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LAB 11 HORMONES AND MALE REPRODUCTION
The interplay among the hypothalamus, pituitary, gonadotropic hormones, sex hormones, and sex organs is highly fascinating and quite complex. In this experiment you will examine the interrelationships of these hormones and organs using rats that are castrated, maintained for 2 weeks (some injected with replacement hormones), and then sacrificed to examine the effects on reproductive organs like the seminal vesicle.
It will be your responsibility to perform the surgical operations, care for the animals, make injections when needed, make the required dissections, and make your group’s results available to the rest of the class on the last day of this experiment.
You are urged to read as much as possible about reproductive physiology so that you can properly explain the results of the experiments. Before you begin, acquaint yourself with the reproductive system in the rat by studying Figures 9.1 (male rats) and 9.2 (female rats) and observing any available demonstration specimens.
I. General Directions for Aseptic Operations
These experiments on the endocrine system are performed on “chronic” animals, that is, animals that are kept alive for an indefinite period following the operation. Rats are generally quite resistant to infection, so successful operations can be performed with a minimum of aseptic techniques; however, it is strongly recommended that all instruments be autoclaved prior to use and keep the procedure as aseptic as possible. See the appendix at the end of this lab for detailed instructions for small mammal surgery.
1. Instruments used in the operation are generally kept in a tray containing 70% alcohol. They sterilized in an autoclave before class and hence are sterile when used to open an incision.
2. As in previous labs, it is best to work in teams of two during the operation, with one person acting as the anesthetist and the other as the surgeon. The anesthetist anesthetizes the rat, clips the hair from the operation site, and swabs the skin with alcohol. It is the anesthetist’s job to watch the animal closely during the operation and regulate the depth of anesthesia (we want our “patients” to live). The surgeon(s) scrubs his hands thoroughly before the operation, performs the operation, and closes the incisions with sutures or wound clips.
3. Weigh the animal to determine the dosage of the anesthesia. Record the weight. Then inject the animal IP with a ketamine-xylazine cocktail. Remember that the dosage is weight dependent. Refer to Appendix B for the recipe and the dosage of K/X.
4. After the operation, place the animals in a box containing clean towels until they are fully conscious. It is often useful to have a lamp placed over the box to help them regain the body heat they lost during the operation. Once they have recovered they will be returned to their clean “home” cage.
5. While still anesthetized, it’s a good idea to number the rats for positive identification later. Tail markings or ear tags are the best means of identification.
Male rats weighting 75 to 100 g are divided into the following categories:
1. Normal rat (control)
2. Normal rat given a subcutaneous injection of chorionic gonadotropin (20 units) daily for the last 12 days
3. Rat castrated 14 days prior to the last lab
4. Rat castrated 14 days prior to the last lab and given subcutaneous injection of 0.1 mg of testosterone daily for the last 12 days (gives the rats two days to recover from surgery).
5. Rat castrated 14 days prior to the last lab and given a subcutaneous injection of chorionic gonadotropin (20 units) daily for the last 10 days
III. Procedure
Record initial and final body weights for all rats in the table in the laboratory report. On the final day you will sacrifice the rats using an overdose of Ketamine I.P. You will carefully dissect out and weigh the seminal vesicles, and record their weight in milligrams per 100 g of body weight. Students will then share their findings to the rest of the class for inclusion in the lab report for this lab.
Removal of Testes (Castration)
1. The intraperitoneal anesthetic cocktail is designed to keep the animal anesthetized for 30-40 min. The animal is unaware of the operation and should not move when the skin is cut. If the animal shows signs of awakening during the operation increase the dose of ketamine/xylazine.
2. Clip the hair along the ventral midline of the scrotum with scisssors, and swab the area first with betadine and then with alcholol. Using fine-pointed scissors make a midline incision about 1.5 cm long through the scrotal skin.
3. Sometimes the testes retract into the abdominal cavity and therefore are not visible in the scrotal sac. Slight pressure on the lower abdominal area will force the testes back into the scrotum. You will note that each testis is surrounded by a translucent membrane called the tunica (Figure 9.3a). Grasp the tunica with forceps and slit it with the scissors to free the testis. When the tunica is pierced, the testes are pushed out one at a time.
4. The testes are raised to expose the underlying blood vessels and tubules. The spermatic chord containing the vas deferens and blood vessels is tied off with heavy surgical thread at the confluence of the blood vessels and epididymis, which is approximately 1cm from the insertion point of the testis.
Once it is tied off, then cut the spermatic cord between the knot and the testis and remove the testis (Figure 9.3b). All deferential vessels and ducts are replaced back into the tunica. Remove the other testis in the same manner. Skin incisions in the scrotum are closed with sutures or two or three stainless steel wound clips. Place the rat in the recovery room box until it regains consciousness. It is sometimes necessary to massage the animal to facilitate recovery.
5. Start giving daily injections to rats two days following surgery for two weeks.
VI. Questions/Data Collection
1. What is the difference between an IV, IP, and SC (subcutaneous) injection?
2. Why did we give gonadotrophin to the normal and castrated rats?
3. What happened to the body weight of the different groups of rats? Explain why you think this occurred.
4. What happened to the relative weight of the seminal vesicle in the different groups of rats? Explain why you this occurred.
5. Is there another experimental design that could be used for this experiment, if so what is it and what additional data could it provide?
6. Generate a graph (SigmaPlot or Excel) that summarizes your findings and include it in your report.
V. Materials:
1. 6 large live male rats, often bald/nude rats are easier to work with 2. Scalpels with # 10 and #11 blades 3. Large and fine forceps 4. Good quality fine and Mayo dissecting scissors 5. Hemostats, large and small 6. Staples for closing incisions 7. medium sized retractors 8. Heat Lamps 9. 100 ml sterile PBS 10. Electric clippers for hair removal 11. Gelfoam sponges 12. Bacitracin antibiotic ointment 14. Probes 16. Latex gloves 15. Cotton and gauze pads, Q-tips 16. Xylazine 20 mg/ml stock, Ketamine 50 mg/ml stock. Normally make up a 1:4 mixture of xylazine to ketamine for injections. This gives a working solution that reflects the 10 mg/kg xylazine and 100 mg/kg of ketamine that is recommended. Next calculate the volume of each drug that is required, add the volumes together and use that volume of the mixture for injection. 17. Tuberculin syringes 18. 50 ml syringe (or 2 oz rubber bulb) and tubing for lung aspirating 19. 50 ml sterile 4.0% dextrose/0.18% NaCl solution 20. plastic tray to hold surgical instruments and alcohol for sterilization 21. 250 mL Pure Corn Oil, sterile 22. Testosterone
Adapted from Experiments in Physiology by Tharp/Woodman, 2008, Ninth Edition Pearson Benjamin Cummings Publisher
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