Compiler: Helga Schulze; with contributions by (in alphabetical order): Eberhard Curio; Colin Groves; Anna Nekaris; Kathrin Petry; Heinz-Adolf Schoon; Roland Plesker; Christian Roos; Ulrike Streicher. The initial draft was based on some publications, for instance Wobeser and Spraker 1980: Post-mortem examination, Munson 2000: Necropsy procedures for wild animals, and others (see references).

This review is partly based on general mammal necropsy guidelines which probably rather consider procedures for larger species. So some of the described procedures may not be adequate for small species like slender lorises, but as far as possible, methods are adapted. If not only a veterinarian examination for diseases and causes of death is planned, but also preservation of specimens for anatomical or taxonomic collections, a choice between identification of health problems and other aims will be necessary because veterinarian examination is usually connected with destruction of structures. This page may be regarded as a list of propositions to chose from for certain purposes.
 

Table of content (including links to other files)

Initial considerations
     Self-protection before and during examination
     Examination of autolytic and decomposed carcasses
     Examination of dead neonates, of embryos found in a female
Some considerations concerning collection and preservation of samples
     Sampling recommandations in case of suspicion of certain diseases (in preparation)
     Review of different kinds of samples, sampling plan (in preparation)
First records, sample collection and measures in the place where an animal has been found
     First general records concerning the specimen (propositions)
     Locality- and environment-related information
     Carcass itself: externally visible features
     Photographic record
     Field report of wildlife death in case of evidence of a possible epidemic
      Records of preservation methods
Necropsy
     External examination of the carcass
     Planning what to preserve, prior to dissection
     Necropsy procedure in the order of appearance / removal of organs
     Review: removal, examination of different organ systems (in preparation)
 
 

Introduction: information which can be obtained from carcasses of animals and other material found in the wild may be very useful for obtaining data for conservation. Therefore, some considerations before starting a necropsy will be useful. In addition, the form of necropsy reports and their possible role as official documents may be important in law suits and should be considered by an appropriate form of records and resulting documents. Therefore preparation of a form sheet prior to necropsy is recommended. See also: information about recording of data, labelling, preservation and storage of carcasses and samples.

Necropsy should take place as soon as possible. If no quick examination is possible, smaller carcasses, after noting all necessary data, should be kept cool, but nor frozen (AZA Prosimian Advisory Group, 2002), if possible. See also below: dealing with autolytic or decomposed carcasses.
A good necropsy procedure is supposed to allow identification and examination of each organ, including opening of all hollow organs. The less experience a prosector has, the more rigidly he / she should adhere to a standard technique to assure that all organs are examined thoroughly. Becoming familiar with the normal appearance of organs will then help to easily recognize abnormalities.
For evaluation of the findings, a cooperation with biologists, specialized pathologists and diagnostic laboratories or experts from Universities or other institutions may be necessary (Wobeser, Spraker 1980).

Equipment: see extra file (in preparation)

First records, sample collection and measures in the place where an animal has been found (in preparation)

Self-protection before and during examination:
Protective clothing:  (in preparation)
Formalin is highly toxic; it should be handles with great care, and hands should be washed thoroughly when work is finished (Rabinowitz et al., 2000).
Procedures in cases of dangerous infectious diseases (in preparation)
   Protective clothing and method for examination of brains in cases of rabies: see below

External examination of the animal (based on Munson 2002; one of us: K. Petry; one of us: H.-A. Schoon; one of us: R. Plesker)
External parasites? (Preservation of some)
Posture, description / photos
Rigor mortis, other signs of death, externally visible signs of decomposition?
Blood visible? If so: clotted, dried?
Wounds? If so: signs of bruising or bleeding into the tissue, indicating that the wounds occurred when the animal was still alive? (Otherwise they might be signs of scavenging). Cause of wounds?
Externally visible: parts of the body missing, changed? Broken bones, missing hair, broken teeth, other signs of trauma?
Hair, skin, nails: changes, missing?
Malformations?
Weight, head and body measurements
Nutritional state: externally visible fat stores, muscle mass? Dehydration?
Sex, external signs of reproductive status:
Males: testes scrotal or inguinal, testes size, scrotal skin patterns, other observation (secretion)
Females: Conditon of the vagina: open, closed, sealed by a skin? A sealed vaginal cleft may occur in juvenile females and in seasonal breeders at certain times of the year, with the vaginal opening closed by a membrane which may look like normal skin with vaginal opening absent (Nagorsen, Peterson, 1980; observation at Ruhr-University). Signs of estrus such as swollen and reddened rims of vaginal opening?. Signs of pregnancy? Mammary glands: signs of lactation? (Can milk be squeezed from the teats?) Nipples looking used? (Nagorsen, Peterson 1980)
Pregnancy? In Loris, pregnancy is usually inconspicuous, even highly pregnant females may show a dilated ribcage rather than an enlarged belly (captive observation at Ruhr-University).
Offspring: babies / infants clinging to the adult found, babies / infants found parked in the proximity?
Condition of the teeth
External examination of eyes, ears before opening the carcass. Blood, pus, parasites in the ears? Blood in the ear and haemorrhage in the skin around the eyes may be signs of a skull base fracture.

Hair samples may be taken for DNA analysis or for a taxonomical reference collection

Samples for microbial examination from externally visible abnormal areas (for instance pus, sample from areas with abscesses, samples from the edge of affected (abnormal) areas most likely to contain organisms) should be taken with sterile instruments and  stored in sterile container (Munson 2000)

Planning what to preserve, prior to dissection
After external examination, a decision is necessary whether preservation of the entire specimen, preservation of parts for non-veterinarian purposes or dissection is most important. This decision must be dependant on importance of destruction of parts for examination (for instance if diagnosis is important because of danger of epidemics or zoonoses) or importance of preservation of parts of the specimen (skull, skin, other) or the whole specimen, for instance for taxonomic purposes.

Dissection
Primates (all species) and small mammals are dissected while lying on the back; they may be fixed in this position. Special devices for laboratory rodents, a soft board, sheet of cardboard or styrofoam covered with plastic foil and four stout pins or hypodermic needles; (Wobeser and Spraker, 1980; Schoon, lecture manuscript).

1) Skin
Preservation of the skin necessary for mounting or preparation of a study skin planned? If so, the inner skin surface with hair roots should not be damaged when removing the skin from the body; otherwise the mounted specimen may later loose hairs. In carcasses beginning to decompose, the hair may already be loose (“slipping” fur), it will then fall out leaving a naked skin unless removal of the skin is done very cautiously. Therefore, tests (trying to plug a few hairs in several parts of the body including the abdomen) prior to skin removal should be done in taxonomically valuable specimens.
Removal of skin for study skins or mounted specimens (in preparation), preparation of a study skin (in preparation), mounting a skin (in preparation).
Removal of skin prior to necropsy may also be useful for other reasons. If necropsy is not done immediately, removal of the skin with insulating fur before cooling or freezing may help to cool the carcass down more quickly. In cases with superficial tumours or lesions supposed to be examined, it may be necessary to leave the parts of skin concerned attached to the body (Schoon, lecture manuscript).
For dissection, the ventral side is opened with an initial skin incision with sterile instruments, usually from the lips along the ventral midline to the perineum, passing the penis and scrotum or clitoris on one side. If the skin is supposed to be preserved as a study skin or mounted specimen, it will be useful not to cut trough too much hair (moistening of the hair may help to prevent it from moving between the scissors); it may be useful to avoid cuts in thinly haired parts of the skin, where after mounting ugly seams might be visible, rather leaving parts of the limbs in the skin. (Vacuum freeze-drying with limbs in the skin may be an adequate method for mounting thinly-haired specimens like slender lorises).
An examination of the inner surface of the removed skin may show wounds hidden under the hair or effusions of blood. In addition, muscles, fat and mammary glands can be examined on this occasion. Mammary tissue is usually whitish in colour, beneath the teats under the skin; presence of milk should be noted (Nagorsen, Peterson 1980). The glands and draining superficial lymph nodes can be incised for examination of the parenchyma (Wobeser, Spraker, 1980).

2) Opening of the body cavities: general
All body cavities (abdomern, chest, heart) must be opened. Technique see below.
Sterile samples of organs for microbial examination should be taken for culture with sterile instruments and stored in sterile containers before organs are handled (Munson, 2000). Such samples will be needed for instance from all abnormal-looking areas, for instance from pus, from areas with abscesses or nodules, from the edge of affected (abnormal) areas most likely to contain organisms.
Small amounts of blood can be collected on filter paper and used in testing for some viral, bacterial and blood parasite DNA or RNA (blood sampling methods: in preparation). Antibodies against some pathogens may also be detected using these samples.

Initial notes. In the opened body cavities, before any organs are removed, the following should be noted:
General condition
Smell
General nutritional condition of the animal, fat deposits
Signs of dehydration?
Condition of the serous membranes (membranes covering the inner surfaces of body cavities), pleural membranes? (Wobeser, Spraker 1980; Schoon, lecture manuscript)
Location of all organs: any organs displaced? Abnormal attachment of any organs to the body cavity? (Munson, 2000).
Abnormal fluids in the pleural, pericardial, abdominal cavity? If so: quantity, colour, smell? Collection of liquid samples. (Examination later in the lab: ascites, tumor cells? What do the granulocytes (white blood cells) look like: juvenile or with polymorphic nuclei? One of us: K. Petry)
See also below: examination of the body cavity after removal of organs
Sections of all lesions and unnormal-looking parts should be collected during following dissection (Munson, 2000).

Opening of the abdomen
The abdominal wall is opened by cutting along the ventral midline from pelvis to sternum. After two additional cuts along the lower rim of ribcage the resulting flaps can be deflected (Wobeser, Spraker 1980; Schoon, lecture manuscript).
Opening of the thorax
If no skeletal preservation for a collection is necessary, the thorax may be opened by cutting through the ribs along both sides (in small mammals with bone scissors, for larger species a saw would be necessary) and removal of the sternum with attached parts of ribcage (Wobeser, Spraker 1980; one of us: R. Plesker; for large mammals, the procedure would be different, see Wobeser, Spraker, 1980 or Munson 2000). Alternative: a cut along the region where the ends of rib bones are connected to cartilage, where cutting is easier and the bony parts of skeleton are less damaged (one of us: K. Petry).
Use of the removed ribs for examination of the stability (ossification) of bones and bone marrow examination: see below, under "examination of muscles and skeleton".
Opening and examination of the oral cavity and throat
Cuts along the inner margins of the mandible are necessary to free the tongue. Then the larynx, hyoid bone and other structures can be freed by cautious traction in cranial direction and cutting down to the soft gums (Palatum molle); the throat organs are freed caudally up to the cranial thoracic opening. Muscles and ligaments at the mandibular joint should be cut through; disarticulation of the mandible and cautious cutting free of the cheek musles and mucosa from the lateral surfaces of the mandibular bone then allows a complete examination of the oral mucosa (Wobeser, Spraker 1980; Schoon, lecture manuscript).
The inside of the mouth, tonsils, and teeth should be examined. Erosions, ulcerations or other lesions on the oral / pharyngeal mucosa, tonsils or any other areas in the mouth? The salivary gland should be examined; the tonsils and several lymph nodes under the skin (at the angle of the jaw and above the larynx) should be removed for histology (Munson, 2000)

Superficial examination of the thoracic viscera
A sample of lung tissue for bacteriology should be taken (Schoon, lecture manuscript)
Examination of tongue, tonsils and the pharyngeal area (Wobeser, Spraker 1980).
External surfaces of the lungs: any lobular differences in the lungs such as different colour, firmness or inflation should be noted (Wobeser, Spraker 1980). Edema, emphysema? (Munson 2000).
Presence and size of thymus? (Wobeser, Spraker 1980)

Checklist: recommendations which fixed tissue samples for histology + other samples should be collected / preserved: in preparation
In carcasses of rare species, preservation of organs in situ for anatomical studies may be useful, with only small samples taken out for examination, causing as little disturbance as possible. If a necropsy is only done for detection of health problems, enough tissue for examinations including bacteriology should be taken (one of us: K. Petry), samples including abnormal areas and surrounding normal areas; Munson (2002) recommends samples no thicker than 1 cm (for good fixation), but long and wide enough to represent different areas of a tissue and possible abnormalities. In small animals, entire organs instead of samples may be collected.

Heart
Before removal of organs, the heart can be opened with sterile tools to take a blood sample for culture (the right atrium is the best location), then additional blood can be taken to obtain serum for serological tests.
Is fluid found in the pericard after opening it? The heart and great vessels should be examined while still attached to the lungs (for easy reorientation in case of some anomalous condition of the vessels). The epicardial surface, size and contour of the heart should be inspected (Wobeser, Spraker 1980). Schoon (lecture manuscript) recommends to take the following measurements: circumference of the heart in the atrioventricular groove (Sulcus coronarius), height of left and right ventricle.
Opening of the heart: usually by following the normal path of blood flow, beginning in the right atrium. A u-shaped incision is made through the right atrio-ventricular valve, following the inter-ventricular septum to the apex, then back to the base of the heart through pulmonary valve. The ventricular wall flap, cut loose this way, can be lifted to expose the ventricle and valves. The left side of the heart can be opened in a similar way, the incision beginning in the left atrium, passing through the atrio-ventricular valve to the apex and back through the aortic valve into the aorta (Wobeser, Spraker 1980; one of us: K. Petry, Schoon, lecture manuscript).
In recently dead animals, the right heart usually contains plasma clots (yellow / tan material), unclotted blood, or clotted blood (Munson, 2000). Postmortal blood clots inside the heart should look smooth; rough-looking blood may indicate a thrombosis (one of us: R. Plesker). See collection of blood or plasma. A yellowish inner surface of the aorta is a sign of jaundice (one of us:: R. Plesker).
Removal of the heart: see below:

Examination, removal of organs. Systematic collection of samples

Removal, examination of the heart
If no changes in the blood vessels have been noticed during opening and first examination, the major bloodvessels can then be cut for removal of the heart, close to the lungs, the aorta and posterior vena cava close to the diaphragm, remaining connected with the heart. Examination of the heart should include thickness of walls (right, left ventricle, septum), the inner surface (papillary muscles) the entire septum muscle (cut), and collection of samples: several sections of ventricle muscle, particularly close to the papillary muscles and rhythmic center (Schoon, lecture manuscript)

Removal of the cranial parts of digestive and respiratory tract and other organs from throat and thorax
The ends of the esophagus should be closed with two adjacent ligatures (= threads tied around it) and then cut between adjacent ligatures to prevent leakage of contents into the body cavities (one of us: K. Petry; Schoon, lecture manuscript; Wobeser, Spraker 1980). It can then be transected at the diaphragma; then tongue, larynx, thyroids, parathyroids, trachea, esophagus, lungs and thymus can be removed (Wobeser, Spraker 1980);
Examination of these parts after removal
The tongue should be examined, and a cross section near tip including both mucosal surfaces should be collected (Munson, 2000). If skeletal preparation is planned, preservation of the bony parts of larynx (hyoid bone) must be considered (Piechocki 1986). For examination, larynx and trachea should be opened by a dorsal longitudinal incision with scissors continueing to the tips of the diaphragmatic portion along the major bronchi and into some smaller bronchi (Wobeser, Spraker 1980; Schoon, lecture manuscript). Exsudate in the airways? (Wobeser, Spraker 1980). Parasites such as trematodes? (Nagorsen, Peterson 1980). Transverse sections from several lobes, including a major bronchus and trachea, should be preserved (Munson, 2000). The bronchial lymph nodes should be examined after an incision (Wobeser, Spraker 1980).
Changes in the lungs such as emphysema are not necessarily a sign of disease, they can also be a consequence of death or euthanasia (one of us: R. Plesker).

The following glands and lymph nodes should be examined:
Thyroid / parathyroids (Munson, 2000)
Lymph nodes (cervical, mediastinal, bronchial, mesenteric and lumbar), cut transversely (Munson, 2000).
Thymus (present only in young animals) (Munson, 2000; one of us: K. Petry)

The diaphragm contains muscle cells, a sample may be collected for muscle examination, see under "muscles" (in preparation)

Liver and gall bladder
The liver may be removed from the body cavity together with the first part of duodenum, cutting the ligament and blood vessel connections to the diaphragm without damaging the latter. Examination whether the bile duct is open is possible by slightly pressing on the gall bladder after opening the duodenum (Schoon, lecture manuscript). This is particularly important if the liver looks yellowish (one of us: R. Plesker). Tha gall bladder should be examined for stones (one of us: R. Plesker). If examination for a Salmonella infection is necessary, the gall bladder should not be opened during dissection (Schoon, lecture manuscript). Trematode parasites may occur in the liver (Nagorsen, Peterson 1980). After lasting environmental distress in captivity, in lorises death due to fatty liver and liver necroses has regularly occurred (data from institutions cooperating with Ruhr-University).
Sections from 3 different areas of the liver including gall bladder should be collected (Munson, 2000); storage of some liver tissue samples for DNA analysis (few g, in plastic bags, frozen) for genetic evaluation are recommended (AZA Prosimian Taxon Advisory Group, 2002).

Spleen: a cross sections including the capsule should be preserved and examined (Schoon, lecture manuscript; Munson, 2000).
An enlarged spleen may indicate a longerlasting infection, duration maybe a week or longer (one of us: R. Plesker)

Gastrointestinal tract
Before removal of the intestine from the abdominal cavity, liver and gall bladder with bile duct leading into the intestine should be examined, and closing of its sections with two adjacent ligatures (= threads tied around it) on either side is recommended, then it can be cut between the ligatures. Places to be closed this way: both ends of esophagus (caudally close to the kardia = entrance of stomach), both ends of the stomach (one of us: K. Petry), duodenum cranial from the ligamentum duodenocolicum = connection between duodenum and colon and anal end = rectum (one of us: K. Petry; Wobeser, Spraker 1980; Schoon, lecture manuscript). Before removal of the intestine, liver and gall bladder with bile duct leading into the intestine should be examined. For removal, the esophagus can be transected at the diaphragma, and tongue and esophagus, lungs and thymus can be removed together with other organs in the head and throat region such as respiratory tract and glands (Wobeser, Spraker 1980). The intestine can be separated from the mesenthery, taken out and spread for examination (Schoon, lecture manuscript), or liver, spleen and digestive system can be taken out as a block. Stomach and intestine remain closed at both ends and are opened last (Wobeser, Spraker 1980).
If a carcass is found in the wild, collection of the content of the entire alimentary tract for food analysis may be useful. Examination of the stomach content alone may not be sufficient for this purpose; in galagos and pottos, after gum-eating it seems that gums are retained in the stomach only for few minutes, so usually no trace of gum is found there, but gum may be found in the caecum (Hladik 1979, partly quoting Charles-Dominique 1971 and 1974). Contents of the digestive tract can be preserved in 5% formalin or 30-40% alcohol (Rabinowitz et al., 2000), or the whole alimentary tract may be preserved in formalin, after injection of formalin into the stomach, for later analysis (one of us: A. Nekaris).

Pancreas: before separating the pancreas from the intestine, the duct leading from the pancres into the duodenum may be examined. Munson (2000) recommends to preserve cross sections from two areas of the pancreas together with the duodenum. This may be a method for larger species. Preservation the of entire pancreas may be more adequate. In this case, curling or shrinking of the soft pancreas tissue can be prevented by spreading the whole organ on tissue paper after removal, cautiously folding the paper around it and depositing the whole package in fixative; the paper will keep the tissue spread out in normal shape during fixation (one of us: R. Plesker).

Examination of intestine:
The examiner must descide whether the entire content of the intestinal tract should be preserved for food analysis or whether other examinations may be more important. Before preserving the intestine or longitudinally opening it for examination, sections from different areas may be closed with thread and removed for later laboratory examination (Schoon, lecture manuscript).

Preservation of samples from the intestine should include multiple sections from different areas, about 3 cm² (Munson, 2000) of the following parts:

• Duodenum (part of intestine following the stomach): White and Edwards recommend to take two sections of the duodenum together with pancreas (in larger animals?); in smaller species like lorises or pottos, preservation of the entire pancreas may make more sense; see below, under pancreas.
• Jejunum
• Ileum (section close to caecum)
• Caecum
• A piece of rectum with content for test for parasites (one of us: R. Plesker) and pancreatic insufficiency (one of us: K. Petry)
• Omentum (Munson, 2000)

Intestinal lymph nodes from the middle of the mesentherium (one of us: R. Plesker)

Urogenital system
After removal of the gastrointestinal tract and liver, the kidneys, adrenals, reproductive organs and rectum / anus can be taken out together, starting with a cut around the kidneys, from cranial via lateral to caudal; the whole system should be cautiously cut loose together with lymph nodes and without damaging the urinary passages. For the caudal part, the ventral part of the pelvis may be removed with bone scissors if no skeletal preparation is planned, starting from the foramina obturatoria. The whole connected system can then be taken out and spread on a flat surface for examination (Schoon, lecture manuscript).
Enlarged adrenals indicate longer-lasting stress prior to death (one of us: R. Plesker). In primates the adrenals in general are larger than in most other mammals; veterinarians not familiar with this may erroneously believe that normal adrenals are enlarged (one of us: R. Plesker). The entire gland should be collected with transverse incision (Munson, 2000).
Kidneys - A longer piece of urinary tract left attached to the left kidney during removal can help to distinguish it from the right one after separating both from the rest (Schoon, lecture manuscript). Samples: cortex and medulla from each kidney. Munson, 2000, recommends two transverse cuts (for large species?); one of us, R. Plesker, cuts the whole kidney logitudinally for examination.
Urinary passages free? (Can be tested with a probe). Urine sample: gravel or crystals present? (One of us: K. Petry)
Urinary bladder, ureters, urethra - cross section of bladder and 2 cm sections of ureter and urethra should be collected (Munson, 2000).
Reproductive organs:
Reproductive tract, male: location of testes (scrotal, inguinal?). Measurements of length and width of both testes for assessment of reproductive status (Nagorsen, Peterson, 1980). Samples from both testes (transversely cut) with epididymis (Munson, 2000); the size of the tubules in the cauda epididymis and presence of sperm can provide information about the reproductive status. Tubules in breeding males may be swollen and visible to the eye, if so, this should be noted (Nagorsen, Peterson, 1980). Entire prostate, transversely cut (Munson, 2000). For taxonomic examination, in males preservation of the external genitalia for examination of penis shape, length and degree of spininess (Bearder et al., 1996) may be useful. The shape of the baculum (os penis) might also be meaningful (Source?)
Reproductive tract, female: sample: entire uterus and ovaries with longitudinal cuts into lumens of uterine horns  (Munson, 2000).

Embryo size measurement (crown-rump length),  taken in natural bent posture (redrawn, changed,  from Nagorsen, Peterson 1980)

.....

Presence, size of embryos? (size: crown-rump length, measured like sitting height, but in natural bent posture).  Possibly resorbed embryos would be conspicuously smaller and underdeveloped when compared with normal ones; if so, the condition should be noted. Embryos may be preserved in formalin or Bouin´s solution. In some mammals (for instance in shrews, rodents and carnivores) placental scars remain in the uterine wall after earlier births, allowing some conclusions concerning reproductive history of the animal; such scars are initially yellow to black pigmented spots on the inside of the uterus, later becoming paler and smaller; in mice they may persist to one year. For simian primates, Benirschke (2002) provides photos and describes them as hyalinized or occasionally calcified scars or plaques persisting for months to more than a year. Both the number of detectable scars and number of sets of scars of different age should then be noted. The examined female can then possibly be classified as nulliparous (signs of breeding success), primiparous (with embryos or one set of placental scars) or multiparous (with embryos and at least one set of placental scars or with more than one set of placental scars) (Nagorsen, Peterson 1980; more information possibly from Hackländer et al., 2001). The reproductive status of partly decomposed animals should not be guessed. (Nagorsen, Peterson 1980)

Still incomplete. More included soon (organ review - eyes ff)

Examination of the body cavity after removal of organs:
Inner side of walls / serosa: reddened? (Sign of inflammation). Deposits (Fibrin, other)? (One of us: R. Plesker)

After necropsy, disinfecting the necropsy site is necessary. The carcass, all remaining tissues and blood soaked dirt or waste should be buried or incinerated. All contaminated paper or plastic materials should be either thoroughly disinfected or incinerated. All blood and residual tissues should be removed from the instruments and tools with soap and water. Then the instruments should be disinfected. Necropsy boots and apron should be cleaned and any contaminated clothing thoroughly washed. The external surfaces of any containers with samples should also be washed. (Munson, 2000)
 

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In: Loris and potto conservation database: field methods

Last amendment: 30 November 2002