INTRODUCTION TO TEST ANIMALS Scoring(BCS) Pharmacology Laboratory Work Instructions A. How to work with Test Animals 1. Everyone, both practitioners and researchers, who work in a laboratory using test animals should read: a. Instructions for maintaining and using test animals When using test animals, you should pay attention to several factors, including age, weight, gender, animal health condition, and heredity. This factor is of concern because the response to a compound often varies for different species and the same animal. Good condition and level of animal health is a priority when using test animals. Test animals are said to be healthy if during the observation period their body weight increases steadily or decreases by no more than 10% and there are no abnormalities in behavior and must be observed one week in a laboratory or animal care center before the test takes place.Test animals or experimental animals: animals thatintentionally maintained and bredto be used as model animals, and also to study and develop various fields of science on a research or laboratory observation scale. Animal models or model animals are animal objects as imitations (imitation) of humans (or other species), which are used to investigate biological or pathobiological phenomena. The purpose of using live animals is as a tool to clarify theories and phenomena that occur in pharmacology lecture material. Test animals are as free as possible from pathogenic microorganisms, because their presence in the animal's body greatly interferes with the reaction in the research examination, so that from a scientific aspect the results cannot be justified. Based on the level of contamination by pathogenic microorganisms, experimental animals are classified into conventional experimental animals,specified pathogen free(SPF) andgnotobiotic. Apart from that, it is important to use test animals with good immune reactions. b. Basics of keeping test animals There are 3 R basic principles that must be applied in health research when using experimental animals, namely:1) Replacement, 2) Reduction,And3) Refinement. Replacementis that the number of experimental animals that need to be used has been calculated carefully,both from previous similar research and literature to answer research questions and cannot be replaced by other living things such as cells or tissue cultures. Replacement is divided into two parts, namely: relative (as far as possible replacing experimental animals by using animal organs/tissue from slaughterhouses or animals from lower orders) and absolute (replacing experimental animals with cell culture, tissue or computer programs). Reduction is defined as using animals in research to a minimum, but still getting optimal results. The minimum number is usually calculated using the Frederer formula, namely (n-1) (t-1) >15, where n is the number of animals required and t is the number of treatment groups. The weakness of this formula is that the fewer research groups, the more animals required, and vice versa. To overcome this, it is necessary to use appropriate statistical designs to obtain valid research results. Refinement is treating experimental animals humanely, caring for animals well, not hurting animals, and minimizing painful treatment so as to ensure the welfare of experimental animals until the end of the research. In research, there are several test animals that are often used, namely mice, rabbits and primates. The problem is that not just any test animal can be used for research. The test animals must meet several criteria so that the test animals can be said to be suitable for the function or disease that is the object of our research. Below are several species of test animals along with their characteristics and how often researchers use them. 2. Treat test animals with love and do not harm them. The basic ethical principle of conducting biomedical research using experimental animals is to treat experimental animals properlyhumaneNOT humanizing animalsbecause animals are NOT humans. 3. How to treat animals: a. Rabbits and Guinea Pigs Never touch a rabbit's ears because the nerves and blood vessels can be affected. b. Rats and Mice Hold these animals by their tails, but be careful not to let them turn around and bite you. Therefore, apart from the tail, also hold the back of the neck near the head with your thumb and pointer. Note: Sometimes you need leather or rubber gloves that are thick enough to protect your hands from animal bites. However, for those who are used to it, it is better without gloves because direct contact with the animal will make it easier to control the animal's movements 4. Reuse animals that have been used To save costs, if possible it is permissible to use more test animalsfrom one time. However, if the animal has been used for a certain period and the drug used in the previous experiment is still in the animal's body, it is possible that the results of the next experiment will provide incorrect data. This is especially true in the case of inductors and enzyme inhibitors. Under this pretext, the animal may only be used again for the next experiment after a minimum period of 14 days. 1. Rodent (rodent) Rodents, classified as mice, have been used as laboratory animals for more than 100 years. Some types of mice have undergone genetic changes to minimize and control extraneous variables that could change research results and for research purposes. Rats are also animalsreproducible so that it is available in sufficient quantities for research that requires many experimental animals. There are various types of mice including: a. MouseBiobreeding These mice are susceptible to type 1 DM, so these mice are widely usedplayed a big role in the discovery of drugs for type 1 DM. b. White ratSprague Dawley strain The main advantages of this animal are calmness and ease of handling (tame). Adult weight is between 250-300 g for females, and 450 – 520 g for males. Life expectancy is between 2.5 – 3.5 years. The tail is longer than the Wistar strain of rats, they breed quickly. These mice are most widely used in biomedical research such as toxicology, efficacy and safety tests, reproductive tests,behaviour/behavior, aging, teratogenic, oncology, nutrition, and other pharmacological tests. Examples of research carried out include studies of maternal and fetal infections, studies of the effects of pre-natal diets high in salt on offspring, studies of the effects of sex and hormonal status on stress-induced memory impairment, studies of the bone-specific ostocalcin gene in mice, and excitability studies.hippocampusduring the estrous cycle in mice. These mice were first produced by livestockSprague Dawley(then becomeSprague Dawley-AnimalCompany) in Madison, Wisconsin in 1925. c. Wistar Strain White Rat The Wistar strain of rat is lighter in weight and more fierce than the Sprague Dawley strain. These mice are widely used in toxicology research, infectious diseases, efficacy testing, and aging. d. Tiny Rats, aka Mice, are different from mice, in that they are mini in size, reproduce very quickly, and have 99% of their genes similar to humans. Therefore, mice are very representative when used as a model for human genetic diseases (congenital). Apart from that, mice are also very easy to genetically engineer to produce suitable models for various human diseases. Apart from that, mice are also more profitable in terms of ease of handling, storage space, and relatively cheaper price. 2. Rabbit Rabbits are also a test animal that is often used besides mice. For example, albino rabbits. This animal is usually used for eye irritation tests because rabbits have fewer tears than other animals and have less pigment in their eyes because their albino color makes the resulting effects easy to observe. Apart from that, rabbits are also widely used to produce polyclonal antibodies. B. HOW TO CODE ANIMALS Often signs are needed to identify animals in a group or cage. So test animals need to be coded. The way to code these animals can use several types of media. Some examples include using a picric acid solution or a simpler method, namely using a marker. C. FEEDING THE TEST ANIMALS TO REDUCE BIOLOGICAL VARIATIONS 1. Animal tests usually provide results with greater deviations compared to in vitro experiments, due to biological variations. So to ensure that variation is minimal, animals of the same species and strain, same age, same sex, are kept in the same conditions. 2. Experimental animals must be fed according to standard food for them and given waterad libitum. 3. To further reduce biological variation, test animals should be fasted overnight before the experiment begins. In this period the animal is only allowed to drink waterad libitum. D. ANIMAL BITE WOUNDS Tetanus immunization is recommended for everyone working with test animals. Wounds that are abrasive in nature or wounds that are quite deep due to animal bites or because of tools that have been used for animal experiments, must be treated as soon as possible according to first aid methods in case of accidents. If the bite victim has never received immunization/immunity against tetanus, he or she must receive prophylactic immunization. E. DESTROY THE TEST ANIMALS 1. The best way to kill is to give an anesthetic overdose. Intravenous injection of barbiturate (sodium pentobarbital 300 mg/mL) for dogs and rabbits, intraperitoneally or intrathoracically for guinea pigs, rats and mice, or by inhalation using chloroform, carbon dioxide, nitrogen etc. in a closed container for all animals mentioned above. 2. The animal is slaughtered, then placed in a plastic bag and wrapped again in paper, placed in plastic, covered and stored in a refrigerator or immediately ashed (incineration).