Prof. Dr. Hamit OKUR

Research is defined as a planned and scientific study carried out to shed light on any subject, to provide a solution to a problem, or to reach certain concepts, theories or laws. In other words, research can also be defined as a planned scientific study to develop or contribute to knowledge that can be generalized. Medical research is carried out under two main headings: clinical and experimental. Clinical studies are studies conducted on patients or volunteers. Although experimental research is research conducted on experimental animals, alternative methods in recent years (tissue cultures, embryo studies, etc.) are also considered experimental research.
Experimental research can be done for different purposes:
To obtain a new biological product (vaccine, hormone, antibody, drug, etc.). to

Measuring the effectiveness of drugs in investigating the cause and treatment of diseases (human or animal) style="font-size:14px;">The importance of research in bringing medicine to this point today is an undeniable fact. Experimental research using animals has played an important role in the development of modern medical treatment and continues to be an essential method to determine new drugs and methods for the treatment of new diseases.
1. EXPERIMENTAL RESEARCH

Experiment is a type of research that includes applying the procedures whose effect will be measured to the subjects under certain rules and conditions, measuring the responses of the subjects to these procedures, and making a decision by comparing the results obtained.
The effect of the procedure is the variable to be measured or compared; drug dosage, etc. or a surgical method.
Subject is the entity such as the individual, animal, embryo, cell, microorganism to which the procedure is applied.
The response of the subjects to the procedure is any physiological, biochemical, metabolic-like changes that may occur in the subjects.
There are basic rules of scientific study. In experimental research, these rules must first be known and followed at every stage of the research. These rules:
Do no harm
Respect

Recording events in full detail
Being honest: The scientist works not to prove that his hypothesis is correct, but to destroy it. Research data should be collected objectively and the results should not be interfered with.
Confidentiality: Especially in research conducted on people, personal information of the research participants must be kept and privacy must be observed. In multidisciplinary studies, those who evaluate the study results should not have knowledge about the groups and intra-group distributions.
Measurability: In order to prove that a study is scientific, the observed events must be measurable and expressed in numbers. 2 Lord Kelvin says exactly this on this subject: “If you cannot describe your observations with numbers, your knowledge is worthless”.
1.1. Standardizing the Experiment

Standardization in animal experiments can be expressed as the animals used do not vary, the feeding and housing conditions of the animals and the procedures performed are the same. The purpose of standardizing animal experimentation is to keep the animal used in the experiment, its environment, housing and feeding conditions, and the method applied constant and under control. In a standardized experiment, the same experiment requires obtaining similar results when the same procedures are applied in a different environment. In order to standardize the experiment, some issues need to be taken into consideration:
1.1.1. Genetic standardization

The primary factor affecting the results of animal experiments is their genetic structure. Because all functions, anatomy and physiology of the individual are controlled by the genetic code. In many experiments, genetic differences between subjects are undesirable. Therefore, the genetic structures of animals need to be standardized. The genotypes of the animals to be used in the experiment must be the same. When experimental animals of different genotypes are used, differences may occur both between groups and within groups. This is a factor that significantly negatively affects the results and reliability of the experiment. Standardization of the genetic structure is only achieved through special breeding methods.
Animals are divided into three main classes in terms of genetics:
Outbred lines with unknown genetic structure
Inbred lines with a known genetic structure. lineages

Transgene and mutant strains whose genetic structure is partially known.
Outbred strains whose genetic structure is unknown
Outbred mice and rats have been frequently used in experimental studies for years. However, in recent years, developments in the production techniques and supply of inbred strains, as well as discussions on the reliability of studies on outbred strains, have brought serious restrictions on the use of outbred strains. There are many drawbacks to using outbred strains in experimental studies:
1. They are phenotypically more variable than inbred lines, so a larger number of animals must be used for statistical interpretation.
2. Serious genetic deviations can occur even within a few generations. For this reason, the validity of the initial features may disappear.

3. Stock names such as Wistar rats or Swiss mice are now considered meaningless. Because Wistar rats obtained from different sources will be different in terms of their characteristics. Therefore, if the research is not reproducible in another laboratory, it will be uncertain whether this may be due to the difference in lineage or other reasons. Some outbred lines are genetically heterogeneous, while others may be close to inbred because their generations may have continued in the same stock for a long time.
For all these reasons, the use of outbred lines in experimental studies is not recommended today.
Inbred lines with a certain genetic structure

It is appropriate to use inbred lines for genetic standardization. Inbred lineage is a form of production by mating offspring born from a mother and father. With this method, genetic heterogeneity is reduced and homogeneity is achieved. It is generally accepted that homogeneity is achieved after the 20th lineage.
Transgenic animals
Transgenic animal is a type of animal whose genetic structure has been changed by gene transfer or gene mutation. In this way, this change in the genetically modified animal can be transferred to subsequent generations. Mice are most commonly used as transgenic animals. There are many transgenic mouse lines available. Gene transfer and gene mutation applications, fertilization mechanisms, embryo manipulations and embryo transfer techniques in mice are very advanced and widely applied. Strains obtained by these methods can be obtained from suitable laboratories for experimental studies.

1.1.2. Microbiological standardization

Laboratory animals to be used in experimental studies should be evaluated in terms of health at certain periods and experiments should be carried out on healthy animals. For this, viral, bacterial and parasitic controls determined by international standards must be applied by animal production laboratories. Many bacteria and viruses can cause latent infections in animals, as well as humans, even if they do not cause clinical symptoms. Latent infections can have significant negative effects on experimental results. For this purpose, it is recommended to use Specific Pathogen-Free (SPF) or Germ-Free (GF) animals, depending on the nature of the study. SPF animals are animals free from specific microorganisms. International microbiological control standards have been established for their housing, nutrition and equipment, and these standards must be complied with. Again, depending on the nature of the experiment, Germ-Free (GF) animals, which are completely free of microorganisms and obtained by hysterectomy and housed in isolators, can also be used. Conventionally bred animals, whose microbiological status is unknown, can be used after a certain quarantine period or after performing the necessary microbiological tests, if appropriate.

1.1.3. Environmental standardization
Laboratory animals are affected by their environment and surroundings. Each animal must be housed in a laboratory environment most suitable for the natural environment in terms of normal development and behavior. For this purpose, guidelines have been created to provide the necessary environment for the shelter, nutrition and reproduction of laboratory animals. Every country has guidelines regarding laboratory animals. And the housing and feeding of laboratory animals must be regulated according to these rules. The structuring of Laboratory Animal Units in the participating countries of the Federation of European Laboratory Animals Science Association (FELASA) and the member countries of the European Union and the stipulated conditions for the animals housed in these units are specified. In this agreement:

The maximum number of animals to be kept in each room and cage, the issues to be observed in the ventilation of the room, temperature and humidity rates, lighting features, sound and noise insulation, how to meet nutritional and water needs, cleaning criteria of cages and rooms, characteristics and training status of animal caretakers are specified in detail and it is recommended that member countries comply with them. The laboratory where the experimental research will be carried out must comply with these criteria and the housing conditions of the animals to be used in the experiment must be standardized according to these criteria.
1.1.4. Nutrition standard

Specific commercial feeds are produced for experimental animals. The content of these feeds and the way the animals are fed affect the development, metabolism and health status of the experimental animals. It affects. For this reason, feed standards specific to each animal species have been determined and it has been recommended that manufacturers produce according to these standards. Differences in the content of feeds and changes in feeding style may cause changes in the test results, which endangers the reliability of the experiment. 3,4,5,6
2. Phases of Experimental Research

In experimental research, the researcher is the qualified person responsible for the implementation of the experiment. As in other countries, researchers who will conduct experimental research in our country must be individuals who have received experimental research training and documented this training. In our country, the standards of this education and the obligation of those who will do experimental work to have received this training It was determined by the regulation on the Working Procedures and Principles of the Animal Experiments Ethics Committees of the Ministry of Environment, which came into force on 06.07.2006. According to this regulation, people who will conduct animal experiments are required to receive at least 80 hours of formal training and have a certificate. Therefore, training is a prerequisite for conducting animal experiments. 7
Experimental research takes place in four main stages:
Planning the experiment
Executing the experiment
Conducting the results evaluation

Publishing
In this article, the planning and execution of the experiment will be briefly discussed.
2.1. Planning the experiment
Planning is the most important part of the experiment. The more labor is spent in a planning process, the less labor is spent in the production process. The planning process goes through certain stages:
Determining the topic
Observation
Collecting Information
Original idea or Introducing the problem determination
Preparation of the research protocol

2.1.1. Determination of the subject: Certain points must be taken into consideration when choosing the subject for the experiment to be carried out.
Rationality: The selected research subject must be rational. On the selected topic The cause and effect relationship must be determined well.
Benefit for the country: It would be a correct choice if the subject to be chosen is aimed at finding a solution to an important problem of humanity as well as the country where the experiment is conducted, and if it is in accordance with the priorities of the country where the experiment will be carried out.
Necessity: When choosing the subject, whether the study to be done is really worth doing, what the contribution of the results may be to medicine. should be questioned.
Applicability: The decision should be made after investigating the extent to which the results obtained on animals are applicable to the human organism.

Repeatability: The experiment must be repeatable in all environments where the same conditions are met.
Continuity: Supporting and continuing the study on the selected subject with future studies is important in terms of preventing unnecessary studies and increasing the scientific weight of the study with subsequent studies.
When choosing a topic, the facilities of the institution where the research will be conducted must be taken into consideration. For cancer research or research involving animals that require special care, the research should be planned after ensuring that the physical and human infrastructure of the research center is sufficient for such a research.
2.1.2. Observation:

An event that attracts the researcher's attention and whose cause is not explained is investigated in detail. As the observations deepen, questions begin to emerge in increasing numbers but becoming more specific. Questions about the observed event and the answers to these questions begin to be investigated. The first step is to accurately detect the observed event. Observation should be completely unprejudiced and the event should be evaluated as it is.
2.1.3. Presenting the original idea or problem:
The problem should be presented in its simplest form. Sometimes the problem may need to be divided into parts and answered separately.
2.1.4. Information gathering:

It is carried out by using literature review or the opinions and thoughts of experienced people on the subject. The initial literature review on the subject to be researched is not sufficient. Normally, a period of two years elapses between the planning and publication of a research. During this period, the literature on that subject should be reviewed from time to time and the latest developments, if any, should be evaluated. However, basic information, classic books and review articles should also be reviewed. It is possible that a topic that is newly observed in the literature may have been studied years ago and become lost among classical knowledge.
2.1.5. Determination of variables:
Tries to find a cause and effect relationship. Variables are divided into two groups: independent and dependent variables.

Dependent variable is the quality that is desired to be changed at the end of the experiment or is observed to change.
Independent variables are the variables that are included in the framework of this experiment but outside the dependent variable.
Controlling variables is one of the basic principles in experimental studies. is someone. The more standard the animals, the fewer animals will need to be used for the experiment. As we mentioned before, for standardization, genetic, microbiological and environmental standards must be complied with. Otherwise, variability will increase. Animals housed in optimal environmental conditions show less variability than animals housed in poor conditions. Mice housed in a single cage are more variable than those housed in groups. When planning an experiment, keeping animals of the same breed, age and weight will also reduce variability. The treatment or method applied should be applied uniformly to all animals.

2.1.6.Establishing a hypothesis:
A hypothesis is a question that can be tested with an experiment. The experiment is conducted to test the validity of the established hypothesis. A hypothesis is an idea or suggestion that will be tried and impossible to accept without adequate testing. If it is known which variables affect the cause-effect relationship, a hypothesis can be established on one or more of them. If more than one variable is effective, it may be difficult to establish an appropriate hypothesis to find out which variable affects the observed situation. In this case, it is necessary to try to find the variables that affect each other independently. If you are an independent single If a variable is found, a hypothesis can be established. The hypothesis to be established must be tested by experiment, must be able to answer the cause and effect relationship, and must be expressed in writing.
2.1.7. Verifying the Hypothesis

To verify whether the hypothesis is correct, observations are continued, the experiment is planned and data is collected. It is necessary to comply with 3 basic rules when doing these things:
No matter how logical a hypothesis is, before its accuracy is proven truth cannot be accepted.
The purpose of verifying a hypothesis is to try to prove that the assumption is wrong, not true. If the assumption cannot be proven false despite all efforts, it can be said to be true.
Association does not always mean cause and effect relationship. The most difficult aspect of scientific research is determining whether the observed relationship is causal or not. For this reason, many inventions in medicine are later determined to be wrong.
2.1.8. Method for realizing the hypothesis:

Looking for observations that contradict the assumption: In order to determine the reality of an assumption, there should be no statistically significant observations that contradict that assumption.
Experiment: It is the most important method in realizing the hypothesis. The basic rule in conducting experiments is to divide the subjects into two groups that are similar at least in terms of the investigated aspects or qualities, apply the factor under investigation to one, and keep the other as a random change group. The experiment deemed appropriate must be suitable for testing the hypothesis. 8.9
2.1.9. Preparation of the research protocol:
Preparation of the research protocol must be done before starting the research and must be signed by all researchers. Preparing a research protocol has many benefits:
1. In carrying out the study It is required for approval from authorized institutions and organizations.

2. Since it allows the referees to evaluate before the study, issues that the researcher missed can be noticed with a critical look.
3. It serves as a document when applying to provide financial support for research.
4. It allows a detailed ethical evaluation of the experiment, especially since experimental animals will be used.
5. It is the most important evidence of the reliability of the experiment.
A research protocol should mainly include the following information:
Title of the project: It is the lifeblood of the project. It should be short, descriptive, instructive and interesting.
The research conductor identity
Identity of the research director
Identities of co-investigators

Indicating the training levels of all personnel who will deal with animals in the experiment and specifying the specificity of the experiment to be carried out before the research Explaining whether they will receive additional training is required.
Summary of the purpose of the experiment: Here, literature information on the subject is given in a few sentences. The missing issues in the literature are stated and the extent to which the missing or new aspect of the subject to be studied in this study will contribute to the subject is stated.

Material and Method: Experimental studies using laboratory animals are extremely important in terms of animal ethical rights. The issue of ethics, which has gained more importance in recent years, is an issue that should be emphasized within the scope of experimental studies. The legal requirement of the European Union and the first thing to consider in experimental studies in our country is that a person who will conduct research on vertebrate animals should know the 3R (Replacement, Reduction and Refinement) issues before conducting the experiment on live animals, and should be prepared for the experiment he/she will conduct. It is a matter of questioning and applying it in experiments.

The first step in the 3R theory should be to investigate whether the Replacement alternative is usable. To do this, first of all, computer programs developed for experimental research should be investigated, and if this is not possible, the use of tissue cultures or a lower life level organism should be considered and investigated. Explanations should be made about whether this study is possible without using experimental animals, such as tissue or organ culture or embryo study, among alternative methods. If a study is to be carried out on experimental animals, the possibility of conducting this study on animals with the lowest mental and social status, such as mice and rats, should first be questioned and, if possible, a study should be planned in these animals.

If replacement is not possible and it is necessary to use live animals The second step is to question the issue of Reduction. Minimizing the number of animals to be used in the experiment should be the main principle of every study. Choosing the appropriate statistical method at the beginning and ultimately interpreting the results well are the basic principles of good scientific work.
The most important factor underlying the progress and progress in medical and biological sciences is the contribution of animal experiments. However, it is very important to get accurate results by using the minimum number of animals that can be used in an experiment, both ethically and economically. 10,11

Statisticians generally do not know the details of the experiment to be performed. The person who will do the experiment does not know statistics. For this reason, if an appropriate statistical method is not applied after the experiment is carried out or if the number of animals in the groups is not sufficient for the statistical method, difficulties in interpreting the results of this experiment are inevitable. A study found that more animals than necessary were used in 50-60% of published experimental studies. It was also determined that inappropriate statistical methods were used in more than 60% of the publications. Before planning the experiment, the researcher should consult a statistician to obtain support regarding the formation of groups and the number of animals in the groups. In order to conduct a suitable statistical study, a sufficient number of animals should be used in each group. In today's world where animal ethical rights have become more important, if more animal use is planned than necessary, the implementation of this study may not be accepted by critics.

Using a Control Group: The purpose of using a control group is to eliminate the effect of unknown or uncontrollable variables and the possibility of coincidence during the experiment. The control group should be identical to the experimental group by all variables except the dependent variable. If the experiment is planned to investigate the effect of a surgical intervention, the control group The same anesthesia and laparotomy should be applied. In this case, the control group is defined as sham. A single group can be both control and experimental, and the pre-surgical values of each animal can be used as controls.

Model animals in the experimental and control groups should be selected to be as uniform as possible in terms of species, age, live weight, genotypic structure, etc. Thus, the possibility of deviations due to differences in model animals between the experimental and control groups is reduced. If the experiment is a repetition of previous experiments, it is necessary to use a control group again in the new experiment. Because there are many variables that we cannot control. Seasonal variables, dietary changes, changes in microflora, personnel and measurement methods are variables that need to be considered in this context, which cannot be claimed to be the same as the previous experiment.

The aim of Animal Welfare (Refinement) is to minimize pain and keep torment and distress at the lowest level. All animals must be kept in the appropriate environment and on the appropriate diet. The care of the animals should be done by trained personnel and the experimental animals should be checked by an experienced veterinary surgeon. Animals to be used in the experiment must not have clinical or subclinical disease. Keeping the animals in a gentle and non-disturbing manner ensures that the animal used in the experiment does not become stressed. Maximum care must be taken to prevent the animals used in the experiment from experiencing pain and suffering. Injections and interventions should be performed in the least painful manner, surgical procedures should be performed with appropriate anesthesia and analgesia, and appropriate postoperative care should be provided. Animals that are in serious pain or are sick should be killed by humane methods rather than waiting for them to die on their own. When the experiment is completed, the animals should be killed humanely by choosing the most appropriate painless method.

In order to prevent errors and negligence that may occur while planning the experiment, it would be appropriate to pay attention to the following points:
1. When planning the study, alternative methods should first be questioned, researched and, if possible, alternative methods should be preferred.
2. It should be stated why live animals will be used in the study and The inadequacy of alternative studies on the subject to be studied should be emphasized.
3. If live animal alternative methods are not possible, the lowest level of animal should be preferred for the study.

4. The material must be described. The purpose of the experiment Suitable species or strains must be determined. After determining the appropriate species/strain, it should be examined whether a special structure (genetic/microbiological) of the relevant species/strain is required. The animal chosen must be sensitive to the substance or method whose effect is to be investigated. It must be proven that the experimental animal to be used in the experiment is suitable for this study. The experiment should be performed on a sufficient number of subjects for the reliability of the results, but should also avoid excessive waste of animals and materials. For this reason, power analysis should be performed.
The cost of the model animal to be chosen must be economical. The general concept here is that animal expenses normally do not exceed 5% of the total project cost. Previous studies on this issue may be guiding.
5. If a painful method is to be applied, it should be explained that alternative methods are inadequate.

6. It should be noted that any discomfort, distress or pain in the animals will be limited or unavoidable in terms of the results of the scientific study. In cases where analgesics, anesthetics and tranquilizers are indicated, it should be explained that they will be used to relieve the animal's pain or discomfort, and the anesthetic, analgesic and sedative substances to be used should be defined.
7. It should be noted that the experiment is not a repetition of another experiment and is an original study.
8. It should be noted that the animals to be used in the experiment have not been used in a major experiment before. If it was used in another experiment, it must be scientifically proven that this experiment does not have a negative effect on the results of the new experiment.

9. The housing conditions of experimental animals must be disclosed. Experimental animals The characteristics of the room where it is housed, temperature and humidity, lighting and ventilation conditions, cage features and the number of groups of animals housed in the cage should be specified.
10. The nutritional status of the experimental animals, the characteristics of the feed given, whether the feed is suitable for the species, the feeding method, and water drinking status should be explained.
11. It should be stated whether any special treatment will be applied to the animals before the experiment
12. The anesthesia method to be applied should be defined,
13. The surgical method and technique to be applied should be defined.
14. The evaluation method of the animal's response to the experiment should be determined.

15.The dose and duration of analgesic to be used during and after the surgical procedure should be specified. If the analgesic substance to be used will affect the results of the procedure, this must be explained scientifically.
16. If infectious agents, compounds or other substances are to be used, these should also be identified.
17. If any physical procedure (such as radiation) is to be applied, the reason and how it will be applied should be explained.
18. If any behavioral change in the animals is expected during the experiment, it should be stated.
19. The type of post-experiment care and who will provide it should be explained.
20. It should be stated whether euthanasia or humane killing will be used at the end of the experiment. One of the appropriate euthanasia methods should be chosen for each species.

21. If the study is to be controlled and implemented, sham groups should be defined and how many animals will be in each group should be stated.
22. How many animals will be used in total
23. Project budget
24. The estimated start time of the project and the duration of the project should be stated.
25. Job descriptions of the research team should be made and their consent should be obtained.
2.2. CONDUCTING THE EXPERIMENT

As with all other studies, experimental studies and research are carried out as a team effort. The project manager is responsible for the management and harmony of the team, like an orchestra conductor. Experimental research is planned and carried out as a multidisciplinary work product. These disciplines are gathered under the main roof such as animal technician, veterinary medicine, surgery and laboratory. People who will take part in the research are determined in advance and work according to a plan. In this plan, the supply and care of experimental animals, veterinary and surgical practices, laboratory procedures, collection and evaluation of results methods should be determined in advance, and the duties of the responsible and executive persons regarding these works should also be recorded in advance. The project manager is responsible for the smooth execution of the project. A diary should be kept for research and daily transactions should be recorded. There should be sealed forms or notebooks in every laboratory, and a form should be kept for each animal and the procedures and daily data regarding that animal should be recorded on this form. In accredited laboratories, these records must be submitted during sudden inspections. If no documents can be shown and the studies are not recorded, the working permit and accreditation certificate of that laboratory may be cancelled. Each member of the research team is obliged to report any disruption related to his/her responsibility to the project manager. The project manager is responsible for reporting and informing the project manager about the progress of the research at regular intervals in line with the previously made plan. If a good plan is not made in advance and the work is not carried out on time and correctly, any disruptions in the execution of the experiment will also affect the results of the experiment.

3. SURGICAL APPLICATIONS IN EXPERIMENTAL RESEARCH
Surgical procedures are performed on experimental animals for a variety of reasons. Today, the basis of the surgical methods and surgical techniques applied in humans lies in the surgical practices performed on experimental animals. In surgical procedures to be performed on experimental animals, the same surgical principles as those applied to humans are valid. To the extent that these surgical rules are followed in experimental studies, the experiment will be reliable and the experimental results will be accurate. The person who will work on the experimental animal must have received appropriate training in asepsis and antisepsis, showing the necessary sensitivity to the tissues, ensuring hemostasis, using appropriate surgical instruments and applying the technique, and suturing techniques. Surgery should be started after obtaining the necessary information about the anatomy and physiology of the animal to be tested and the medications to be administered.

3.1. Health status of the animals and adaptation
If the animals to be used in the experiment are taken from another unit, a certain period of time must be waited for adaptation to the new unit. Detailed information about the health status, population information and identity of the animals must be available when they are accepted to the new unit.
Animals lose 10-20% of their body weight during transportation. It may take 48 hours or more for them to return to normal. Therefore, if animals are brought from another place, these animals must adapt to the new environment. During this period, the animals are observed and evaluated carefully and in detail by trained personnel in terms of behavior, feed and water consumption, social adaptation and disease findings.

Before surgery, animals should be re-evaluated in terms of health. The healthy animal is active, behaves normally, He continues to feed and is well hydrated. The results of studies conducted on sick animals will not be reliable.
3.2. Temperature of the operating room
Animals lose heat during surgery. The smaller the animal, the faster it loses heat. For small rodents, this can reach 10 degrees in the first 10 minutes, which is dangerous. The operating room or the environment where the animal is located should be heated with heaters such as radiant heaters, warming blankets, and the temperature of the animal should be monitored with a monitor during surgery. Hypothermia may rapidly develop in the anesthetized animal, which may cause mortality and morbidity after the procedure.
3.3. Sound and noise

Noise is the most important disturbing stress factor in laboratories. Noise affects the physiological state of animals. Research laboratories should be quiet at all times.
3.4. Pre-operative preparation
In large animals, feeding is stopped in the evening the day before. He may be allowed to drink water. Water drinking is also stopped 2-3 hours before the surgery.
There is no need to stop feeding and drinking water before the surgery in rodents and small animals. It is not right to starve small rodents such as rats and mice for more than 2-3 hours, especially due to their fast metabolism.
3.5. Surgical procedure time:

Surgery performed late in the day may not provide sufficient time for appropriate postoperative observation. Similarly, surgeries performed on weekends also cause less attention. This depends somewhat on the type of surgery performed. In long-lasting and major surgical operations should pay attention to this situation. In terms of standardization of the experiment, the same procedures should be performed on all animals at the same time of day. Daily hormonal variations in animals, as in humans, may make the surgical stress response different.
3.6. Asepsis and antisepsis

In surgical procedures performed on animals, it is necessary to comply with the rules of asepsis and antisepsis applied to humans. There is always the possibility of microbiological contamination in surgical procedures. Here, in addition to the process, the tools used, the person and the environment are also effective factors. For these reasons, people who will perform surgery on animals must know the general asepsis and antisepsis rules and apply these rules during surgical procedures on animals. Antiseptic surgery is especially necessary in large animals. After appropriate anesthesia is applied to the animal, the hair in the surgery area is shaved. The incision area and the adjacent area must be clean of hair. Hairs, In addition to bacterial contamination, it is a factor that irritates the wound and delays wound healing. The person who will perform the procedure should wash his hands in accordance with the surgical procedure, wear sterile gloves, clean the area where he will operate with antiseptic solution and begin the surgical procedure. Although there is a general belief that small rodents such as mice and rats are more resistant to infections, aseptic surgery should also be applied in surgical procedures on these animals. Standards must also be observed regarding the cleanliness and ventilation of the operating room. The instruments to be used in surgery must be sterile and separate sterile materials must be used for each animal. Antibiotic prophylaxis is usually not required. However, antibiotics may be administered for prophylaxis or treatment in long-lasting surgical procedures or in special cases.

3.7. Anesthesia

The purpose of anesthesia is to keep the animal motionless during the procedure, to use appropriate muscle relaxants when necessary, and to prevent the animal from feeling pain during and after the procedure. Appropriate anesthetic should be administered before surgery and the anesthesia status should be checked throughout the procedure. The appropriate one among intravenous and inhalation anesthesia methods should be preferred, and if respiratory depression is required, the need for a ventilator should be provided. Suitable endotracheal tubes and suitable anesthesia devices are also available for small rodents such as mice and rats. Anesthesia technique varies depending on the type of animal to be used in the experiment, the type and duration of the surgical procedure to be performed, and the experience of the researcher. During anesthesia, the vital functions of the animals (heart rate, respiration, body temperature, etc.) should be monitored and necessary interventions should be made in case of changes. For both ethical and scientific reasons, the pain and discomfort of animals should be eliminated or minimized with appropriate anesthesia and analgesia during painful procedures.

3.8. Maintenance during the experiment

If the animal's eyes are open, the cornea should be prevented from drying out by applying a suitable eye ointment or by covering the eyes with tape. It is sufficient for long surgeries and the postoperative period Hydration and nutrition should be provided. During surgery, animals lose significant fluid through evaporation, blood loss and respiratory tract. If the surgical procedure will take a long time, Ringer Lactate or saline solution should be given intravenously at a rate of 10-15ml/kg/hour. In small rodents, if vascular access is not established, this amount can be safely administered intraperitoneally or subcutaneously. However, in cases of excessive bleeding, since these amounts need to be given quickly and in sufficient amounts, it would be appropriate to open a suitable vascular access and perform infusion from there. Care should be taken to ensure that the liquids given are not cold and are warmed to body temperature. Especially in laparotomy surgeries, care should be taken not to dry the tissues during the surgery. Therefore, warm serum should be dripped from time to time. Small rodents remove suture material with their teeth. For this reason, when closing the skin, if the incision is within reach of the animal's teeth, either subcutaneous suturing or metal clips should be used.

3.9. Waking up from anesthesia and post-surgical care

Monitoring practices during surgery should continue during the awakening period. During waking up, animals should be kept in a dry and warm place where they can breathe comfortably. Hypothermia should be avoided here too. The room temperature should be higher than the normal maintenance room temperature. Respiratory depression can often develop after surgery and can be a cause of death if not noticed. Therefore, in cases of respiratory monitoring and depression, oxygen support or medical intervention may be required. In addition to preventing pain in the post-surgical period, it is also important to prevent stress. The recovery room should be quiet and dimly lit. Care should be taken to ensure that the animal's bottom is dry. A cold and noisy environment on a wet floor can be a cause of stress in the postoperative period. After the procedure, the animals must be kept in individual cages and have a comfortable awakening period. After surgery, animals may not take oral fluids for a certain period of time, which can sometimes be 12-24 hours. Loss of 15-20% of the animals' circulating blood volume can cause hypovolemic shock. Water consumption of animals should be monitored and if they do not drink enough fluid, this need should be met by gavage or subcutaneous, intraperitoneal route. In many species, the 24-hour maintenance fluid requirement is 40ml/kg.

3.10. Pain
Pain is an event that results from stimuli that damage the tissue or have the possibility of damaging the tissue and is perceived differently by each individual. We can predict what causes pain in animals based on the situations that cause pain in humans. For example, pneumonia and osteomyelitis are very painful in humans. It is certain that it will cause pain in animals in the same way. Likewise, any type of surgical intervention will cause pain in animals as well as in humans. Animals are also sensitive to all kinds of painful interventions. In this case, restlessness, abnormal They show abnormal posture, abnormal behavior patterns, abnormal voice and unusual aggression. In the simplest way, the researcher should ask himself the question "Would I feel pain during such an intervention?" and if the answer is yes, he should give the animal an appropriate analgesic substance.

The fact that animals are more resistant to pain than humans does not mean that they do not need analgesics. If the procedure to be performed on a laboratory animal is a painful procedure, the animal's pain must be relieved.
Stimulus can cause pain in different ways, and as a result of this pain, animals may react in different ways: Pain It can also cause behavioral findings such as abnormal appearance, posture, and immobility, as well as species-specific vocalizations, depression, and other behavioral changes. The animal's response to a painful stimulus may take different forms:
Unobservable pain: The situation in which there is no change in the animal's behavior
Mild pain: Pain that changes but does not disrupt normal behavior. In this case, the animal A slowdown in movements may occur.

Serious pain: Pain that significantly alters normal behavior. In this case, inactivity such as shrinking into a corner and not paying attention to the environment may be observed.
Chronic pain or stress: This situation is even more difficult to evaluate. Lack of optimal environmental conditions or chronic disease causes adaptation problems and this is a cause of stress. Stress negatively affects the animal's physiology, and the results of studies conducted on stressed animals are not safe. Conditions such as apathy and unresponsiveness to stimuli are symptoms of chronic distress. Animals also transmit pain and stress to other animals. Therefore, such animals should be kept in a separate environment. The personnel responsible for animal care or the researcher should evaluate the animals in this respect during their daily observations. The analgesia method to be applied should be specified in detail in the research protocol. The appropriate use of anesthetics and analgesics in research animals is a scientific and ethical obligation.

3.11. Euthanasia
Euthanasia is the conscious termination of life in a painless and stress-free manner. At the end of the procedure, if the animals' lives are to be terminated, a painless and stress-free euthanasia should be applied and an environment should be created to prevent other animals from seeing the euthanasia. If the animal is in pain, has an incurable disease, and is not required to live for the purpose of the experiment, it is recommended that the animal be terminated.
The following clinical/pathological changes in the animal under investigation require euthanasia indication. Observation of signs of dehydration
He shows difficulty in breathing and cannot be corrected
He begins to harm himself
Permanent and significant decrease in body temperature (<34 Co)
Increase in tumor size (>10% of body weight or ~2 cm3 for mouse, >10% of body weight or ~40 cm3 for rat), necrosis and bleeding
Two main types of euthanasia can be applied: chemical and physical. In chemical methods, methods such as high dose anesthetic agent administration, CO2 application, and in physical methods, methods such as cervical dislocation and decapitation can be applied.
The following points should be taken into consideration when performing euthanasia:
It should be carried out in a separate environment from other animals.
Death should be carried out quickly, without pain or suffering.
Consciousness must be lost as quickly as possible.
The chosen method must be appropriate, applicable and irreversible.
The method must cause minimal psychological stress for both the animal and the practitioner.
Dead animals, residues and biological materials generated at the end of the work should be disposed of in a way that does not harm human and environmental health. In this regard, the principles in the Medical Waste Control Regulation must be followed.
4. FACTORS AFFECTING THE ACCURACY OF THE RESEARCH:
Not receiving the necessary training
Starting research with bias
Ethical rules failure to comply
Failure to study a sufficient number of subjects
Not using a control group
Not applying the principle of not making aware
Failure to find a measure appropriate to the characteristics of the subject to be examined
Failure to properly select independent variables that may affect the dependent variable to be examined
Using inappropriate statistical techniques
Making a decision by comparing incomparable situations
Failure to interpret the results correctly
Failure to collect data in accordance with the data collection principle and the characteristics that the data should have
All these issues mentioned in this article should not be considered as a compelling and hindering factor for scientific research. When the production of scientific knowledge, which requires a very difficult and long-term effort, is carried out in parallel with scientific and ethical values, science workers will be deemed to have fulfilled both their social and academic responsibilities. Second Ed.Blackwell Science Ltd., 1998, pages 10-34
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