1) Hormonal induction fails to produce sperm or sperm of poor quality.
2) Large quantities of sperm are desired.
3) Time or other constraints prevent the use of hormonal induction.

Testis can be excised (removal of body part = ablation) from anurans recently dead through natural mortality or from euthanized animals (see euthanasia).

Alternatively, testes can be sampled surgically - leaving the animal healthy and vital and in a state to reproduce by the expression of sperm, either through hormonal induction or through environmental entrainment.

Where sperm of threatened or difficult to obtain males is required, or where hormonal induction with these species fails, one testis or preferably parts of testis may be removed surgically. Using appropriate anaesthesia (see anaesthesia), a testis may be exposed through a 5mm (0.2in) dorsolateral incision and a part or the whole testis may be excised. No procedures for homeostasis (stopping bleeding) are needed and the incision may be closed with simple surgical stitches.

The excision of testis from dead anurans.

All anuran tissue and cells, including somatic and germ cells must be kept as cold as possible above freezing. The best way to do this is to use an ice slurry in an insulated bin, with the bin stored in a fridge. Samples are placed directly in the slurry in Eppendorf tubes, cryotubes, vials, test tubes, ziplock bags etc. Ice itself is unsuitable for the storage of live tissue as the specimens may freeze if the ice is at a low temperature. The process of bringing the ice to near freezing point by the use of water is caller 'tempering'. An ice slurry prevents the freezing of specimens and keeps them close but above 0ºC, that is at the maximum potential 'degree days' of storage.

Degree Days: An important principle used for tissue storage is the concept of "Degree Days". This principle is based on the rate of the decomposition of tissues and is frequently used in food preservation for establishing refrigeration storage times under different refrigeration regimes. The principle works on a inverse law of time and temperature; which is considered as Celsius (Fahrenheit to Celsius conversion table) For example if an item will store at a certain quality for 10 days at 4ºC, it will store for 5 days at 8ºC, or for 20 days at 2ºC. The important aspect to notice when it comes to tissue preservation is that a change from - lets say - a refrigeration temperature of 4ºC to a temperature in an ice slurry of 1ºC will result in FOUR TIMES the storage period. Keeping the sample at 20ºC for 10 minutes is the same as keeping it at 200 minutes (2hr 20min) at 1ºC in ice slurry.

Anuran sperm is also immobilized at low temperature. Nevertheless, testes and sperm samples (unless in solutions at or above 200mM) should be kept as close to 1ºC to prevent activation.

† Because of variance between the times of sampling sperm and the time of the initiation of different experimental procedures, the results of experiments in which sperm was not held in an ice slurry after sampling and/or between experimental procedures should be viewed very cautiously.

Excising Testis

For the complete excision of the testes the toad or frog should be euthanised by the injection of 1 ml MS-222 at 0.4% (w/v) per10 g of animal weight (see MS-222). When the animal does not respond to toe pinching, and by not blinking when the eye is touched it is in deep anaesthesia. At this stage is pituitaries are not to be sampled the animals can be pithed, and then the heart removed during dissections. If the pituitaries are to be sampled the heart should be excised after anesthetization.

Instruments: The instruments need for the excision of the testes consist of a pair of surgical scissors, blunt forceps, and curved forceps. The needle nosed forceps are for the excision of pituitaries. The toad shown is a male - identified by the black vocal sack - Fowlers Toad (Bufo fowleri) from Tennessee, USA.

Initial incision: The toad is placed on its back and a piece of skin snipped off from its lower stomach. The stomach wall is exposed enabling the opening of the stomach.

Opening of stomach: An incision is made with the scissors forward along the length of the abdomen to the sternum. This incision exposes the right liver.

Stomach organs exposed: A cut is made across the body below the ribs and near the cloaca to exposing the heart, with both livers above the intestines, kidneys and testis.

Right testis exposed: Gently expose the right testis - which lies above the kidney and is close to and parallel to the backbone. Be cautious the testis may be confused with lobes of the fat bodies. The testis is cylindrical and fixed along its length. The fat bodies form a loose lobed structure originating from the near the anterior medial portion of the testis. The toad in the image does not have visible fat bodies. At this stage it can be helpful to dry the abdominal cavity with a tissue, as besides blood etc. a large amount of water may be present from lymph sacs and water storage organs.

† The testes of frogs are generally not relatively large, as elongate, or conspicuously white as those of toads (Bufo spp.). Frog testes are often relatively smaller, spherical, and grey, and can be difficult to distinguish from other organs particularly the gall bladder. Puncturing the gall bladder by mistake is very bad. The gall bladder is usually green and filled with a particularly virulent digestive fluid which detrimentally affects any tissues it makes contact with.

Gripping right testis with curved forceps: The right testis is gripped under its lower (dorsal) face by the curved forceps. Sometimes it can be removed at this stage simply by lifting it of the kidney. However, difficulties in detaching the testes can demand the use of scissors. In this example the left testes is in two pieces, a common situation in toads. (Above)

Removing one part of the right testis: Removing the right testes with curved forceps and scissors. In this example the right testes is one continuous piece. When removed the testes should be rolled on a piece of paper to remove blood and other tissue. It is very important to remove blood, which if present will quickly destroy sperm. (Above)

Left testis exposed: Similarly as with the right testes the right testes is exposed by carefully displacing the organs. In this case the left testes is in one piece.(Above)