GB1569012A - Method of increasing the feed efficiency of an animal by feeding it with an enzyme extract from streptomyces - Google Patents

Description

(54) A METHOD OF INCREASING THE FEED EFFICIENCY OF AN ANIMAL BY FEEDING IT WITH AN ENZYME EXTRACT FROM STREPTOMYCES (71) We, W. R. GRACE & CO., a Corporation organized and existing under the laws of the State of Connecticut, United States of America, of 1114 Avenue of the Americas, New York, New York 10036, United States of America, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to improving the feed efficiency of non-human animals.

For many years, the poultry industry has been interested in methods of increasing the feed efficiency of chickens, turkeys and other forms of poultry. Because of the large amounts of feed consumed by poultry and the small margin of return on capital invested in raising them, even increases as small as 1% are significant economically. The "feed efficiency" is the amount of weight gain in relation to the amount of feed consumed. Feed efficiency is also important for other animals raised as food.

A variety of additives have been suggested for use in raising animal feed efficiency. For example the fermentation products having enzymatic activity of the bacteria Streptomyces fradiae 'are useful in reducing the viscosity of intestinal mucus to a level between trypsin and chymotrypsin. Animals receiving feed containing the enzymatic extract are said to experience an increase in feed efficiency. Apparently the enzyme extract permits nutrients of digested food to pass more easily through the mucous membrane which covers the wall of the intestine.

It has been said that excessive reduction in mucus viscosity should be avoided because in this situation a counter-reaction is engendered, i.e. mucus production is actually stimulated.

Further useful enzymes must not be inhibited by trypsin inhibitors.

We have found, according to the present invention, that the feed efficiency in non-human animals, e.g. cattle as well as non-ruminant animals such as swine and poultry can be increased by feeding controlled amounts of an enzyme fraction having the characteristics set out below obtained by culturing Streptomyces griseus in a fermentation medium. The fraction has approximately half the proteolytic activity of the enzyme obtained from Streptomyces fradiae on an equal weight basis. It possesses a high capacity to reduce mucus viscosity approximately 3 times that of the Streptomyces fradiae enzyme. The "mucinase" activity of the enzyme fraction is, unlike theStreptomycesfradiae enzyme, inhibited by trypsin inhibitor.

The preferred enzyme fraction for use in the present invention is obtained from the species of S. griseus micro-organism having the culture collection number NRRL 5747; a representa tive culture has been placed on deposit at the U. S. Department of Agriculture, Northern Regional Research Laboratory, 1815 North University Street, Peoria, Illinois 61604, and is available from that depository. Details, including the taxonomy, of this species are to be found in United States Patent No. 3,868,448 to which reference should be made for further details.

The enzymatic activity of the fraction is measured in relation to its effect on gastric mucin.

Thus, one unit of enzyme activity is defined as that amount of enzyme fraction which, when added to one ml of water, and that solution added to one ml of Prepared Solution of Gastric Mucin (as hereinafter defined), reduces the viscosity of the said Prepared Solution of Gastric Mucin by 50% in two hours at 390C. The centrifuged broth from the fermentation medium generally contains 0.3 to 1.0 units of enzyme fraction/mg broth.

The enzyme fraction for use in the present invention has the following characteristics: (a 1 g of enzyme fraction represents about 500 units of enzyme activity as defined above; b said extract is insoluble in acidic acetone; and (c) storage of the dry enzyme fraction at 109OF (43"C) for 28 weeks or at 1220F (50"C) for 8 weeks results in less than 10% activity decrease, United States Patent No. 3,868,448 gives details of the production and isolation of this enzyme fraction and it also indicates how the enzyme activity can be determined.The "Prepared Solution of Gastric Mucin" is a 7.5% (w/w) solution of gastric mucin, bacteriological in 0.2 M phosphate buffer, pH 7.0; it can be prepared by heating the suspension to about 50"C until it dissolves, then cooling to 250C.

In the isolation of the enzyme fraction, the fermentation broth is first centrifuged to remove all insoluble material including cells. This centrifuged broth is a solution of acetoneinsoluble enzyme fraction and may be used as such to improve feed efficiency. However, the broth is dilute and generally contains only 0.3 to 1.0 units of enzyme fraction/ml. of broth. It would be necessary to handle large volumes of broth to provide the activity level described below. Accordingly, the use of broth is not preferred, and it is advantageous to further process the broth to remove water by centrifugation and the pH of the remaining supernatant solution adjusted to 5.5 with glacial acetic acid with subsequent processing as described in United States Patent No. 3,868,448.

The following table sets forth typical daily amounts of enzyme fraction for administration to various types of animals to obtain increased feed efficiencies.

Swine Chickens Turkeys Cattle Average Daily Amount 40-400 units 2-20 units 4-40 units 160-16QO units The daily feeding level does, of course, depend on the size of the animal. The above ranges are average levels and reflect the fact that lower levels will generally be fed initially with more fraction being fed as the animal grows and eats more. The 2-20 daily unit range for chickens takes into account that chicks would only be expected to eat about 100 grams of food during their first week. As the bird grows the amount of feed and enzyme fraction consumed will increase. A preferred average daily range is from 4-10 units during the first several, say 3, weeks after hatching. For turkeys it is preferred to feed about 6-15 units per day for the first 3 weeks following hatching.For pigs it is preferred to feed about 80-300 units per day for the first 4 weeks after the animal is weaned. For cattle the corresponding level is about 320-1200 units per day for the first 7 weeks after weaning.

Another factor contributing greatly to the success of the feeding programme is to begin feeding the enzyme fraction as soon as possible, i.e. for birds as soon as possible after they have hatched and for mammals as soon as possible after weaning. For chickens it has been found that delays of a week, for example, can greatly decrease the improvement in weight gain. Therefore, for birds such as chickens and turkeys feeding of the enzyme fraction should preferably begin 1 to 2 days after hatching. For pigs, cattle and similar animals, the fraction should be administered within 1 to 7 days after the animal is weaned, and generally continu ously thereafter until the desired weight level is obtained.

Chicks are generally fed a powdered "mash". In preparing the mash a powdered premix generally sold in 20-pound bags is employed. From 0.05 - 0.4 pounds of the enzyme fraction should be employed for every 20 pounds of the premix, i.e. the ratio by weight of enzyme fraction to premix (enzyme fraction plus all other materials) is from 0.0025 to 0.0200. As the chicks mature they are switched to "crumbles". Here again the 0.05 - 0.4 pound range is satisfactory.

In forming premix, the enzyme fraction can be mixed with, for example, vitamins, minerals, antibiotics and other additives together with an orally ingestible carrier such as a feed material, dried grains, alfalfa, corn meal, citrus meal, fermentation residues, ground oyster shells, wheat shorts, mollasses solubles, corn cob meal, antibiotic mycelia and vermiculite.

The premix can be further processed by conventional techniques to form crumbles, e.g. by blending in liquids to form a paste, drying and crushing.

Premixes, prepared in a similar manner, can also be employed for other animals; they are generally sold in bags averaging 20-50 pounds. On a weight basis such premixes will usually contain 0.1 to 1% by weight of enzyme fraction.

Powdered enzyme fraction can also be added directly to the animal feed, typically in an amount from 0.001 to 0.1 %by weight for chickens, pigs, cattle and turkey. Feeds commonly used for poultry, pigs, cattle and other animals can be used, for example corn meal, alfalfa, dried grains (e.g. wheat, corn, oats, barley or rye), citrus meal, wheat shorts, and mollasses solubles. It should be noted that because of the protease inhibiting action of trypsin inhibitors, certain materials, e.g. soybean meal and soya grits should not be mixed with the enzymatic fraction unless they are first heat-treated to deactivate the inhibitor.

The following Examples further illustrate the present invention; Examples 1 and 2 are included to illustrate the preparation and properties of the extract.

EXAMPLE 1 Pro tease Activity his Example illustrates the protease activity of the enzyme fraction in comparison with other enzymes. The procedure employed in determining protease activity is as follows.

SUBSTRATE 1.0% Casein in 0.1 M sodium phosphate buffer adjusted to pH 7.0 (This solution should be heated with stirring until Casein is completely dissolved, then cool.) (Freeze in small vials.) REAGENT: 5.0% Trichloroacetic acid in distilled (d) H20 (w/v). "TCA" STANDARD: 0.1 mg ficin/1.0 ml d H20 (enz. solution) PROCEDURE: 9.0 ml substrate 1.0 ml enzyme solution Combine in 25 ml Erlenmeyer flasks and place in the shaker bath at 370C.

Take 1.0 ml samples at 10 min. intervals (include a zero time sample) and place in centrifuge tubes containing 3.0 ml TCA, to quench the reaction.

Let all samples stand for approximately 20 minutes, then centrifuge at 19,000 rpm for 35 minutes.

Pour off clear layer and read the optical density (OD) by spectrophotometer at 280 m,u using water as a reference.

CALCULATIONS: Protease Units = OD*/min. X 103 *OD sample - OD zero time The following table sets forth the protease activity of the enzymes.

Table I O.D. O.D.

15 min 30 min O.D. (Inhibitor1 O.D. (Inhibitor1 Sample (1.5 min) Present) (30 min) present) 1. Fradiase3(1.0 mg/ml) 0.169 0.172 0.386 0.480 2. Fradiase (2.5 mg/ml) 0.550 0.578 1.03 1.02 3. Fradiase (5.0 mg/ml) 0.912 0.860 1.31 1.24 4. Mucinase2(2.5 mg/ml) 0.301 0.320 0.486 0.478 5. Trypsin (0.5 mg/ml) 0.368 - 0.463 6. Trypsin (2.5 mg/ml) 0.502 - 0.548 7. Chymotryspin (2.5 mg/ml) 1.09 - 1.39 8. Maxatase4(2.5 mg/ml) 0.341 - 0.606 - I Soybean trypsin inhibitor was present in amounts by weight equal to the weight of enzyme employed.

2 Mucinase is the enzyme fraction used in the present invention prepared as described above.

3 Fradiase is the known enzyme preparation obtained from S. Fradiae.

4 A proteolytic enzyme sold by Pfizer.

In runs 1, 2 and 3 the protease activity of fradiase was not inhibited by the presence of soybean trypsin inhibitor. Similarly run 4 demonstrates that the protease activity of the enzyme fraction of the present invention is also not inhibited by the presence of the trysin inhibitor. Comparison of the optical density measurements of runs 2 and 3 with run 4 indicates that on a weight basis the proteolytic activity of fradiase is approximately twice that of the enzymatic fraction of the invention.

Example 2 Effect of Pro tease Inhibitor on Mucin Viscosity The per cent decrease in mucin viscosity due to enzymatic activity was determined as described above for samples of mucinase and fradiase. Several of the samples contained soybean trypsin inhibitor. In each run the average drain time for two samples T1 l and T2) was taken. The "% Viscosity Decrease" was calculated using the formula given in United States Patent No. 3,868,448. In the formula the "Time of Blank" shown in the denominator was 1.6 seconds. Units of enzyme activity in the sample were determined by consulting Table 3 of United States Patent No. 3,868,448. The results are set forth in the following table. Table 2 Time Viscosity Units/ Sample Amount T1 T2 Avg. Decrease mg.

1. Mucinase 3.0 mg. 7.3 6.3 6.5 67.1% 2.3/3= 0.77 2. Mucinase & Soybean Trypsin Inhibitor 3.0 mg.* 10.2 14.6 12.4 27.5% 0.27/3= 0.09 3. Fradiase 0.3 mg. 12.5 10.3 11.4 34.2% 0.34/0.3= 1.10 4. Fradiase 3.0 mg. 9.2 10.6 9.9 44.3% 0.7/3.0= 0.23 5. Fradiase 30.0 mg. 5.3 5.7 5.5 73.8% 3.1/30= 0.10 6. Fradiase & Soybean Trypsin Inhibitor 0.3 mg.* 9.2 11.1 10.2 42.3% 0.6/0.3= 2.0 7. Fradiase & Inhibitor 3.0 mg.* 9.8 10.8 10.2 42.3% 0.6/3= 0.2 8. Fradiase & Inhibitor 30.0 mg.* 11.0 10.6 10.8 38.3% 0.43/30= 0.01 9. Water & Mucinase Fraction (Control) 3.0 mg. 19.0 14.0 16.5 *In runs 2, 6, 7 and 8 trypsin soybean inhibitor was employed on an equal weight basis with qthe amount of enzyme.

X From Table 2 it is apparent that soybean trypsin inhibitor seriously affected the activity of 'mucinase", i.e., comparison of runs 1 and 2 shows that the activity was reduced from 67.1% to 27.5%. For fradiase, comparison of runs 3, 4, 5 with 6, 7 and 8 indicates that enzyme activity was not greatly decreased by the inhibitor. Run 1 shows that mucinase contains approximately 0.77 units of enzyme activity for reducing mucin viscosity. By contrast run 4 indicates that fradiase contains about 0.23 units per mg. of viscosity reducing activity.

Example 3 - Chicken Feeding Trial A chicken trial of two groups of 60 birds each (4 pens of 15 birds for the first four weeks and 2 pens of 30 birds for the second four weeks) was run for 56 days using a commercial diet consisting of 20% crude protein. For one group, the enzyme fraction in the form of a dry powder was added at a level of 0.04%of the feed. Approximately 0.2 units of enzyme activity were employed for each gram of feed. For the other group (control), no additives were added to the diet.

Feed and water were offered ad libitum. Weight gain and feed efficiency was determined.

After 56 days, the results were: Group I, 0.04% enzyme fraction: Improvement over Group II (Control) Average Daily Gain: 35.1 gm 4.3 % Feed Efficiency: 2.26 3.42 % Group II, Control Average Daily Gain: 33.7 gm -- Feed Efficiency: 2.34 - - Example 4 - Chicken Feeding Trial A second trial was conducted similar to Example 3, except 45 chickens per treatment were used, two levels of the enzyme fraction were used, and the additive was withdrawn after 49 days. From the 49th to 56th days, the birds were fed equal amounts of identical commercial rations. After 56 days, the results were: Group I, 0.044% enzyme fraction: Improvement over Group III (Control) Average Daily Gain: 35.9 gm 1.9 % Feed Efficiency: 2.50 2.7 % Group Il,0.022% enzyme fraction: Average Daily Gain: 36.0 gm 2.2 % Feed Efficiency: 2.49 3.1 % Group Ill, Control Average Daily Gain: 35.2 gm - Feed Efficiency: 2.57 - Example 5 - Chicken Feeding Trial A third trial was carried out following the procedure of Example 3 except that the birds were assigned directly to greenhouse pens as follows: 6 pens of 50 birds each per treatment.

The level of enzyme fraction was 0.01% rather than 0.04%. The results were as follows: Group I, 0.01% enzyme fraction: Improvement over Group II (Control) Average Daily Gain: 35.2 gm 2.3 % Feed Efficiency: 2.55 3.4 % Group II, Control Average Daily Gain: 34.4 gm - Feed Efficiency: 2.64 - Example 6 - Turkey Feeding Trial Twenty-four turkeys were divided into two groups of 12 each. A first group was fed ad libitum water and a commercial turkey ration containing 0.01% by weight of enzymatic fraction. To ensure proper mixing, the enzymatic fraction was admixed with a small amount of feed to form a premix. The premix was then diluted with untreated quantities of turkey ration. In the finished ration, the dosage level was about 0.5 units of enzyme activity per gram of ration. The trial was continued four weeks.Water and feed (minus enzyme fraction) were also fed ad libitum to the control group during the 4 weeks of the trial.

Average daily weight gain and feed efficiency were measured weekly after the second week with the following results: Average Daily Weight Gain Number of (gms) Feed Efficiency Birds Period (Weeks) 2 3 4 2 3 4 Diet 1 15.48 20.26 25.94 1.45 1.63 1.70 12 Basal Ration Diet 2 16.68 21.18 26.15 1.40 1.57 1.67 12 Basal Ration + 0.01% enzyme fraction Example 7 Comparison of "Mucinase" vs. "Fradiase" Activity in Chickens To compare mucinase and fradiase levels in vivo, a chicken trial (8 weeks) was conducted as in Example 3. This trial consisted of 60 birds (4 pens of 15 birds for the first four weeks and 2 pens of 30 birds each for the second four weeks) per dietary treatment. The treatments consisted of a control diet and two rations containing 0.04% by weight of fradiase of mucinase. The diets were kept refrigerated prior to use.

Feed and water was offered ad libitum. Weight gain and feed efficiency were measured in approximately two week periods. In the data set forth in the following table, a 51-day set of totals is reported as additional fradiase was not available to complete a 56-day trial. The control and mucinase treatments were then carried out to 56 days (8 weeks) and totals for these are presented.

Table 3 To@als 14th @ 27th @ 42nd @ 51@@ @ to % 56th % Totals/ % ADG* Day In@@case Day In@@case Day In@@case Day In@@case Date Incrcase Day incrcase Trial Incrcase Control 16.00 --- 29.98 --- 41.05 --- 46.76 --- 33.45 --- 46.98 --- 33.67 -- Mucinase 16.67 4.0 31.11 3.63 42.61 3.66 49.40 5.34 34.95 4.29 48.09 2.31 35.11 4.10 Fradiase 16.73 4.4 30.09 0.3@ 42.79 4.07 47.61 1.79 34.31 2.51 --- --- FE** Control 1.67 --- 2.06 --- 2.59 --- 2.94 --- 2.32 --- 3.28 --- 2.34 -- Mucinase 1.64 1.8 2.02 1.94 2.50 3.47 2.80 4.76 2.24 3.24 3.16 3.66 2.26 3.42 Fradiase 1.61 3.6 2.07 0.48 2.49 3.86 2.90 1.36 2.27 1.94 --- -- *ADG = Averape Daily Weight Gain **FE = Feed @ffi@iency fital Example 8 - Swine Feeding Trial A comparative study using two lots of 10 pigs each (total of 20 animals) was run for 42 days.

During this period the animals were fed on an ad libitum basis. The control group of 10 pigs was fed a commercial ration containing at least 18% crude protein, 3.5% crude fat, 4.5% crude fibre, penicillin (50 grams/ton), 0.011% sulfamethazine and chloratetracycline (100 grams/ton). To evaluate the present invention the other group of 10 pigs received the same ration containing in addition 0.01% by weight of the enzyme fraction.

The fraction was mixed into the ration as a dry powder.

The study was begun shortly after weaning when the pigs were about 4 weeks old. It has been found that the efficacy of the enzyme fraction is increased if use is begun as soon as Control Enzyme Fraction Group Initial Number of Animals 10 10 Initial Weight (Ibs/head) 23.6 + 0.6 23.7 + 1.6 Final Weight (Ibs/head) 67.0 89.1 Gain (total for 42 days - lbs/head) 43.4 + 0.01 65.5 + 1.7 Daily Gain (Ibs/head) 1.03 + 0.01 1.56 + 0.04 Feed Intake (Ibs/head) 114.9 + 6.0 144.6 + 3.9 Feed Efficiency (Feed Intake/Gain) 2.65 + 0.21 2.21 + 0.04 Mortality (%) 30 0 possible after weaning.

During the study three pigs in the control group died. All pigs in both groups contracted "scours" although the symptoms were much less severe in the group receiving the enzyme fraction.

The results of the comparative study are set forth below. Data for Final Weight, Gain, Daily Gain, Feed Intake and Feed Efficiency are averages based on the surviving pigs without regard for the data from the three pigs that died.

By comparing "Feed Efficiency" for the two groups, it is clear that the pigs fed with the enzyme fraction used about 0.44 Ibs. of feed less than the control group for each pound gained.

WHAT WE CLAIM IS: 1. A method of increasing the feed efficiency of a non-human animal which comprises feeding it with an extract obtained by culturing Streptomyces griseus in a fermentation medium, said extract being an enzyme fraction possessing the following characteristics: (a) 1 g of the dried enzyme fraction represents about 500 units, 1 unit being that amount of enzyme fraction which when dissolved in 1 ml of water reduces the viscosity of 1 ml of prepared gastric mucin (as hereinbefore defined) by 50% in 2 hours at 390C; (b) said fraction is insoluble in acidic acetone; and (c) storage of the dry enzyme fraction at 1090F (43"C) for 28 weeks or at 122OF (50"C) for 8 weeks results in at most 10% reduction in activity.

2. A method according to claim 1 in which the species of Streptomyces griseus is that identified by the NRRL culture collection number 5747.

3. A method according to claim 1 or 2 in which the extract is fed mixed with the animal feed.

4. A method according to any one of the preceding claims in which the extract is fed daily for at least 3 weeks.

5. A method according to any one of the preceding claims in which the animal is a species of cattle.

6. A method according to claim 5 in which the extract is fed in an amount of 320 to 1200 units per day for 7 weeks, the first administration being not more than 7 days after weaning.

7. A method according to any one of claims 1 to 4 in which the animal is a pig.

8. A method according to claim 7 in which the extract is fed in an amount of 80 to 300 units per day for 4 weeks, the first administration being not more than 7 days after weaning.

9. A method according to any one of claims 1 to 4 in which the animal is a species of poultry.

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (15)

**WARNING** start of CLMS field may overlap end of DESC **. Example 8 - Swine Feeding Trial A comparative study using two lots of 10 pigs each (total of 20 animals) was run for 42 days. During this period the animals were fed on an ad libitum basis. The control group of 10 pigs was fed a commercial ration containing at least 18% crude protein, 3.5% crude fat, 4.5% crude fibre, penicillin (50 grams/ton), 0.011% sulfamethazine and chloratetracycline (100 grams/ton). To evaluate the present invention the other group of 10 pigs received the same ration containing in addition 0.01% by weight of the enzyme fraction. The fraction was mixed into the ration as a dry powder. The study was begun shortly after weaning when the pigs were about 4 weeks old. It has been found that the efficacy of the enzyme fraction is increased if use is begun as soon as Control Enzyme Fraction Group Initial Number of Animals 10 10 Initial Weight (Ibs/head) 23.6 + 0.6 23.7 + 1.6 Final Weight (Ibs/head) 67.0 89.1 Gain (total for 42 days - lbs/head) 43.4 + 0.01 65.5 + 1.7 Daily Gain (Ibs/head) 1.03 + 0.01 1.56 + 0.04 Feed Intake (Ibs/head) 114.9 + 6.0 144.6 + 3.9 Feed Efficiency (Feed Intake/Gain) 2.65 + 0.21 2.21 + 0.04 Mortality (%) 30 0 possible after weaning. During the study three pigs in the control group died. All pigs in both groups contracted "scours" although the symptoms were much less severe in the group receiving the enzyme fraction. The results of the comparative study are set forth below. Data for Final Weight, Gain, Daily Gain, Feed Intake and Feed Efficiency are averages based on the surviving pigs without regard for the data from the three pigs that died. By comparing "Feed Efficiency" for the two groups, it is clear that the pigs fed with the enzyme fraction used about 0.44 Ibs. of feed less than the control group for each pound gained. WHAT WE CLAIM IS:

1. A method of increasing the feed efficiency of a non-human animal which comprises feeding it with an extract obtained by culturing Streptomyces griseus in a fermentation medium, said extract being an enzyme fraction possessing the following characteristics: (a) 1 g of the dried enzyme fraction represents about 500 units, 1 unit being that amount of enzyme fraction which when dissolved in 1 ml of water reduces the viscosity of 1 ml of prepared gastric mucin (as hereinbefore defined) by 50% in 2 hours at 390C; (b) said fraction is insoluble in acidic acetone; and (c) storage of the dry enzyme fraction at 1090F (43"C) for 28 weeks or at 122OF (50"C) for 8 weeks results in at most 10% reduction in activity.

2. A method according to claim 1 in which the species of Streptomyces griseus is that identified by the NRRL culture collection number 5747.

3. A method according to claim 1 or 2 in which the extract is fed mixed with the animal feed.

4. A method according to any one of the preceding claims in which the extract is fed daily for at least 3 weeks.

5. A method according to any one of the preceding claims in which the animal is a species of cattle.

6. A method according to claim 5 in which the extract is fed in an amount of 320 to 1200 units per day for 7 weeks, the first administration being not more than 7 days after weaning.

7. A method according to any one of claims 1 to 4 in which the animal is a pig.

8. A method according to claim 7 in which the extract is fed in an amount of 80 to 300 units per day for 4 weeks, the first administration being not more than 7 days after weaning.

9. A method according to any one of claims 1 to 4 in which the animal is a species of poultry.

10. A method according to claim 9 in which the animal is a turkey.

11. A method according to claim 10 in which the extract is fed in an amount of 6 to 15 units per day for 3 weeks, the first administration being no more than 2 days after hatching.

12. A method according to claim 9 in which the animal is a chicken.

13. A method according to claim 12 in which the extract is fed in an amount from 4 to 10 units per day for the 3 weeks following the hatching, the first administration being more than 2 days after hatching.

14. A method according to claim 1 substantially as hereinbefore described.

15. A method according to claim 1 substantially as described in any one of Examples 4 to 9.