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Problems of large scale production
FdCosmet. Toxicol. Vol. 3, pp. 223-228. Pergamon Press 1965. Printed in Grea/Britain
Problems of Large Scale Production W. HEINE Zentralinstitut fiir l/ersuchstierzucht, Lettow-Vorbeck, A llee 57, 3, Hannover-Linden, W. Germany
Please allow me to limit my remarks to experiences encountered in our institute and related to the production of mice and rats. In large scale production we have to consider two demands: (1) the animal bred must be of a quality suitable for the research to be carried out; (2) the production must be organized economically. Both demands are always present in large scale production and therefore dictate the methods chosen. It is the task of the manager of such a unit to coordinate the scientific necessities with the most economic methods, so that neither the one nor the other side becomes neglected. It is hardly necessary to emphasize that this is not an easy task. The laboratory animal as a measuring instrument shows inaccurate values if by the stress of the experiment, especially in long-term experiments, latent diseases become active. These diseases force the organism or the animal measuring instrument to reactions that are not caused by the experimental treatment. Modern animal experiments require, more than ever, animals that are healthy and clean, that is, free from the known pathogenic micro-organisms for that species. In large scale production these conditions can only be attained if the animal is free from pathogenic microbes from the outset.Therapeutic measures of the conventional kind, undertaken to improve the quality of the animals, are both time consuming and expensive in large scale production. The value of such treatment is always in doubt because it is impossible to work in the animal room to such exact conditions as in a laboratory. In any case therapeutic treatment is inadvisable, as a laboratory animal that has been treated with a drug can yield false experimental results. We have tried in recent years to free our animals from ecto- and endoparasites (Heine, 1962). Our stocks of mice and rats were infected with mites of the species Myobia and Mycoptes and with lice of the species Polyplax. As endoparasites we found pinworms of the species Syphacia and Aspiculuris. To free the colonies of ectoparasites, animals were dipped at three 11-day intervals in a 0.5% solution of Alodan. A total of 5583 breeding rats was dipped individually and 4010 mice together with their progency were treated in a special dipping-pot. All the animals in one breeding room had to be dipped in one session as there were no clean reserve rooms. After treatment, the animals could not be put into clean cages since only the required number of reserve cages for the weekly routine cleaning was available. Further the current production could not be disturbed. The condition imposed after the dip was that no female carrying parasites with eggs was allowed to remain in the animal rooms. Since the end of treatment in January 1962 we have not seen any ectoparasites. We have tried several times to free our mice from pinworms. We have only succeeded partially, although today there are excellent medicaments against pinworms among the piperazine preparations. Our animals received piperazine hexahydrate at 1% in their E
223
224
W. HEINE
drinking water on 3 alternate weeks. Before treatment commenced the animals in clean cages were transferred into steam-cleaned rooms. Several weeks elapsed before we found pinworm eggs again and we have reason to suppose that after the treatment the mice were at first free from worms. The animal rooms may have been free of pinworm eggs but we were not able to clean the equipment rooms thoroughly. Probably worm eggs were carried into the animal rooms after treatment causing re-infection. We have not undertaken any further treatment for the improvement of our animal colonies since the failure of the pinworm treatment, because of the expenses incurred. Besides, we cannot be certain that animals passing into the hands of the experimenter are free of drug residues. The euUing of all unhealthy animals which should be carried out in all colonies can help to maintain but not improve the health of the colony. It is much more likely that culling can lead to strains which are resistant against certain microbes, and whose resistance breaks down during.the experiment. The reaction which then becomes apparent can be misinterpreted as a result of the experiment. In our rat colony we have been trying for years to eliminate sick animals in order to improve the condition of the colony. Our rats carry the virus of the bronchiectasis and pleuropneumonia-like organism (PPLO) like most conventional rat colonies. We have only partially succeeded in eliminating these viruses and as therapy is out of the question we are discarding all the rat colonies for the time being and intend to rear a new colony from a nucleus developed and reared by the isolator technique. As mentioned above, large scale production will be started with an animal colony which is already free from known pathogenic microbes for this species. This technique is the best available at present from the scientific and economic viewpoints. Foster (1961) has described a breeding colony built up by this method which is more profitable than a conventional colony. Under these conditions one is practising prophylactic measures by maintaining a barrier between the healthy stock and possible surrounding contamination. This requires expensive equipment but the cost of partial or total loss of the colony due to insufficient safety precautions would be even greater. For example in 1960 we saw changes in the tails and paws of 6 mice maintained in one of our isolated mouse rooms. These signs resembled mouse-pox and we killed the whole colony of 12,000 animals at a minimal cost of 11,000 DM. We believe that we have achieved an optimal separation for a large scale production unit in our new animal houses. Contact with the outer world is maintained by (1) one or more autoclaves or by the disinfection tank; (2) air filters which must retain 99 ~ of particles 1 t~ or more in size; (3) four-doored shower tracts; only one of the doors can be opened at once. Infections carried by the nose, mouth, and throat of personnel remains uncontrolled. The building has no outside window except the room housing the air conditioning equipment and the canteen. The staff wear sterilized clothes and work for 4 hr in the building and after a change of clothing leave the clean area to eat in the canteen or take advantage of toilet facilities. Then the staff take another shower before re-entering the working area for a further 4-hr shift. The toilet in the clean inner part of the animal house is sealed up and is only used in an emergency. All doors of the building are made of iron and are air tight. Soiled material and animals are passed out of the unit through the double-ended autoclave without sterilization after which the autoclave is sterilized. Used bedding is stored in a central silo from which it is automatically burnt thereby providing part of the heat for the building. We think that these measures permit us to keep the animals healthy. A very difficult problem of large scale production, at least in Germany, is the demand for ~lualified animal staff. This lack and economic reasons compel us to apply extensive mechan-
PROBLEMS OF LARGE SCALE PRODUCTION
225
ization and rationalization of animal husbandry. The great number of cages and animals multiply each handling many times. For instance, to abolish one handling of about 2 sec in 1000 cages daily, saves over 150 working hours a year. Cage and bottle washing machines are today essential equipment. The mechanical aspects, for instance the vacuum transportation of pellets, soiled and clean bedding is now established in laboratory animal farms. Automatic feeding and drinking are also employed. As part of this scheme we, in conjunction with the company Bayer-Werke, have developed new cages and bottles made from the polycarbonate plastics macrolon both of which are now well known on the continent. This material is almost unbreakable and can be autoclaved. The cages can be stacked with the bedding inside without the walls touching each other. The bottles have a wide neck and in place of the drinking tube, which is always difficult to clean, a conical stainless-steel cap. Brock, Battling & Wilk (1962) of Asta-Werke have designed a very good automatic watering system involving the use of a pressure-reducing valve which is attached to an ordinary water pipe. By means of a spring it is possible to increase the pressure in the system momentarily so that the drinking valves are flushed out. The low pressure pipe runs above the cages and by means of short plastics tubes connect to the drinking valves which enter the cages like a normal drinking tube. In order to replace wood shavings Wilk (personal communication in 1963) of the Thomae company, in collaboration with the manufacturers of paper napkins, has developed a bedding material consisting of several layers of cellulose to fit the bottom of the cage. At present we are testing 6000 of these sheets. Previous experience with cellulose showed an increase in dirt and smell in the animal house, thus making the animals more difficult to control. It is very important to find out how high the productivity of the animal colony is. The breeder always tries to raise the productivity of the individual animal, so that the colony has a high average productivity. The animals must be mated in an optimal manner in order to achieve the highest production in relation to the space available. The usual method o f assessing productivity of a colony is to estimate the number of young weaned per female per week (y/f/w). This method of estimation is relatively simple but is, however, not quite exact. Only the number of animals weaned in a certain time is recorded. It is possible that a litter of 8 is heavier than one of 12. The mother of 8 would in reality have achieved more than the mother of 12, although on the above basis the latter would be scored higher. These considerations caused Haakh (1960) to develop a new way of estimating productivity. He does not compare the number of animals weaned but the weight of the young animals on day 21 after birth. This method not only takes into account fertility but also lactation and other factors influencing the growth of the young. We measured the productivity of a small colony of 51 pairs of the non-inbred mouse strain N M R I employing permanent mating over a period of 162 days. Measurements were made by the method of Haakh (1960) and also by estimating values of y/f/w. We found values of 2.76 g/day and 1.89 y/f/w. Further matings were made using 48 pairs from litters of the females which showed the best performance. Brother-sister matings were avoided. In this case the values were 2.99 g/day and 1.94 y/f/w at 126 days. A second selection of 49 young pairs were mated yielding values of 3.10 g/day and 2-13 y/f/w. Our results accord with tohse obtained by Haakh (1960). This method of assessment has two disadvantages: first it involves a large volume of written work and second, weighing must be carried out on day 21 even if this falls on a Sunday. This prevents from the outset its application to larger colonies.
226
w. HEINE
In large colonies, especially, it is necessary to regulate productivity but the customary methods of estimating the number of weaned for a specific time is to be preferred to the method of Haakh (1960). At the same time one should take care that these measurements are carried out on a large sample. From a colony of several thousand cages a sample of 500-800 with a mating system of 4 females to 1 male (4F:IM) will give information on 2000-3000 females and the associated males. This sample is sufficiently large to be representative of the whole colony and not lead to inbreeding. The new breeding stock is selected from the control cages the animals of which have an above average performance. By this system only the control sample has record cards but not the bulk of the colony which has been derived from the controls. A single record card is kept for all animals mated on a single day with notes of date of mating, number of animals mated and the number of young weaned. The productivity of single cages is not recorded by this method. It is only necessary to remove the animals that have failed to produce young leaving the remainder for the breeding period. As part of the colony is continually under inspection and the poor breeders are eliminated one would expect the numbers of the latter to be gradually reduced. We prefer this control of a part of the whole colony to the control of a separate small nucleus. As already mentioned, the important factor is the productivity of the colony with reference to space rather than that of individual animals. We found in our N M R I mouse strain that the mating ratio of 1M:IF for 50 cages yielded an average of 1-98 y/f/w, while under the same conditions 36 cages each containing 1M:2F gave 1-35 y/f/w; 43 cages with a 1M:3F ratio gave 1.12 y/f/w and 25 cages with a 1M:4F ratio gave 1 y/f/w. This demonstrates that the greater the number of females in a cage of a given size, the lower will be the value of y/f/w, due to increased losses during rearing. These results suggest that the best performance could be gained by breeding 1M:I F in cages which are comparatively small, thereby obtaining the highest production per female in the smallest space. We, however, feel that the space required per female is less when caged together than when caged individually. This supposition awaits experimental confirmation. We have recently extended the breeding period of our N M R I mouse strain from 6 to 8 months. With 570 females caged in a ratio of 1M :3F (190 cages) the values of y/f/w after 6 and 8 months were 1-16 and 1.19 respectively. With 760 females caged IM :4F (190 cages) the corresponding values were I. l and 1.13 y/f/w. This work indicates that one can extend the breeding period to 8 months. In terms of total numbers produced the results can be expressed as follows: 190 cages (1M :3F) gave 678 animals per week and a total of 23,062 in 8 months; 190 cages (1M:4F) gave 589 animals per week, and a total of 29,199 in 8 months. Comparable data for rats are not available since we have discarded our rat colonies for the time being, as reported above. But one thing ought to be mentioned that fcrmerly we mated our rats in group cages and separated the individuals for parturition. This prevented the practice of postpartem mating and led to long time intervals between litters. In the wild the lactating rat is not separated from the population and postpartem mating is probably practised. Therefore there seems little reason to separate the sexes in our breeding colony. Subjectively, we feel that permanent pairing is more productive than intermittent pairing in rats as we have already seen in mice. The increased soiling of the cage by the male is offset by the greater productivity. The objective of the animal breeder is to provide the experimenter with an animal with
PROBLEMS OF LARGE SCALE PRODUCTION
227
which he can obtain reliable and reproducable results. In use, the experimental animal is a measuring instrument and like all measuring instruments can be calibrated by establishing zero points or known standards. Absolute calibration of an experimental animal or colony however, is impossible, as living organisms are far more complicated than even an electronic computer. But we must try to approach the exactness of a graduated measuring instrument and to standardize the animals as far as possible, by genetic and environmental control. It seems to me, however, that we have not paid sufficient attention to a very important aspect of our work. We should all ensure that animals of a given strain possess consistent and well-defined characteristics. Allow me to cite some examples. The Wistar rat or Sprague-Dawley rat is claimed to be a well established entity. For most animal experimenters the name Wistar rat implies an experimental animal with a special characteristic. It often happens that laboratories obtain contrasting results from similar experiments using Wistar rats because the animals were derived from different colonies. Thus the Wistar rat is not a well-defined entity. Another example is afforded by experiences gained in our institute where one of the mouse strains bred is C57BL/6. These animals were originally obtained from the Jackson Memorial Laboratories (JaxLab), Barharbor, USA. They have since then been bred by exact brothersister mating. One can follow their pedigree from generation to generation but despite this inbreeding I cannot imagine that our line still possesses the same characteristics as the original. Yet a further line of this strain is maintained by a well-known commercial breeder in the USA whose colony originated from a caesarean-derived nucleus. These animals must surely carry less microbes than conventional lines reared in JaxLab and in our own laboratories and consequently may react differently. Experimenters working with these various lines may well obtain different results. In some cases a suffix is added to the strain identification to indicate the origin for example C57BL6/ Jax for the JaxLab line and C57BL/6Jax/Han for the line maintained at Hanover. In my experience the experimenter is not aware of the differences which may exist between these lines and tends to disregard the suffix. In my opinion the maintenance of lines with the same name at various places can only lead to confusion. As I have already mentioned, we can never succeed in calibrating an experimental animal. Neither can we succeed in keeping absolutely constant the characteristics of one strain over long periods. As we cannot calibrate the animal or prevent variation we must attempt to standardize strains throughout the world. My recommendations for doing this are: (1) Agreement must be reached for standardizing the breeding conditions for each strain; (2) the basis of each strain should be maintained at one centre from which other breeders could obtain breeding stock. This stock should be replaced at specified intervals. This would ensure consistency of strains and if the parent strain varied with time all the lines maintained would show similar changes. Although I realise that this is the ideal I think that international co-operation between breeders could achieve this end. I shall conclude by mentioning the important points once again. If a chemist requires a substance for his analysis, he can obtain it anywhere in the world to a defined purity. He can measure the quantity exactly and obtain results with universally-accepted consistency. The animal experimenter will never be able to work so exactly as he uses the most complicated material that exists, the living organism. As we cannot calibrate this organism we should make provision for it to be as consistent as possible. A given animal strain must be
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k e p t a n d b r e d u n d e r the same c o n d i t i o n s t h r o u g h o u t the world. O n l y in this w a y can we h o p e to o b t a i n c o m p a r a b l e a n d r e p r o d u c a b l e results in a n i m a l e x p e r i m e n t a t i o n . REFERENCES Brock, N., Battling, H. & Wilk, W. (1962). Zur Ern/ihrung der Laboratoriumstiere. 3. Mitteilung: Technische Probleme und Effahrungen bei der Hertstellung und Dalreichung der Altromin-Standarddiiiten fiir Versuchstiere. Arzneimittel-Forsch. 5, 507. Foster, H. L. (1961). The development of specific pathogen free and germ free animals. Biomed. Purview 1, 77. Haakh, U. (1960). Zur Zucht und Haltung der weissen Maus. IV. Das Leistungsprinzip in der Versuchstierzucht. ZentbL VetMed. 7, 715. Heine, W. (1962). Zur Ektoparasitenbek~impfung bei Maus und Ratte. Z. VersuchstierK. 2, 1.
Problems of Large Scale Production W. HEINE Zentralinstitut fiir l/ersuchstierzucht, Lettow-Vorbeck, A llee 57, 3, Hannover-Linden, W. Germany
Please allow me to limit my remarks to experiences encountered in our institute and related to the production of mice and rats. In large scale production we have to consider two demands: (1) the animal bred must be of a quality suitable for the research to be carried out; (2) the production must be organized economically. Both demands are always present in large scale production and therefore dictate the methods chosen. It is the task of the manager of such a unit to coordinate the scientific necessities with the most economic methods, so that neither the one nor the other side becomes neglected. It is hardly necessary to emphasize that this is not an easy task. The laboratory animal as a measuring instrument shows inaccurate values if by the stress of the experiment, especially in long-term experiments, latent diseases become active. These diseases force the organism or the animal measuring instrument to reactions that are not caused by the experimental treatment. Modern animal experiments require, more than ever, animals that are healthy and clean, that is, free from the known pathogenic micro-organisms for that species. In large scale production these conditions can only be attained if the animal is free from pathogenic microbes from the outset.Therapeutic measures of the conventional kind, undertaken to improve the quality of the animals, are both time consuming and expensive in large scale production. The value of such treatment is always in doubt because it is impossible to work in the animal room to such exact conditions as in a laboratory. In any case therapeutic treatment is inadvisable, as a laboratory animal that has been treated with a drug can yield false experimental results. We have tried in recent years to free our animals from ecto- and endoparasites (Heine, 1962). Our stocks of mice and rats were infected with mites of the species Myobia and Mycoptes and with lice of the species Polyplax. As endoparasites we found pinworms of the species Syphacia and Aspiculuris. To free the colonies of ectoparasites, animals were dipped at three 11-day intervals in a 0.5% solution of Alodan. A total of 5583 breeding rats was dipped individually and 4010 mice together with their progency were treated in a special dipping-pot. All the animals in one breeding room had to be dipped in one session as there were no clean reserve rooms. After treatment, the animals could not be put into clean cages since only the required number of reserve cages for the weekly routine cleaning was available. Further the current production could not be disturbed. The condition imposed after the dip was that no female carrying parasites with eggs was allowed to remain in the animal rooms. Since the end of treatment in January 1962 we have not seen any ectoparasites. We have tried several times to free our mice from pinworms. We have only succeeded partially, although today there are excellent medicaments against pinworms among the piperazine preparations. Our animals received piperazine hexahydrate at 1% in their E
223
224
W. HEINE
drinking water on 3 alternate weeks. Before treatment commenced the animals in clean cages were transferred into steam-cleaned rooms. Several weeks elapsed before we found pinworm eggs again and we have reason to suppose that after the treatment the mice were at first free from worms. The animal rooms may have been free of pinworm eggs but we were not able to clean the equipment rooms thoroughly. Probably worm eggs were carried into the animal rooms after treatment causing re-infection. We have not undertaken any further treatment for the improvement of our animal colonies since the failure of the pinworm treatment, because of the expenses incurred. Besides, we cannot be certain that animals passing into the hands of the experimenter are free of drug residues. The euUing of all unhealthy animals which should be carried out in all colonies can help to maintain but not improve the health of the colony. It is much more likely that culling can lead to strains which are resistant against certain microbes, and whose resistance breaks down during.the experiment. The reaction which then becomes apparent can be misinterpreted as a result of the experiment. In our rat colony we have been trying for years to eliminate sick animals in order to improve the condition of the colony. Our rats carry the virus of the bronchiectasis and pleuropneumonia-like organism (PPLO) like most conventional rat colonies. We have only partially succeeded in eliminating these viruses and as therapy is out of the question we are discarding all the rat colonies for the time being and intend to rear a new colony from a nucleus developed and reared by the isolator technique. As mentioned above, large scale production will be started with an animal colony which is already free from known pathogenic microbes for this species. This technique is the best available at present from the scientific and economic viewpoints. Foster (1961) has described a breeding colony built up by this method which is more profitable than a conventional colony. Under these conditions one is practising prophylactic measures by maintaining a barrier between the healthy stock and possible surrounding contamination. This requires expensive equipment but the cost of partial or total loss of the colony due to insufficient safety precautions would be even greater. For example in 1960 we saw changes in the tails and paws of 6 mice maintained in one of our isolated mouse rooms. These signs resembled mouse-pox and we killed the whole colony of 12,000 animals at a minimal cost of 11,000 DM. We believe that we have achieved an optimal separation for a large scale production unit in our new animal houses. Contact with the outer world is maintained by (1) one or more autoclaves or by the disinfection tank; (2) air filters which must retain 99 ~ of particles 1 t~ or more in size; (3) four-doored shower tracts; only one of the doors can be opened at once. Infections carried by the nose, mouth, and throat of personnel remains uncontrolled. The building has no outside window except the room housing the air conditioning equipment and the canteen. The staff wear sterilized clothes and work for 4 hr in the building and after a change of clothing leave the clean area to eat in the canteen or take advantage of toilet facilities. Then the staff take another shower before re-entering the working area for a further 4-hr shift. The toilet in the clean inner part of the animal house is sealed up and is only used in an emergency. All doors of the building are made of iron and are air tight. Soiled material and animals are passed out of the unit through the double-ended autoclave without sterilization after which the autoclave is sterilized. Used bedding is stored in a central silo from which it is automatically burnt thereby providing part of the heat for the building. We think that these measures permit us to keep the animals healthy. A very difficult problem of large scale production, at least in Germany, is the demand for ~lualified animal staff. This lack and economic reasons compel us to apply extensive mechan-
PROBLEMS OF LARGE SCALE PRODUCTION
225
ization and rationalization of animal husbandry. The great number of cages and animals multiply each handling many times. For instance, to abolish one handling of about 2 sec in 1000 cages daily, saves over 150 working hours a year. Cage and bottle washing machines are today essential equipment. The mechanical aspects, for instance the vacuum transportation of pellets, soiled and clean bedding is now established in laboratory animal farms. Automatic feeding and drinking are also employed. As part of this scheme we, in conjunction with the company Bayer-Werke, have developed new cages and bottles made from the polycarbonate plastics macrolon both of which are now well known on the continent. This material is almost unbreakable and can be autoclaved. The cages can be stacked with the bedding inside without the walls touching each other. The bottles have a wide neck and in place of the drinking tube, which is always difficult to clean, a conical stainless-steel cap. Brock, Battling & Wilk (1962) of Asta-Werke have designed a very good automatic watering system involving the use of a pressure-reducing valve which is attached to an ordinary water pipe. By means of a spring it is possible to increase the pressure in the system momentarily so that the drinking valves are flushed out. The low pressure pipe runs above the cages and by means of short plastics tubes connect to the drinking valves which enter the cages like a normal drinking tube. In order to replace wood shavings Wilk (personal communication in 1963) of the Thomae company, in collaboration with the manufacturers of paper napkins, has developed a bedding material consisting of several layers of cellulose to fit the bottom of the cage. At present we are testing 6000 of these sheets. Previous experience with cellulose showed an increase in dirt and smell in the animal house, thus making the animals more difficult to control. It is very important to find out how high the productivity of the animal colony is. The breeder always tries to raise the productivity of the individual animal, so that the colony has a high average productivity. The animals must be mated in an optimal manner in order to achieve the highest production in relation to the space available. The usual method o f assessing productivity of a colony is to estimate the number of young weaned per female per week (y/f/w). This method of estimation is relatively simple but is, however, not quite exact. Only the number of animals weaned in a certain time is recorded. It is possible that a litter of 8 is heavier than one of 12. The mother of 8 would in reality have achieved more than the mother of 12, although on the above basis the latter would be scored higher. These considerations caused Haakh (1960) to develop a new way of estimating productivity. He does not compare the number of animals weaned but the weight of the young animals on day 21 after birth. This method not only takes into account fertility but also lactation and other factors influencing the growth of the young. We measured the productivity of a small colony of 51 pairs of the non-inbred mouse strain N M R I employing permanent mating over a period of 162 days. Measurements were made by the method of Haakh (1960) and also by estimating values of y/f/w. We found values of 2.76 g/day and 1.89 y/f/w. Further matings were made using 48 pairs from litters of the females which showed the best performance. Brother-sister matings were avoided. In this case the values were 2.99 g/day and 1.94 y/f/w at 126 days. A second selection of 49 young pairs were mated yielding values of 3.10 g/day and 2-13 y/f/w. Our results accord with tohse obtained by Haakh (1960). This method of assessment has two disadvantages: first it involves a large volume of written work and second, weighing must be carried out on day 21 even if this falls on a Sunday. This prevents from the outset its application to larger colonies.
226
w. HEINE
In large colonies, especially, it is necessary to regulate productivity but the customary methods of estimating the number of weaned for a specific time is to be preferred to the method of Haakh (1960). At the same time one should take care that these measurements are carried out on a large sample. From a colony of several thousand cages a sample of 500-800 with a mating system of 4 females to 1 male (4F:IM) will give information on 2000-3000 females and the associated males. This sample is sufficiently large to be representative of the whole colony and not lead to inbreeding. The new breeding stock is selected from the control cages the animals of which have an above average performance. By this system only the control sample has record cards but not the bulk of the colony which has been derived from the controls. A single record card is kept for all animals mated on a single day with notes of date of mating, number of animals mated and the number of young weaned. The productivity of single cages is not recorded by this method. It is only necessary to remove the animals that have failed to produce young leaving the remainder for the breeding period. As part of the colony is continually under inspection and the poor breeders are eliminated one would expect the numbers of the latter to be gradually reduced. We prefer this control of a part of the whole colony to the control of a separate small nucleus. As already mentioned, the important factor is the productivity of the colony with reference to space rather than that of individual animals. We found in our N M R I mouse strain that the mating ratio of 1M:IF for 50 cages yielded an average of 1-98 y/f/w, while under the same conditions 36 cages each containing 1M:2F gave 1-35 y/f/w; 43 cages with a 1M:3F ratio gave 1.12 y/f/w and 25 cages with a 1M:4F ratio gave 1 y/f/w. This demonstrates that the greater the number of females in a cage of a given size, the lower will be the value of y/f/w, due to increased losses during rearing. These results suggest that the best performance could be gained by breeding 1M:I F in cages which are comparatively small, thereby obtaining the highest production per female in the smallest space. We, however, feel that the space required per female is less when caged together than when caged individually. This supposition awaits experimental confirmation. We have recently extended the breeding period of our N M R I mouse strain from 6 to 8 months. With 570 females caged in a ratio of 1M :3F (190 cages) the values of y/f/w after 6 and 8 months were 1-16 and 1.19 respectively. With 760 females caged IM :4F (190 cages) the corresponding values were I. l and 1.13 y/f/w. This work indicates that one can extend the breeding period to 8 months. In terms of total numbers produced the results can be expressed as follows: 190 cages (1M :3F) gave 678 animals per week and a total of 23,062 in 8 months; 190 cages (1M:4F) gave 589 animals per week, and a total of 29,199 in 8 months. Comparable data for rats are not available since we have discarded our rat colonies for the time being, as reported above. But one thing ought to be mentioned that fcrmerly we mated our rats in group cages and separated the individuals for parturition. This prevented the practice of postpartem mating and led to long time intervals between litters. In the wild the lactating rat is not separated from the population and postpartem mating is probably practised. Therefore there seems little reason to separate the sexes in our breeding colony. Subjectively, we feel that permanent pairing is more productive than intermittent pairing in rats as we have already seen in mice. The increased soiling of the cage by the male is offset by the greater productivity. The objective of the animal breeder is to provide the experimenter with an animal with
PROBLEMS OF LARGE SCALE PRODUCTION
227
which he can obtain reliable and reproducable results. In use, the experimental animal is a measuring instrument and like all measuring instruments can be calibrated by establishing zero points or known standards. Absolute calibration of an experimental animal or colony however, is impossible, as living organisms are far more complicated than even an electronic computer. But we must try to approach the exactness of a graduated measuring instrument and to standardize the animals as far as possible, by genetic and environmental control. It seems to me, however, that we have not paid sufficient attention to a very important aspect of our work. We should all ensure that animals of a given strain possess consistent and well-defined characteristics. Allow me to cite some examples. The Wistar rat or Sprague-Dawley rat is claimed to be a well established entity. For most animal experimenters the name Wistar rat implies an experimental animal with a special characteristic. It often happens that laboratories obtain contrasting results from similar experiments using Wistar rats because the animals were derived from different colonies. Thus the Wistar rat is not a well-defined entity. Another example is afforded by experiences gained in our institute where one of the mouse strains bred is C57BL/6. These animals were originally obtained from the Jackson Memorial Laboratories (JaxLab), Barharbor, USA. They have since then been bred by exact brothersister mating. One can follow their pedigree from generation to generation but despite this inbreeding I cannot imagine that our line still possesses the same characteristics as the original. Yet a further line of this strain is maintained by a well-known commercial breeder in the USA whose colony originated from a caesarean-derived nucleus. These animals must surely carry less microbes than conventional lines reared in JaxLab and in our own laboratories and consequently may react differently. Experimenters working with these various lines may well obtain different results. In some cases a suffix is added to the strain identification to indicate the origin for example C57BL6/ Jax for the JaxLab line and C57BL/6Jax/Han for the line maintained at Hanover. In my experience the experimenter is not aware of the differences which may exist between these lines and tends to disregard the suffix. In my opinion the maintenance of lines with the same name at various places can only lead to confusion. As I have already mentioned, we can never succeed in calibrating an experimental animal. Neither can we succeed in keeping absolutely constant the characteristics of one strain over long periods. As we cannot calibrate the animal or prevent variation we must attempt to standardize strains throughout the world. My recommendations for doing this are: (1) Agreement must be reached for standardizing the breeding conditions for each strain; (2) the basis of each strain should be maintained at one centre from which other breeders could obtain breeding stock. This stock should be replaced at specified intervals. This would ensure consistency of strains and if the parent strain varied with time all the lines maintained would show similar changes. Although I realise that this is the ideal I think that international co-operation between breeders could achieve this end. I shall conclude by mentioning the important points once again. If a chemist requires a substance for his analysis, he can obtain it anywhere in the world to a defined purity. He can measure the quantity exactly and obtain results with universally-accepted consistency. The animal experimenter will never be able to work so exactly as he uses the most complicated material that exists, the living organism. As we cannot calibrate this organism we should make provision for it to be as consistent as possible. A given animal strain must be
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k e p t a n d b r e d u n d e r the same c o n d i t i o n s t h r o u g h o u t the world. O n l y in this w a y can we h o p e to o b t a i n c o m p a r a b l e a n d r e p r o d u c a b l e results in a n i m a l e x p e r i m e n t a t i o n . REFERENCES Brock, N., Battling, H. & Wilk, W. (1962). Zur Ern/ihrung der Laboratoriumstiere. 3. Mitteilung: Technische Probleme und Effahrungen bei der Hertstellung und Dalreichung der Altromin-Standarddiiiten fiir Versuchstiere. Arzneimittel-Forsch. 5, 507. Foster, H. L. (1961). The development of specific pathogen free and germ free animals. Biomed. Purview 1, 77. Haakh, U. (1960). Zur Zucht und Haltung der weissen Maus. IV. Das Leistungsprinzip in der Versuchstierzucht. ZentbL VetMed. 7, 715. Heine, W. (1962). Zur Ektoparasitenbek~impfung bei Maus und Ratte. Z. VersuchstierK. 2, 1.