JP2579475B2 - How to prevent nitrate poisoning in herbivores - Google Patents

Description

The present invention relates to a method for preventing nitrate poisoning (methemoglobinemia) in herbivores, and can be used for maintaining the health of herbivores in the field of dairy farming.

(Prior Art) In breeding herbivores such as cows, horses, pigs, and goats, pasture, greening crops and silage are widely used as feed, but a large amount of nitrate nitrogen is contained therein. Some contain. It is known that when a diet containing a large amount of nitrate nitrogen is constantly fed, nitrate poisoning, that is, various metabolic disorders and sudden death called so-called “Pockley disease” are likely to occur.

That is, for example, in the case of a dairy cow, the nitrate nitrogen contained in the fed food is reduced to the nitrite nitrogen by the action of microflora, which is a bacterium in the rumen (ruminal stomach), and then into the stomach. It is thought that it is reduced in the blood and finally changed to ammonia, but the oxygen released during these reductions is converted to vitamins such as vitamins A, D, E and K, which are oxidizable vitamins. It is likely to cause metabolic disorders such as reproductive disorders, postpartum stagnation, inability to stand up before and after childbirth, mastitis, blood milk, etc. In addition, nitrite nitrogen absorbed into the blood combines with oxyhemoglobin in erythrocytes to become metemoglobin having no oxygen-carrying ability, resulting in anoxia, resulting in reduced systemic function, and this anoxia is severe. Becomes Pokkuri disease is sudden death in case, also ammonia finally generated not only damage the cells directly, would impose a heavy burden on the liver resulting in detoxification.

The reason why grasses contain a large amount of nitrate nitrogen is that nitrogen taken up for its growth is stored mainly in the stem, and partly in leaves, in the form of nitrate nitrogen until photosynthesis. For example, when referring to pasture, a large amount of nitrogen fertilizer is applied for the purpose of large-scale harvesting, and manure is dumped on the pasture due to rationalization by multiple breeding and lack of manure dumping sites. As a result, the nitrogen concentration in the pasture is cumulatively excessive. This means that the herbivore absorbs the nitrate nitrogen formed by the grass and the like for its growth through the feeding of the grass and the like, and if the amount is excessive, nitrate poisoning occurs in the herbivore. Prevention has become a major concern for dairy farmers because of this causal relationship and because the occurrence of nitrate poisoning, as described above, places an excessive financial burden on dairy farmers. .

The onset of nitrate poisoning can be roughly estimated by measuring the amount of methemoglobin in the blood. It is not clear how much the amount of methemoglobin will be lethal, depending on the individual, and it depends on the oxygen demand of the individual at that time. It is said that 90% of the methemoglobin was converted to methemoglobin, so a methemoglobin content of 50% is a reasonable threshold for the danger zone [Shinozaki, "Livestock Research" Vol. 29 No. 3 No. 3
375 pages (1975) and Miyazaki "Livestock Consultant" No. 141
Section 47 (September 1976)].

However, conventional measures to prevent nitrate poisoning include sampling food such as grass and quantifying the amount of nitrate nitrogen to give a substance with a small amount of nitrate nitrogen. In addition, only a mixture of wild grasses and the like having a small amount of nitrate nitrogen only reduced the overall intake of nitrate nitrogen. For example, in the case of feed stored in silos, nitrate nitrogen is water-soluble and is often dissolved in the water stored at the bottom. Measures such as measuring the content of water, if the amount is high
Specifically, the following countermeasures are to be taken [Agriculture, Forestry and Fisheries Technology Council Office, “Practical Technical Report” No. 34 (Causes of nitrate poisoning in cattle and control measures, page 10)].

a) Avoid feeding for 3-5 days if the rainfall is followed by a rapid drought due to the high nitrate content in the forage crop; b) Withhold cutting and grazing until the nitrate content falls; c ) Give only the upper part with low nitrate content, d) devise a slow feeding rate, e) gradually increase the dose over 1-2 weeks without free eating, f) feed less G) do not feed diseased or frail animals with presumed high nitrate content feed crops; h) at time intervals, e.g., every 24 hours for presumed high nitrate content feed crops I) Forage crops presumed to have high nitrate content should be given in combination with forage with low nitrate content. J) Forage crops presumed to have high nitrate content should be given in large quantities at once. K) feed with high-energy feed for feed crops presumed to have high nitrate content; l) feed silage for feed crops presumed to have high nitrate content; m) limit the amount of non-protein nitrogen added; n) add iodine to feed crops presumed to have high nitrate content; o) feed crops presumed to have high nitrate content Is added with vitamin A, p) Avoid sudden changes in feed, do not add urea when providing green crops, q) Avoid antibiotics when providing feed crops that are presumed to have high nitrate content .

In addition, as a countermeasure for the appearance of symptoms considered to be due to nitrate poisoning, administration of vitamin A and sugar is effective.For example, administration of sugar temporarily reduces blood methemoglobin concentration. In some cases, this can cause a sudden rise, which can lead to lethality of the animal. Therefore, the only practical countermeasures are the intravenous administration of a 2% methylene blue saline solution or the combined use of this methylene blue administration and epinephrine administration. [Miyazaki "Livestock Consultant", No. 141, p. 47 (September 1976)
Month)]]].

(Problems to be Solved by the Invention and Object of the Invention) There is a problem in that any measures conventionally adopted to prevent nitrate poisoning are passive. There is a fateful causal relationship between nitrate nitrogen contained in grasses and the like and nitrate poisoning of herbivores, and grasses are a finite resource and there is no nitrate nitrogen, or It is not always possible to always supply those whose content is extremely low,
Therefore, it is not possible to reliably prevent the generation of nitrate due to feed.

On the other hand, intravenous administration of methylene blue, which has been conventionally used to treat nitrate poisoning, tends to be avoided by animal owners, especially in the case of beasts, because methylene blue is a basic dye by nature. There is a problem in showing. In other words, in the case of nitrate poisoning, the animal is in a state of breath amami so that it is necessary to administer methylene blue, and the administration does not necessarily guarantee the survival of the animal,
If it dies, it cannot be sold as meat.On the other hand, even if its administration avoids poisoning death, the meat will be colored and its value as meat will decrease, so it will be brought to the slaughterhouse while it is alive and eaten as meat It is up to the animal owner to decide whether to sell or bet on treatment with methylene blue.

Accordingly, an object of the present invention is to provide a method for positively preventing the occurrence of nitrate poisoning in herbivores,
More specifically, it is an object of the present invention to provide a method for preventing nitrate poisoning by positively detoxifying nitrate nitrogen inevitably ingested by feeding on grass and the like.

(Means and Actions for Solving the Problems and Achieving the Object) The present invention basically aims to solve the above problems by administering denitrifying bacteria to herbivores.

Various types of denitrifying bacteria are known,
Some of them are used for sewage treatment, etc., but they are generally pathogenic, and it has never been considered to use denitrifying bacteria in relation to living organisms. I didn't come.

Therefore, the inventor of the present invention has studied the use of photosynthetic bacteria, which are generally considered to have low toxicity.
f Microbiology ", Vol. 108, pages 265-269 (1976)
Toshio SATOH is a photosynthetic bacterium with excellent denitrification ability
Have found what has been reported. According to this document, seven photosynthetic bacteria (1L 101-1L 107 strains) were obtained, of which 1L 101, 102, 103, 104, 106 and 107 strains belonged to Rhodopseudomonas sphaeroides (bacteria belonging to Rhodopseudomonas sphaeroides). Yes, the 1L 105 strain is considered to be a bacterium belonging to Rhodopseudomonas capusulata (Rhodopseudomonas capschulata), and details regarding the 1L 106 strain are disclosed. In the middle of the right column of page 266, in this paper, "1L 106 generates a violent gas when anaerobic cultivation is performed in a versal medium containing 0.2% KNO ₃ with or without illumination. The inventor noted that the collected gas was identified by gas chromatography. Most of the collected gas was nitrogen, and H ₂ , NO and N ₂ O were not detected. ” The reason is that nitrate itself, which is ingested by predation of grasses and the like, does not harm herbivores, but nitrite and ammonia generated by its decomposition (reduction) are responsible for nitrate poisoning in herbivores. This is because photosynthetic bacteria (1L 106 strains) were considered to have the function of directly converting harmless nitrogen gas without releasing ingested nitrate as harmful nitrite or ammonia.

Since the above research paper has been published by a research group at Tokyo Metropolitan University, visit the representative, Toshio Sato, to confirm that it has no pathogenicity,
Rhodopseudomonas sphaeroides forma sp.denitrifican
s (Rhodopsonamonas sphaeroides forma sp. denitrificans) 1L 106 strain was provided with a denitrifying (photosynthetic) bacterium. The inventors have found that the amount of hemoglobin is significantly reduced, and completed the present invention.

That is, the method for preventing nitrate poisoning of herbivores according to the present invention comprises the above-mentioned photosynthetic bacterium Rhodopseudomonas sphaeroides forma sp.
It is characterized in that denitrificans is administered to herbivores.

In the method of the present invention, administration of the denitrifying bacteria can be performed by oral administration of the culture solution. Culture solution 1000ml
The amount of bacterial cells inside is about 3 g by wet weight, about 1/5 by dry weight
-1/6, and about 500-1000 ml of the culture solution is sufficient.

The Rhodopseudomonas sphaeroides forma sp. Denitrificans 1L 106 strain is a Rhodobacter sphaeroides f.sp. denitrificans (Rhodob
Deposited as acter sphaeroides f.sp.denitrificans) and deposited at the Institute of Microorganisms and Industrial Technology, the Ministry of International Trade and Industry of Japan.

In addition, although it is under study whether the administered denitrifying bacteria can be established, it has not yet been clarified due to individual differences among target animals.

(Effects of the Invention) According to the method of the present invention, it is possible to lower the blood methemoglobin concentration of a herbivore simply by administering a denitrifying bacterium, and further to prevent nitrate intoxication in herbivore. . Therefore, the method of the present invention has the advantage that its implementation is extremely easy.

(Examples) Next, a dairy cow of the Holstein breed was selected as a herbivore, and a water quality test was performed on the well water that was drinking water, a nitrate content measurement test was performed on various diets,
The present invention will be described in further detail in connection with a culture test of a denitrifying bacterium and an administration test of a denitrifying bacterium.

Test Example 1 (Water water quality test) As a result of conducting a water quality test of well water, which is drinking water for dairy cows, by a conventional method and examining the contents of nitrite nitrogen and nitrate nitrogen, the former was not detected. The latter content is only 1.445p
pm.

Test Example 2 (Nitrate Nitrogen Content in Diet) Beef pulp, oats, corn silage, Sudan grass hay, hay cube, etc. are added to dairy cows in addition to the compound feed and straw as appropriate. The results of examining the content of potassium nitrate by the method were as shown in Table 1 below.

Test Example 3 (Culture of Denitrifying Bacteria) a) Pre-culture The denitrifying bacteria Rhodopseudomonas sphaeroides forma sp.
Denitrificans (1L, 106 strains) was inoculated into the following liquid medium and illuminated at 30 ° C with light of about 3000 lux (60W
(In the case of an incandescent lamp, separate it from the light source by about 5-10 mc).

Liquid culture medium for pre-culture Yeast extract 0.3% Casamino acid 0.2% KNO ₃ 0.2% Distilled water Remaining b) Main culture The culture solution after completion of the above pre-culture is added to the following liquid culture medium for main culture in about 1 to 50 (volume ratio). And incubate for about 24 hours under the same conditions as in the preculture. In addition, in the case of the main culture, it is preferable that the culture flask is almost completely filled with the pre-culture liquid and the main culture medium in order to make the anaerobic conditions sufficient, so that the remaining space of the culture flask is preferably reduced as much as possible. It was thought that strict consideration was not necessary because nitrogen gas was released as the culture progressed.

The amount of cells in the obtained culture solution was about 3 g in wet weight on average per 1000 ml of culture solution, and about 1 / 5-1 in dry weight.
/ 6.

Liquid culture medium for main culture Versal salt solution (Note 1) 100 ml Sodium maleate 3 g Vitamin solution (Note 2) 1 ml (NH ₄ ) ₂ SO ₄ 1 g KNO ₃ 1 g Yeast extract 0.1 g The above mixture was sterilized. The sterilized mixture is prepared by adding 15 ml of a phosphate solution (Note 3), and then adding distilled water to make a total volume of 1 liter.

(Note 1) Preparation of versal salt solution MgSO ₄ .7H ₂ O 2.0 g CaCl ₂ .2H ₂ O 0.75 g FeSO ₄ .7H ₂ O 118.0 mg and EDTA (4H) 200.0 m are dissolved in distilled water 800 ml and the whole amount is dissolved. Make 990ml and pH
Adjust to 6.8, then add 10 ml of trace element solution (Note 4) to make the total volume 1 liter.

(Note 2) Composition of vitamin solution Niacin 1 g Thiamine hydrochloride 1 g Biotin 0.04 g Distilled water balance 1 liter (Note 3) Preparation of phosphate solution Add 40 g of K ₂ HPO _{4 to} 40 g of KH ₂ PO ₄ , and then add It is prepared by adding distilled water to make the total volume 1 liter and then subjecting it to a sterilization treatment.

(Note 4) Composition of trace element solution _{MnSO 4 · 4H 2 O 2.1g H} 3 BO 3 2.8g Cu (NO 3) 2 · 3H 2 O 40.0mg ZnSO 4 · 7H 2 O 240.0mg Na 2 MoO 4 · 2H 2 _{O 750.0mg Co (NO 3) 2} · 6H 2 O 248.0mg ( dose studies denitrifying bacteria) distilled water balance total 1 liter example a) test period 1986 December 2 - December 18 b) experimental animals Three Holstein dairy cows with good health conditions were selected, two of which were used as test animals, and the remaining one was used as a control animal.

c) Feed The test animals and control animals were fed the same quality and the same amount of feed.
The main feeds given per day during the test period were as shown in Table 2 below.

d) Test drug (denitrifying bacteria) The culture solution obtained in the main culture in Test Example 2 was used as it was.

e) Administration day, dose and administration method of study drug Two hours after feeding the morning feed on a predetermined day (December 4, 10, 16, and 17) (December 16 and 17) 1 hour after the day), 500 ml of the culture medium for the test animal No. 1 and
Forcibly administer 1000ml orally. Of course, no test drug is administered to the control animal (No. 3).

f) Test method For test animals, 2 hours after administration of the test drug (12
After 1 hour on the 16th and 17th of the month), about 7 ml of venous blood was collected from all the animals including the control animals by vacuum blood collection tube, stored frozen, and then the total hemoglobin and methemoglobin were measured by a conventional method. did.

g) Results and discussion The results were as shown in Table 3 below (in the table, MHb and THb mean methemoglobin and total hemoglobin, respectively, and are underlined in the column of "Date of blood collection"). Indicates that the test day is the day of administration of the test drug, a denitrifying bacterial culture.)

Nitrate taken into the stomach by feeding and water supply is reduced to nitrite by the action of bacteria in the stomach,
Some of them bind to hemoglobin in the blood and become methemoglobin without oxygen transport ability.When the amount increases, the function of compensating for this in healthy animals tends to increase and the total hemoglobin amount tends to increase. As the amount of methemoglobin decreases, the amount of total hemoglobin also tends to decrease, and the data on control animals without administration of the denitrifying bacterial culture reflect these trends. On the other hand, in the test animals, not only does the blood methemoglobin amount decrease due to the administration of the denitrifying bacterial culture solution, but even if the methemoglobin amount decreases, the total hemoglobin amount tends to increase, which indicates that the methemoglobin amount It is thought that administration of the denitrifying bacterial culture has an inherent effect of enriching blood oxygen, not as a compensation for the oxygen deficiency associated with the increase in oxygen.

Claims (2)

(57) [Claims] 1. A photosynthetic bacterium having the ability to denitrify, Rhodopsmonas sphaeroides forma sp.
-A method for preventing nitrate poisoning in herbivores, which comprises administering denitrificans to herbivores. 2. The method for preventing nitrate poisoning in herbivores according to claim 1, wherein said photosynthetic bacterium is orally administered in the form of a culture solution thereof.