SU1625317A3 - Method of obtaining preparation for feeding ruminants - Google Patents

Abstract

The invention relates to a composition for improving the efficiency of ruminant feed utilization, which comprises one or more microbial cultures, which are capable of adjusting the weight ratio of acetic acid to propionic acid produced during fermentation of energy-producing nutrients in the rumen of an animal to an optimum value, preferably to 1.5-4.0:1, and of growing in the rumen and persisting there at least for 60 days. The cultures may optionally be in admixture with carriers, diluents, and preserving agents conventionally used in animal husbandry and nutritive and/or other substances conventionally administered to ruminants. According to another aspect of the invention there is provided a process for the preparation of microbial cultures used as active ingredient in the above composition. The invention further relates to a process for the preparation and use of said compositions.

Description

This invention relates to agricultural biotechnology and can be used in animal husbandry for feeding animals.

The aim of the invention is to increase productivity.

The goal is achieved by obtaining a preparation capable of changing the ratio of acetic and propionic acids in the gastric juice of the rumen of ruminants in the range (1.5-4.0): 1. At the same time, the strain Proplontbacterlum sp. Is used as a biologically active ingredient in the preparation. HNCMB No. 00287 or strain Veilonella sp. HNCMB N 00288, or strain Bifidobacterium sp. HNCMB # 00289. Strains Propionlbacterium sp., Veilonella sp., Bifidobacterium sp. isolated from the rumen of ruminants, selected for the ability to biosynthesis of volatile fatty acids

lot and deposited in the National Collection of Microorganisms at the Hungarian State Institute of Health under the number 00287 and / or 00288 and / or 00289.5

To increase the yield of ruminant animals, it is preferable to use as a biologically active ingredient a culture of microorganisms that maintains the ratio of acetic and 10 propionic acids in the range (2.0-3.5): 1, for example 2: 1. For dairy products, the optimum ratio of ususic and propionic acids is about 3.0: 1, for a pregnancy period of 4.0: 1, while 15 when raising heifers {2.0-3.0): 1. Example 1

A. Selection of microorganism strains isolated from the rumen of animals fed with hay.

- Ovez is fed with hay for a month, then a sample is taken through the closure fistula operatively inserted into the rumen. The sample immediately after sampling is selected on a special nutrient medium.

To obtain a nutrient medium, 7.5 ml of saline I is used, containing:

Dikaliyygidrofosfat, m0,630

Distilled water, ml Up to 100 7.5 ml of saline solution II, containing: Sodium chloride, g1,2

Ammonium sulfate, g1,2

Potassium dihydrofoyifat, g 0.6 35

Calcium chloride, 0.12

Magnesium sulfate heptahydrate, G0.25

Distilled water, ml Up to 100 and 40

0.01% solution of rezazurin, ml 0,1

Agar (bakto), g2,5

Scar fluid, ml10.0

Glucose, g0.05 45

Celibiosis, g0,05

Cysteine hydrochloride monohydrate, g0.05

8% sodium carbonate solution, ml 5.0 50

Distilled water, ml Up to 100 Designation of nutrient medium RGCA. The sample taken from the rumen is filtered through a loose gauze filter, and the filtrate is then stored at -20 ° in CO atmosphere. 55 PH of the nutrient medium is set to 6.8, then the medium is sterilized. Sterilization is carried out in a C02 atmosphere.

Pre-medium is diluted with a sterile mixture of the following composition:

Saline solution I ml 7.5

Saline solution II ml 7.5

Cysteine hydrochloride monohydrate, g0.05

Sodium carbonate, g0,3

0.1% solution of resazurin, ml 0.1 Distilled water, ml Up to 100 Designation of the mixture HB. The cultures are incubated for 120 hours under anaerobic conditions at 35 ° C.

Then, a separate colonies are sown nutrient medium containing hay extract with the following composition,%:

Saline solution 15.0

Saline solution II15,0

0.1% solution of resazurin0, 1 Tripton I 421.5 Yeast extract 0.5 Cicatricial fluid 10.0 Sodium carbonate 0.4 Cysteine hydrochloride monohydrate 0.05 Hay extract 10.0 Designation RGCF. To prepare the hay extract, cut into small pieces of a part of a cycnes-plants are digged in water, boiled and then filtered. The filter residue is added to the medium before sterilization.

The pH of the nutrient medium is set to 6.5 using carotid acid, then the medium is sterilized.

Sterilized in test tubes nutrient medium with a volume of 5 ml seeded with a suspension of cells obtained from isolated colonies grown on solid nutrient medium RGCA. Cultures were incubated for 5 days at 35 ° C under anaerobic conditions. The growth is monitored by microscopy, then the culture is selected on a nutrient medium RGCA of the indicated composition. However, glucose and cellulose are not added to the medium, but 2% bacto-cellulose is used instead.

The cultures were incubated at 35 ° C for 120 hours, then the individual colonies, which developed on cell-capable cells, were isolated by centrifugation in a CO atmosphere.

B. Genetic labeling of bacteria produced.

Genetic labeling is carried out using EschericWa coll Plasmid p.1011.

Plasmid DNA is isolated and dissolved in a mixture of the following composition, mmol: Calcium Chloride 75

Magnesium chloride Tris-hydrochloride buffer, pH 7.5

ten

(Tris) - (hydroxymethyl) - {aminomethane) -hydrochloride.

The cells obtained in section A are suspended in a mixture of the following composition, mmol: Calcium chloride 75

Magnesium Chloride 5

Tris-hydrochloride buffer, pH 7,510

Cysteine hydrochloride monohydrate1

Sodium thiosulfate 1

The suspension contains 5 x 109 cells per milliliter. The cell suspension is diluted in a 1: 1 ratio (0.1 µg / µl) with a mixture containing p.1011 plasmid DNA, then incubated at 4 ° C for 60 minutes. Next, incubation was continued for 2 minutes at 41 ° C, after which the culture was applied to a dense nutrient medium containing cellulose as a carbon source, with kanamycin B added to 500 µg / ml before sterilization. Cultures were incubated for 120 hours at 35 ° C and under anaerobic conditions x, then colonies capable of growing in the presence of 500 μg / ml kanamycin B are examined.

Plasmid p1011 carries genes that determine resistance to kanamycin and chloramphenicol and the ori region, which provides for the replication of the plasmid in E. coli cells. Since the DNA plasmid is able to replicate only in E. coli, it is lost after entering other bacteria, but is fully or partially integrated into the chromosomes, i.e. genetic recombination occurs. The gene embedded in the chromosomes appears in a favorable case and provides resistance to kanamycin and chloramphenicol.

In this way, kanamycin-resistant B colonies are obtained, the transfer rate of which is 3 X10.

Some resistant colonies isolate and determine the antibiotic sensitivity of the original strains and Kanamycin B (KmR) resistant strains. The following results were obtained:

Microorganisms Minimum inhibitory growth concentration of kanamycin B, µg / ml Scar content31

Source strains4.0-7.5

Genetically labeled KmR strains: Hh-GVOK1-1,8 500

27250

91 500

1231000

142 500

Strain Hh-GVOK1-1-123 / K is applied to cellulose-containing nutrient medium R GCA with the addition of 1000, 5000 and 10000 µg / ml kanamycin B. The cultures are incubated for 168 hours at 35 ° C under anaerobic conditions, and then grown in the presence of 10,000 µg / ml antibiotic strains. Thus, the selected strain, designated as Hh-GYOK-1-123 Sz, is synthesized with propionic acid. The bacterium is gram-positive and rod-shaped. Glucose and starch are fermented by this bacterium into propionic and acetic acids, and some butyric acid and carbon dioxide are also formed. On complex nutrient media, young microorganisms of rod-shaped cells appear, which later turn into polymorphic forms of various sizes and contents, lacking cilia and often expanding at the apex, becoming wider. The body also grows well under anaerobic and semi-anaobic conditions, it is optionally anaerobic. Therefore, the strain designated as Hh-GVOK1-1-123 Sz (as well as the strain Hh-GYOK 1-48a derived from it) belongs to the genus Propionibacterium. Strain Propionibacterium sp. (Hh-GYOK-1-123 Sz) is deposited in the National Collection of Microorganisms at the Hungarian State Institute of Health under the number 00287.

B. The introduction of genetically marked microorganisms into the scar.

Strain Propionibacterium sp. HNCMB No. 00287 seeded with sterile nutrient medium RGCFa of the following composition, ml:

Potassium hydrophosphate 0.3% 45

Ammonium sulfate

Sodium chloride

Sulfate Monohydrate

magni 45

Calcium chloride dihydrate

Potassium dihydrophosphate

Bakto Cellulose

Agar (bzkto) 65

Yeast Extract20

Hydrochloride monohydrate

cysteine20

Sodium thiosulfate 20

Sodium carbonate

Scar fluid20

The culture is diluted under anaerobic conditions in 500 ml flasks containing 150 ml of the culture medium. After 48 hours, growth is controlled, then the culture is mixed into the feed of sheep receiving hay. Before feeding and in the following days, samples of 50-200 ml are taken through a fistula inserted by operation into the scar.

After appropriate dilution, the samples are applied to RGCA nutrient media without kanamycin and other antibiotics. Cultures were incubated for 72 hours at 35 ° C under anaerobic conditions, then the number of bacterial colonies developed was determined. The animal is orally administered with 340 mp culture containing 4.7 x 10 bacteria per milliliter. The results are presented in table. one.

From the data table. 1 it follows that the introduced microorganism exists in the rumen for a long time and multiplies,

140 ml of Propionibacterium sp. Culture containing 2 x 10 cells per milliliter are also orally administered to the animal. The results are presented in table. 2

From the data table. 2, it follows that the introduced microorganism is present in a significant amount in the rumen, and also multiplies.

Example 2

A. Selection of strains of microorganisms isolated from the rumen of animals fed with cereal products.

The culture is obtained similarly to p. A of Example 1 with the difference that the initial sample is taken from the rumen of animals fed with cereal products. Separate isolates sow the HaRGCF medium containing powdered grain (2%) instead of hay. Cultures grown in liquid RGCF-cpe are applied to dense RGCA-cpe-AV, and colonies developed from cells that assimilate grain products are isolated from this nutrient medium.

B, Genetic labeling of starch destructive bacteria.

Perform an anapogic step. B of Example 1.

Resistance to Kanamycin. In some transformed and KmR strains, the following:

Microorganism Minimum growth inhibition of kanamycin B, µg / ml

Stomach contents31

Baseline w gamma1.8

Genetically labeled Km strains: Hh-GYOK1-2-4 14 Ab 37

81

250 250 250 125

The selected strain designated as Hh-GYOK1-2-14Ab, fixed, gram-negative, does not produce

pigments.

It grows well under anaerobic conditions at 37 ° C. Spherical cells appear in large numbers and are located one next to the other. Their average value

is 0.3-0.4 µm. It sprays carbon to acid and carbon dioxide. On complex nutrient media forms hydrogen sulfide, does not dilute gelatin, does not cause hemolysis. Therefore, the strain designated as Hh-GVOK1-2-14Ab belongs to the genus Veilonella.

Strain Vellonella sp. (Hh-GV OK1-2-14UB) deposited under number 00288 in the National Collection of Microorganisms

at the Hungarian State Institute of Health.

B. The introduction of genetically marked microorganisms into the scar.

Determined as in Example 1 of Example 1 with the difference that the strain Veilonella sp, (Hh-GYOK1-2-14Ab) is used and sterile RGCFa medium containing 1.8% soluble starch instead of bactocellulose is inoculated. Then culture

mixed into the feed of animals receiving grain. Before administration and then daily, a sample is taken through the fistula. 310 ml of a culture containing a total of 7.1 x 105 bacteria was orally administered to the animal.

The number of cells contained in the KmR samples is given in Table. 3

From tab. 3, it can be seen that the introduced microorganism remains alive for a long time and multiplies.

. PRI me R 3.

A, Selection of strains of microorganisms isolated 1.z rumen animals fed molasses.

The culture is obtained similarly to point A of Example 1 with the difference that the initial sample is taken from the rumen of animals fed with molasses. Separate isolates are plated on R GCF medium containing glucose instead of yeast extract. Cultures developed in a liquid nutrient medium are applied to an R GCA medium. On the same nutrient medium sowed colonies that have developed from cells capable of absorbing molasses.

In this way, microorganisms derived from the rumen are obtained that can metabolize molasses.

B. Genetic labeling using molasses bacteria.

Carry out similarly to point B of Example 1.

The resistance of some KPA light to kanamycin B is as follows:

Microorganism The minimal overwhelming increase in the concentration of kanamycin B, µg / ml.

The contents of the rumen31

Source strains7,5

Genetically mayor

Cased CT Strains: NP-SUOK1-3-2250

14,500

34250

81 Me500

132500

The selected strain designated as Hh-GVOK1-3-81 Me, optionally anaerobic, gram-positive. Cells are rod-shaped, immobile. The ends of the sticks are squeezed. Forms acetic acid, ferments glucose to lactic acid. The sticks grow on different nutrient media, rarely separately, in most cases in the form of chains. Does not form pigment. Therefore, the strain belongs to the genus Bifidobacterium. Strain Bifidobacterium sp. (Hh-GVOK1- 3-81 Me) was deposited under number 00289 in the National Microbial Assembly at the Hungarian State Institute of Public Health.

B. The introduction of genetically marked cells into the scar.

Proceed according to paragraph. In example 1 with the same difference that the strain Bifidobacterium sp. HNCMB No. 00289 (Hh-GYOK-3-81 Me) are seeded with R GCF-a medium containing 1.8% glucose instead of cellulose. The culture is admixed to feed of animals receiving molasses. Before feeding and then daily through the fistula introduced into the rumen, samples are taken. The animal is orally administered 380 ml of a culture that contains 1.6 x 107 cells per milliliter.

The number of Cdc cell samples contained is given in Table. four.

From the data table. 4, it can be seen that the introduced microorganism has been in the rumen of the animal for a long time.

Example 4. Changes in the ratio of volatile fatty acids produced in the rumen when using the bacteria obtained.

Two sheep receive a complex feed (a mixture of hay and sheep feed), then after 14 days a sample is taken through the fistula. 2 liters of cicatricial juice are filtered through a multi-layer gauze, the filtrate is separated. The pieces remaining on the gauze are suspended in 1 l of physiological buffer, then, after mixing, they are also filtered. Both filtrates are combined, allowed to stand, after 1 h the absorbent solids are removed, only liquids are used for the study. The composition of the physiological buffer is as follows, g / l:

Disodium hydrogen phosphate (sodium hydrogen phosphate 0.316

Potassium dihydrophosphate 0.152

Sodium bicarbonate 2,260

Potassium Chloride 0.375

Magnesium sulfate 0.112

Calcium chloride dihydrate 0.050

Iron (II) sulfate heptahydrate 0.08

Magnesium sulfate monohydrate 0.004

Zinc Sulphate Heptahydrate 0.004 Copper Sulphate Pentahydrate 0.002 Cobalt (II) Hexahydrate 0.001 The pH of the mixture is controlled and, if necessary, adjusted to 7.2 using a dilute aqueous solution of hydrochloric acid or sodium hydroxide solution.

The resulting mixture in a 1: 1 ratio is diluted with the above physiological buffer, and 4 g of the feed mixture is added to 1 l of the solution. 30 ml of the resulting suspension was filled into 100 ml Erlenmeyer flasks, then 200 portions of the previously prepared nutrient medium were sterilized, 200 further portions were not sterilized, and were seeded with bacteria prepared in Examples 1-3,

The studied bacteria are incubated at 37 ° C under anaerobic conditions for 48 hours on R GCA + CG-nutrient media of the following composition: 1%:

Saline I15

Saline solution II15

The solution of trace elements0,3

Yeast extract 0,5

Filtered rumen fluid 10.0

Sodium carbonate 0.4

Cysteine hydrochloride monohydrate 0.05

Sodium thiosulfate 0.008

Cellulose (bacto) 0.3

Glucose2,0 The composition of the solution of trace elements the following mg:

Zinc chloride40

Copper (It) chloride dihydrate 10

Sodium tetraborate decahydrate 10

Ammonium molybdate tetrahydrate 10

Iron Trichloride Hexahydrate200

Tetrahydrate chloride mar-

Ganza (II) 10

Free from ions, ml. Up to 1000 Cultures are seeded with previously prepared nutrient media in Erlenmeyer flasks, as indicated above. Sowing is carried out at a ratio of 2 ml of culture per 50 MJI nutrient medium. The non-sterile culture is also sown.

Sowing is incubated for 40 hours under anaerobic conditions, then reproduction is stopped with 10% concentrated formic acid and the volatile fatty acid content is examined. For this, the culture is filtered through gauze and then centrifuged in a rotor with a speed of 4000 for 15 minutes. The mixture is filtered again and then, to determine the fatty acids with 2-5 C atoms, are introduced into the separation column. The gas chromatograph equipped with plasma ionization the detector. The temperature of the column is 150 ° C. Dividing column 2 m long, glass tube with an inner diameter of 4 mm; filling: 1C% ethylene glycol adipate + 2% orthophosphoric acid on a strong silica gel (0.2-0.3 mm). Injector temperature 190 ° C. The flow rate of Na is 50 ml / min. Flow rate At 50 ml / min. Air flow rate 200 ml / min. Paper speed 160 cm / h. Chromatography time 20 min. Amount of sample 1 m ;; l.

For each sample, 4 parallel measurements were made.

The standard solution contains acetic, propionic, iso-butyric, butyric, isovaleric and valeric acids.

The results are presented in Table. 5. In table. 5 the following notation is used:

+ S is sown after sterilization; NS is seeded from a tube containing the live flora of the scar;

a - a small amount;

B - negative control: content in the nutrient medium (rumen fluid.

physiological buffer and feed) volatile fatty acids (average of 12 measurements);

c - positive control: the content in the containing the initial bacteria of the rumen and the incubated culture (nutrient medium from the rumen liquid, physiological buffer and feed) of volatile fatty acids (average of 12 measurements); d - similarly with, but in the nutrient medium with the addition of 5 ppm monenzins (sodium salt), average of 6 measurements;

e - as with, but in a nutrient medium with the addition of 100 ppm of monentsin (sodium salt), the average of 6 measurements.

5 From the data table. 5 that when introducing the proposed microorganism cultures, the ratio of volatile fatty acids formed in the rumen may vary widely. Using the received

0 from the culture Hh-GVOK-48a strain, for example, the amount of propionic acid produced can be increased, while the amount of the microorganism Hh-GVOK1-109e is increased

5 acetic acid produced. An increase in the number of individual fatty acids formed can also be observed on nutrient media that do not contain any other microorganisms (S) or in

Presence of normal rumen bacteria (NS). In the experimental system with monencin sodium salt used, the acetic acid / propionic acid ratio (d, e) is reduced by 1 -2 tenths.

5 Example 5. Preparation of a bacterial preparation for feeding animals.

The introduced bacterium is grown on R GCA + CG-nutrient media of the composition indicated in Example 4 under anaerobic conditions.

0 After growing, the cells are removed in a known manner, for example, by filtration or centrifugation. Separated lightly suspended in physiological buffer (see example 4) and subjected to drying

5 by freezing. The lyophilized bacterial preparation is collected and, under known conditions, in the form of the corresponding preparation BVOD .. T (set) animals. Breeding culture of microorganisms

It can also be carried out on other known nutrient media, for example, on nutrient media containing glucose, starch, etc. as a carbon source, and inorganic salts as a nitrogen source.

The drug can be administered in a mixture with feed, drinking water, alone or together with other biological active substances, for example with antibiotics, vitamins.

In addition to the lyophilized preparation, other products can also be prepared. The microorganism can also be injected after displacing the mass of bacteria separated by centrifugation or filtration with appropriate carriers or diluents, for example, calcium carbonate, grain crops, premix or other feedstuffs.

Depending on the feed used and on the direction of production, it is determined which of the microorganisms that are prepared using the proposed method and favorably influencing the rumen flora of microorganisms and in what quantity. If it is necessary to reduce the particular acetic acid — propionic acid, then, for example, a culture of the strain Proplonibacterium sp. HNCMB N2 00287, if you need to increase this quotient, then give, for example, the culture of the microorganism Bifidobacterium sp HNCMB No. 00289 (Hn-SUOK1-3-81Me). If nitrogen-binding bacteria are introduced, the amount of organic nitrogen source administered to ruminants can be reduced. The culture was introduced in such an amount that about 5 X 102 and 5 X Yu7 cells functioned in 1 ml of the rumen content.

Example 6

A. Preparation of the dry frozen preparation.

The wet bacterial paste OE-950, weighing 100 g, obtained in accordance with Example 5 by centrifugation, is lyophilized in a freeze dryer. Then, the resulting 54 g of the product is thoroughly mixed with 486 g of wheat bran and placed in a bag that protects the bacteria (impermeable to bacteria). A preparation containing 10% of the biologically active substance is obtained.

B. Preparation of a preparation containing wheat bran as a carrier.

100 g obtained in accordance with Example 5 by centrifuging the wet paste of the microorganism are uniformly mixed with 100 g or 900 g of wheat bran, then placed in a bag or container that protects the bacteria. In this way, a preparation containing 5 or 10% of the biologically active substance is obtained.

B. Preparation of a preparation containing bantonite as a carrier.

100 kg of bentonite powder are sterilized at 160 ° C for 2 hours, then after cooling to room temperature, placed in a mixer. The mixer is powered by a liquid dispenser.

10 kg of the suspension of microorganisms obtained in accordance with Example 5 are sprayed. The resulting suspension is dried at 30 ° C to a moisture content of 6-8% and placed in bags (containers) that protect the bacteria.

5 g of the product is stored at 4 ° C, at room temperature or at 37 ° C, samples are taken at day zero, day 20, day 51 and day 72,

then the preparation is suspended in sterile HB mixtures and, after appropriate dilution, seeded onto RGCA agar. Cultivation was carried out for 48 hours under anaerobic conditions, then the number of grown colonies was determined.

The results are shown in Table. 6. From the data table. 6, it can be seen that the number of bacteria in the product containing bentonite is almost unchanged.

G. Obtaining a drug containing attapulgite as a carrier.

100 kg of colloidal attapulgite are sterilized for 2 hours at 160 ° C, then, after cooling to room temperature, are placed in

mixer.

The mixer is activated and, through the nozzle of the liquid dispenser, the sprayed microorganism suspension obtained in accordance with Example 5 in the amount of

10 kg sprayed on attapulgite.

The resulting dispersion is dried at 30 ° C to a moisture content of 6% and placed in bags (tanks) that protect the bacteria.

5 g of the product is stored at 4 ° C, with

room temperature or at 37 ° C, then at zero, the 20th, the 51st and on the 72nd day take samples. The drug is suspended in a sterile HB mixture and after the corresponding

dilutions are plated on RGCA agar. The cultivation was carried out for 46 hours under anaerobic conditions, then the number of grown colonies was determined.

The results are shown in Table. 7

From the data table. 7 shows that the number

the bacteria in the product containing attapulgite does not change.

Example 7, the nutrient medium RGCA + CG specified in example 4 of the composition is seeded with a culture of the strain Propionibacterium sp. HNCMB N2 00287 (Hh-GYOK1-1-123Sz). The cultivation is carried out in two fermenters with a useful volume of 5 liters at 37 ° C under anaerobic conditions. Fermentation begins with seeding 10 ml of the same inoculum prepared on a nutrient medium and continues for 48 hours. Then the cells are separated by centrifugation in a rotor with a speed of 5000 / min and the resulting centrifuged wet sediment in an amount of 53 g is thoroughly mixed with 4 kg of corn meal. The mixture was divided into 8 portions and introduced to 8 sheep. The animals are first forced to fast one day.

Animals receive the drug at the age of 3 months. 8 animals serve as controls. After giving the drug, continue feeding with hay, the weight of animals is determined weekly. Food: meadow hay ad libitum (600-900 g / day) and 100 g / day of mineral salt and vitamin supplements in a carrier of corn meal.

The results are shown in Table. eight.

From the above data, the average daily weight gain of one animal is calculated. The results are shown in Table. 9.

In animals of the control group, with a poor feed over a period of 35 days, a weight gain of 360 g was observed; in treated with the strain Propionibacterium sp. HNCMB Nb 00287 (Hh-GVOK1-1-123Sz) animals weight gain 2620 g; in animals treated with Hp-6UOK1-48a strain, weight gain was 3875 g. From the data presented, it can be seen that the preparations obtained using the proposed method significantly increase the weight gain of sheep.

Claims (1)

Claims for obtaining a preparation for feeding ruminants, including mixing a culture of microorganisms with targeted additives, characterized in that, in order to increase productivity, the cultures of microorganisms use a strain of Propionibacterium sp. HNCMB N2 00287, or strain Vellonella sp. HNCMB No. 00288, or strain Bifldobacterium sp. HNCMB Ns 00289. Table 1 table 2 17 Wrong sampling. 1625317 18 Table 3 T table 4 Table 5 Continued table. five Table 6 Table That blitz 8 Continued table. eight Table 9