Materials / Reagents Required :

            10 % Neutral Buffered Formalin (NBF), Davidson’s fluid (1 litre), Modified Davidson’s fluid (1 litre), Normal saline (10 litre), 70 % Ethanol (1 litre), different sizes of scissors, forceps, scalpel, spatula, bone cutter, cardboard paper, etc.

Safety Precautions :

            Protective equipments including laboratory coats, gloves, apron, etc., should be worn during euthanasia, necropsy as well as during the preparation of the solutions.

Euthanasia :

• The identification of the animals received at the necropsy room shall be verified by the ear tag. The ear tag along with some portion of the ear shall be preserved along with the other organs of the same animal.
• The animal identification number, study number and other details shall be recorded. The animal shall be transferred from the cage to the cotton bed of the euthanasia chamber.
• The internal pressure of the CO2 cylinder shall be checked by its pressure gauge. CO2 gas shall be released into the euthanasia chamber by opening the delivery valve of the CO2 and the regulator shall be adjusted so as to arrive at the required pressure of 1.5 kg/sq.cm.
• For sedation, the animals shall be exposed for 1 to 1½ minutes. Exsanguination shall be accomplished by severing the femoral arteries, abdominal aorta or by incising the skin at the neck region.
• Organ weights shall be recorded, if needed. Normal saline shall be kept ready during the necropsy to keep the organs moist. The fur of the animal shall be moistened with alcohol to prevent the fur from getting adhered into the tissue samples.
• Skin along with mammary gland shall be collected first. Two rows of teats shall be located lateral to the mid-line from the cervical to the inguinal region. This piece of tissue shall be then placed on a piece of light cardboard paper (fur side up) and into fixative.
• The eyes are then removed by grasping the adnexa with forceps in cutting into the socket of the globe and placed in a tissue cassette for further fixation. The brain is then exteriorized by removing the calvarium. Shallow cuts shall be made with the bone cutting forceps to circumscribe the calvarium so as to prevent damage to the brain tissue. The brain is removed using a small blunt spatula.
• The pituitary gland is removed along with its underlying skull bones and is fixed as such. The gland is removed from the bones after fixation. The abdominal and thoracic cavities are opened with a ventral mid-line incision upto the jaw. The skin and musculature are reflected to expose the xyphoid cartilage.
• The thoracic cavity is opened by a cut through the diaphragm and the ribs about half inch lateral to the mid-line. The tissues of the abdominal and thoracic cavities and the ventral cervical region are observed for their gross appearance. The adrenal gland and mesentric lymph nodes are first collected in a tissue cassette as they may be easily lost.
• The gastro-intestinal (GI) tract as a single structure, liver, pancreas and spleen are removed. The kidneys are removed. The thoracic viscera including trachea, thyroid gland, para-thyroid gland, esophagus, heart, thymus and lungs are removed as a single structure.
• The thymus will be separated, placed over the cardboard and then fixed. The heart will be separated and the excess blood in the heart will be removed by gentle squeezing. The testes and epididymis are exposed by applying gentle pressure over the scrotal sac, separated and dissected out carefully.
• The seminal vesicles with coagulating gland, prostate gland and urinary bladder will be dissected out, placed over a cardboard paper and fixed. If the urinary bladder is not distended, it will be infused with 10 % NBF.
• The female reproduce system including the ovaries, uterus and vagina will be dissected out carefully and fixed. The ovaries alone shall be kept in tissue cassettes as they may be easily lost and then, are fixed.
• The skeletal muscle shall be collected along with the sciatic nerve by excising and flipping over the thigh muscles. The sciatic nerve will be placed on the cardboard paper, stapled and transferred to the fixative containing jar.
• The femur with the joint and tibia intact will be removed from the right hind limb. The left hind limb will be preserved as a whole.
• The necropsy tools and their accessories will be rinsed with water thoroughly and then disinfected using the approved disinfectant solution. Then, they will be cleaned with dry cloth and allowed for air drying.
• After the completion of the necropsy, the floor of the necropsy room and the necropsy table will be cleaned with water and then with soap solution. Finally, they will be mopped with the approved disinfectant solution.