performing a sampling method, a detection index and a detection method according to the method of the example 3, and comparing the influences of different treatments on the selenium content in milk, the milk components, the selenium content in blood and the antioxidant index in blood after feeding the normal cows for 120 days.
From experimental results, after the selenium-rich corn silage prepared in example 3 is added with the selenium-rich functional nutrition package in example 6, the selenium content in milk is increased from 1.99 mug/100 g of a control group to 7.93 mug/100 g, and is increased by 262.85%, and the selenium-rich content standard of liquid food specified in the pre-packaged food nutrition labeling convention is reached; the difference of other indexes is not obvious. In conclusion, the selenium-rich corn silage is matched with the selenium-rich functional nutrition bag for feeding, so that the selenium content in milk can be improved, and the health of the cow organism is not influenced.
The change of selenium content in milk is shown in Table 7.
TABLE 7 Simultaneous feeding of selenium corn silage and functional nutritional milk change in selenium content (ug/100 g)
All milk indexes and blood antioxidant capacity indexes are average values of sampling detection within 120 days, the milk indexes have no significant difference from a control group, the blood selenium and antioxidant capacity (T-AOC) is higher than that of the control group, the Malondialdehyde (MDA) content is significantly lower than that of the control group, the result is similar to that of the experimental example 2, and the specific table is shown in Table 8.
TABLE 8 influence of simultaneous feeding of selenium-enriched corn silage and functional nutritional packets on various milk and blood indices
Experimental example 5 stability experiment
In order to further verify the processing stability of the selenium-enriched milk product provided by the invention, a group of sample stability experiments in shelf life are specially designed. The raw milk of the dairy cow fed with the selenium-rich corn silage and the functional nutrition bag at the same time is sterilized and filled to obtain the selenium-rich milk of the experimental example.
As can be seen from Table 9, after the selenium-enriched milk of the experimental example is packaged into a product through pasteurization treatment, the selenium content in the milk is slightly lost through the processing technology treatment, and is reduced from 7.62 mug/100 g to 7.57 mug/100 g, but the difference is not obvious. The selenium content of the product is basically kept stable in the shelf life and is maintained in the standard range of the selenium content of the liquid food of 7.5 mug/100 g specified in the general rules of prepackaged food nutrition labels, which indicates that the loss of the product is less influenced by the processing technology. The specific results are shown in Table 9.
TABLE 9 Effect of processing on the stability of milk selenium content in products
In conclusion, the selenium-enriched corn silage is matched with the selenium-enriched functional nutrition bag for feeding, so that the selenium content in milk can be improved, and the health of the cow organism is not influenced; on the other hand, the method ensures that the selenium content and structure enriched in the raw milk are relatively stable, the content loss of the selenium is less influenced by the treatment of the batroxobin processing technology, and particularly, the content of the corresponding functional product in the processing production is relatively stable in the shelf life.
Possibility of industrial application
According to the invention, selenium-enriched milk can be produced by feeding the lactating cow with the selenium-enriched corn silage and/or the selenium-enriched functional nutrition bag, the product has good stability and no influence on the health of the cow body, and the selenium-enriched and vitamin E-enriched raw milk is formed by metabolism of the cow body, is easier to be absorbed by a human body compared with a product added by an exogenous source, and is easier to be accepted by consumers. Therefore, the method has high social value and economic value, is easy to operate and has high practicability.
Claims (10)
1. A feeding method for improving selenium content in milk is characterized in that selenium-rich silage corns are fed to a lactating cow, wherein the selenium-rich silage corns are obtained by spraying an organic selenium-rich foliar fertilizer on the corns in a corn jointing stage to a large horn mouth stage, and performing sealed fermentation after harvesting.
2. A feeding method for improving selenium content in milk is characterized in that dairy cows are fed with daily ration added with a selenium-rich functional nutrition bag, wherein the selenium-rich functional nutrition bag contains inorganic selenium and organic selenium.
3. A feeding method for improving selenium content in milk is characterized in that dairy cows are fed with selenium-rich silage corns, and daily ration added with a selenium-rich functional nutrition bag is fed, the selenium-rich silage corns are obtained by spraying an organic selenium-rich foliar fertilizer on the corns in the corn jointing stage to the large horn mouth stage and performing sealed fermentation after harvesting, and the selenium-rich functional nutrition bag contains inorganic selenium and organic selenium.
4. A preparation method of selenium-rich silage corns comprises the steps of spraying an organic selenium-rich foliar fertilizer on the corns in the jointing stage to the milk stage and 7-14 leaves, and performing sealed fermentation for 60-180 days after harvesting, wherein the organic selenium content concentration of the organic selenium-rich foliar fertilizer is 4-10 g/L, and the spraying dosage is 200 ml/mu each time.
5. The method for preparing selenium-enriched silage corn according to claim 4, comprising the following steps:
sowing: selecting corn varieties with the seed germination rate of more than 95 percent and the growth period of 95-110 days, and sowing the corn varieties according to the density of 4000-5000 plants/mu;
spraying: and (3) taking 200ml of organic selenium leaf fertilizer with the organic selenium concentration of 4-10 g/L and adding 10-20 kg of water when 7-10 leaves exist in the corn jointing stage-large horn mouth stage, and uniformly spraying the organic selenium leaf fertilizer on corn plants with the land amount of one mu by using a plant protection operation machine.
6. The preparation method of the selenium-rich silage corns as claimed in claim 4 or 5, characterized in that 200ml of organic selenium leaf fertilizer with concentration of 4-10 g/L is taken and mixed with 1-2 kg of water to be sprayed on one mu of corn in the number of leaves 10-14 in the male-drawing period to the milk-maturing period of the corns by using a plant protection unmanned aerial vehicle.
7. Use of selenium-enriched silage corn obtained by the method of making selenium-enriched silage corn of any one of claims 4-6 to feed lactating cows at a feed rate of 20-25 kg/head/day.
8. A selenium-rich functional nutritional bag comprises inorganic selenium and organic selenium, wherein the selenium-rich functional nutritional bag comprises 10-50 parts by weight of inorganic selenium and 20-90 parts by weight of organic selenium relative to 100 parts by weight of the total weight of the selenium-rich functional nutritional bag,
preferably, the vitamin E further comprises 10-30 parts by weight of vitamin E.
9. The use of the selenium-enriched functional nutritional supplement of claim 8, which is used for feeding lactating cows in an amount of 1g to 5g per head per day.
10. Use according to claim 9, characterized in that it is further supplemented with a yeast culture in an amount of between 100 and 150 g/head/day.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011197978.3A CN114431186A (en) | 2020-10-30 | 2020-10-30 | Feeding method for increasing selenium content in milk |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011197978.3A CN114431186A (en) | 2020-10-30 | 2020-10-30 | Feeding method for increasing selenium content in milk |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114431186A true CN114431186A (en) | 2022-05-06 |
Family
ID=81358077
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011197978.3A Pending CN114431186A (en) | 2020-10-30 | 2020-10-30 | Feeding method for increasing selenium content in milk |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114431186A (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1341374A (en) * | 2000-09-04 | 2002-03-27 | 吴敦虎 | Bioconverted selenium-enriched milk and its production method |
| CN102598972A (en) * | 2012-01-10 | 2012-07-25 | 上海博琛生物科技有限公司 | Selenium-enriched corn stalks and production method thereof |
| CN104431316A (en) * | 2014-11-07 | 2015-03-25 | 安徽玉龙新材料科技有限公司 | Method for producing selenium-enriched silage by utilizing selenium-enriched crop straws |
| CN105104738A (en) * | 2015-09-03 | 2015-12-02 | 黑龙江大庄园农牧业联合科技有限公司 | Feed for cow and processing method thereof |
| CN105941869A (en) * | 2016-05-26 | 2016-09-21 | 辽宁绿源生物肥业有限公司 | Selenium-enriched feed additive and production process thereof |
| CN111742885A (en) * | 2020-07-13 | 2020-10-09 | 北部湾大学 | Continuous production method of various selenium-rich products based on substance circulation mode |
| CN111802519A (en) * | 2020-07-17 | 2020-10-23 | 广西壮族自治区水牛研究所 | Dairy cow breeding method for producing selenium-rich milk |
-
2020
- 2020-10-30 CN CN202011197978.3A patent/CN114431186A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1341374A (en) * | 2000-09-04 | 2002-03-27 | 吴敦虎 | Bioconverted selenium-enriched milk and its production method |
| CN102598972A (en) * | 2012-01-10 | 2012-07-25 | 上海博琛生物科技有限公司 | Selenium-enriched corn stalks and production method thereof |
| CN104431316A (en) * | 2014-11-07 | 2015-03-25 | 安徽玉龙新材料科技有限公司 | Method for producing selenium-enriched silage by utilizing selenium-enriched crop straws |
| CN105104738A (en) * | 2015-09-03 | 2015-12-02 | 黑龙江大庄园农牧业联合科技有限公司 | Feed for cow and processing method thereof |
| CN105941869A (en) * | 2016-05-26 | 2016-09-21 | 辽宁绿源生物肥业有限公司 | Selenium-enriched feed additive and production process thereof |
| CN111742885A (en) * | 2020-07-13 | 2020-10-09 | 北部湾大学 | Continuous production method of various selenium-rich products based on substance circulation mode |
| CN111802519A (en) * | 2020-07-17 | 2020-10-23 | 广西壮族自治区水牛研究所 | Dairy cow breeding method for producing selenium-rich milk |
Non-Patent Citations (2)
| Title |
|---|
| 刘海波等: "《畜禽养殖与疾病防控》", 云南科技出版社, pages: 144 - 145 * |
| 庄安宁;梁稼烨;王若勇;时国峰;辛亚平;: "添加酵母硒生产富硒牛奶的研究", no. 05, pages 26 - 28 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101225000B (en) | Growth regulator promoting healthy growth of animal plant and preparation method thereof | |
| KR20060047555A (en) | Fruit tree culture solution containing selenium and culturing method of fruit tree containing selenium | |
| Mauricio et al. | Feed ruminants using Tithonia diversifolia as forage | |
| Naik et al. | Effect of feeding hydroponics maize fodder replacing maize of concentrate mixture partially on digestibility of nutrients and milk production in lactating cows | |
| CN107788506A (en) | Selenium-enriched hericium erinaceus powder and its production method and its purposes as selenium fortification agent | |
| CN105475642B (en) | A kind of milk cow forage | |
| CN102038105A (en) | Method for producing harmless selenium-rich black earth pork by utilizing selenium-rich astragalus sinicus secondary conversion technology | |
| CN107047984B (en) | High-calcium aromatic biological compound feed, preparation method thereof and feeding method of milch goats | |
| CN109824386A (en) | A kind of polyglutamic acid protein peptides Nano-selenium leaf fertilizer | |
| CN102037894A (en) | Method for breeding and screening astragalus sinicus varieties with high selenium content | |
| Singh et al. | Effect of feeding micro-nutrient fertilized oat hay based diets on nutrient utilization and mineral balance in growing lambs | |
| CN106538849A (en) | A kind of compoiste fermented microbial inoculum of millet straw and its ensiling millet straw feedstuff and method of preparation | |
| CN114431186A (en) | Feeding method for increasing selenium content in milk | |
| CN110150120A (en) | Alpha-aminobutyric acid content and the production method of yield is improved in a kind of enrichment beans bean sprout | |
| CN102067951A (en) | Method for producing pollution-free selenium-enriched duck and duck egg by second conversion technology of selenium-enriched Chinese milk vetch | |
| Promkot et al. | Cassava root fermented with yeast improved feed digestibility in Brahman beef cattle | |
| Das et al. | Effect of feeding micronutrient fertilized sorghum hay based diet on nutrient utilization and mineral balance in sheep | |
| CN108419835A (en) | A kind of production method of pure natural DHA raw milks and its application | |
| CN109748694A (en) | A kind of grape young fruit period Water soluble fertilizer | |
| CN107021833A (en) | A kind of selenium-rich gold needle mushroom culture medium | |
| KR101377708B1 (en) | Fermented solution of by-product of conifer and manufacturing method of the same, feed and water including the fermented solution | |
| Sherasia et al. | Evaluation of feeding practices and certain minerals status of lactating buffaloes in coastal zone of western India | |
| Boufennara et al. | Comparing Chemical Composition and Digestibility of Pedicels and Palm Leaves as a Source for Livestock Feeding by in vitro and in situ Techniques | |
| CN112471341A (en) | Daily ration for rapidly fattening Angus bull and preparation method thereof | |
| Gunun et al. | Effect of Mao (Antidesma thwaitesianum Mull. Arg.) seed supplementation on in vitro rumen protozoal population and digestibility using a gas production technique |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |