The necropsy based fish health assessment is used to assess the health of wild fishes for routine monitoring, specific studies comparing sites and as a basis for tissue collection for more in-depth analysis. Fish are constantly exposed to a combination of stressors such as complex mixtures of chemicals, pathogens and parasites. These combinations of stressors can lead to adverse effects such as reduced disease resistance, cancer, and reproductive failure.
The techniques demonstrated allow for a standardized evaluation of visible abnormalities and the collection of blood and tissues for histopathology, otoliths for age and tissues for other downstream analyses. First we will demonstrate the routine necropsy based assessment with blood, otolith and tissue collection for histopathology using one fish. Collection of anterior kidney for immune function assays and liver or other tissue for gene expression are additions to this method that we will also demonstrate.
It is important to collect tissues as quickly as possible to avoid any post mortem changes. So we generally have an assembly line process with two to three people collecting tissues and someone recording the data. Heather Walsh and Cheyenne Smith, PhD students from West Virginia University and Ryan Braham, a recent PhD graduate, will be demonstrating the various steps in the process.
After collecting and euthanizing fish according to institutional guidelines, weigh and measure the fish and obtain a blood sample. Next measure the total length of the fish from the tip of the snout to the end of the tail. Insert a three CC syringe with a 22 or 23 gauge needle, previously coated with heparin, anterior to the caudal area below the lateral line.
Angle the need upwards until it hits the spine and withdraw it slightly to obtain a blood sample. Prior to transferring the blood sample to a collection tube, remove the needle and place it in a sharps bin. For day trips, blood can be held on wet ice until return to the laboratory.
Then it is centrifuged to remove the plasma which was stored at minus 80 degrees Celsius until analyzed. If not returning to the lab each day, a centrifuge and dry ice storage is necessary. Next, document any external abnormalities or lesions on the body, fins, eyes, and gills of the fish.
Inspect the fish for external parasites such as leeches, grubs, trematode metacercarial cysts, and gill parasites. Then use scissors to open the abdominal cavity from the anal area to the operculum. Remove the flap of muscle to expose the internal organs.
Examine the internal organs and document general and focal discolorations, presence of raised areas, cysts, parasites and size abnormalities. Next, to remove the liver, sever the hepatic artery and the connective tissue of the anterior end. Gently lift the liver out of the carcass and trim any adhesions and connections to intestines and adipose fat taking care not to puncture the gallbladder.
Weigh the liver. Then obtain the hepatosomatic index as outlined in the text protocol. Next remove the gonads and weigh them.
Carefully, without pulling, cut out small tissue samples. Place the tissue samples from one fish in the same container. Next, cut at least five, three to four millimeter thick pieces from various regions of the liver and place them in the fixative container.
Depending on the size, place a whole gonad or pieces of a gonad in the fixative container. Then place smaller whole organs and pieces of other larger organs in the fixative container. If any abnormal tissues are observed preserve an additional adjacent piece of normal tissue.
Numerous structures such as scales, fin rays and otoliths may be used to age fish. Here we are using otoliths as they are the most accurate. First, cut through the gill isthmus and bend the head backwards.
Then to locate the prootic bulla strip away connective and muscular tissues around the inferior portions of the neurocranium. Use a bone cutter to crack the prootic bulla and expose the otoliths. The otoliths should be visible to the naked eye.
Place the otoliths in a labeled, uncapped vial and store them at room temperature until age analysis can be performed. Exposure to numerous chemicals and other environmental stressors can cause immunosuppression. Lymphocytes, macrophages and granulocytes can be isolated from this tissue and in the laboratory their ability to kill pathogens, produce antibodies, respond to tumor cells ad other functions can be measured.
Using sterile scissors, scalpel and forceps remove the anterior kidney tissue. Place the anterior kidney sample in the appropriate media to keep the cells alive. Use a sterile handheld tissue grinder to homogenize the sample into single cell suspensions.
And hold the samples on wet ice. For RNA preservation, place two to three small pieces of liver in the appropriate preservative at a 10 to one ratio of preservative to tissue. In this study, white suckers were collected from three Great Lakes areas of concern, or AOCs, and the reference site Kewanee River.
The percent of fish with external tumors ranged from 15.5%at the St.Louis AOC to 58%at the Milwaukee AOC. Several types of external lesions were observed including discreet white spots, slightly raised mucoid lesions, and large papillomatous lesions. Many of the discrete white spots and mucoid lesions observed in this study, particularly at the Kewanee reference site, were hyperplastic lesions as opposed to neoplasia.
Histopathological analysis also identified liver tumors that were not identifiable with visual observation. Once mastered, this technique can be done in 15 to 20 minutes per fish if it is performed properly. While attempting this procedure, it's important to know what is normal for your particular species and to be as consistent and accurate as possible in describing abnormalities.
Include size, color, texture and location. Photo documentation is useful. This technique can be used for any fish species and is useful for documenting fish health both spatially and temporally to better understand the adverse effects of environmental stressors and identify invasive diseases.
