TITLE: Vezina Lab Mouse Castration Protocol
AUTHORS: Chad Vezina

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Vezina Lab Mouse Castration Protocol 3/15/2010</div></div>

[STEPS]
?. [2 weeks prior to experiment]
Contact RARC (animal resource center) and reserve Isoflurane vaporizer and glass bead sterilizer.
?. [2 days prior to experiment]
Collect the following supplies and ensure that isofluorane and buprenorphine are in date: Betadine scrub, 32 oz (*RARC pharmacy)Surgical drape, non-sterile, 38.5 in X 100 yd (VWR 34300-532)Procedure face masks, 2 Ply with Earloops, 50/bpx (*RARC pharmacy)Sterile gloves, 50/Box (*RARC pharmacy)Sterile wooden applicators with cotton tips (Fisher 14-959-91)Isoflurane (FORANE), 250 mL (*RARC pharmacy)Buprenorphine, 1 mL of a 0.3 mg/mL solutionKetoprofen, 100 mg/mL solution X 50 mL (*RARC pharmacy)Sterile saline solution (for diluting buprenorphine and/or Ketoprofen to an appropriate working solution), 0.9%, 250 mL (*RARC pharmacy)Sterile empty serum vial (for diluted buprenorphine and/or ketoprofen), 10 mL X 25/box (*RARC pharmacy)1 mL syringe, 100/box (*RARC pharmacy)26 G X 1/2 in length syringe needle, 100/Box (Fisher 14-826-15)Suture, Vicryl 4-0, absorbable polyglactin 910,6/pk or 36/box (*RARC Pharmacy)Artificial tears, 1/8 oz tube (*RARC pharmacy)Mouse clippers (WAHL brand, Walmart)2-count Hemostat, straight, serrated tip (Fisher 08-907)Blunt-pointed dissecting forceps, 5 inch (Fisher 08-890)Sharp pointed dissection scissors, 4.5 inch (Fisher 08-940)Gauze, 2 X 2 in, 12-ply, 200/bag (*RARC pharmacy)
?. [1 day prior to experiment]
Autoclave surgical equipment: Place 4.5-inch scissors, two hemostats, and one dissecting scissors into an autoclave bag. Seal bag and complete autoclave cycle.
?. [1 day prior to experiment]
To prepare 0.02 mg/mL Buprenorphine working solution from a 3 mg/mL stock solution, into a sterile septated bottle, add 0.33 mL of Buprenorphine stock (0.3 mg/mL) to 4.67 mL saline.Remember to mark down the use of Buprenorphine stock in the controlled substances log. The expiration date should be listed on the bottle and is the earliest date of either the buprenorphine expiration date or the saline expiration date.
?. [1 day prior to experiment]
Review Vezina Lab Animal Care and use protocol sections that pertain to castration and analgesia.
?. [1 day prior to experiment]
Pick up vaporizer and glass bead sterilizer from RARC (372 Enzyme Institute Building).
?. [1 day prior to experiment]
Send the following email message to svmvets@rarc.wisc.edu:To whom it may concern: we will be performing castrations on a batch of [insert number] mice on [state date of surgery] in accordance with our approved animal protocol.The mice will be housed in AHABs room B23A/B and their cage cards will be marked with a label stating the date of the surgery.We anticipate some bleeding at the surgical site that should clear within a few days of the surgery.”
?. [Day of experiment]
Clear items from surgery area.
?. [Day of experiment]
Treat bench surface with 10% bleach for 10 min.
?. [Day of experiment]
Mop bleach from bench surface and spray with 100% ethanol and air dry.
?. [Day of experiment]
Bring animals upstairs.
?. [Day of experiment]
15 minutes prior to surgery, inject animals with 0.1 mg/kg buprenorphine (volume = microliters of 0.02 mg/mL buprenorphine per gram of mouse).
?. [Day of experiment]
Lay down sterile drape.
?. [Day of experiment]
Enter surgery start time and other information into surgical log.
?. [Day of experiment]
Scrub hands and gown up.
?. [Day of experiment]
Arrange tools in surgical space.
?. [Day of experiment]
Set isoflurane vaporizer and turn it on (set mixer to 2% and flow rate to 1 liter/minute).
?. [Day of experiment]
Induce mouse anesthesia and pinch toe to confirm unresponsiveness.
?. [Day of experiment]
Add artificial tears to mouse.
?. [Day of experiment]
Shave scrotal area.
?. [Day of experiment]
Use betaine scrub to prep scrotal area.
?. [Day of experiment]
Use scissors to make a vertical incision halfway between anus and penis.
?. [Day of experiment]
Use forceps to gently grab tissue surrounding testicle and make a small incision.
?. [Day of experiment]
Use forceps to exteriorize testis, epididymis, and fat pad.
?. [Day of experiment]
Use suture material to ligate the major testicular vessel.
?. [Day of experiment]
Remove testis and repeat this procedure for other testis
?. [Day of experiment]
Close the scrotal incision with a single suture.
?. [Day of experiment]
Remove mouse from vaporizer and return to cage.House at a density of one mouse per cage until wound is healed.Watch closely until coordination returns.
?. [Day of experiment]
Enter surgery end time into surgical log.
?. [Daily for 1 week after surgery]
Inspect mice and record date, time, and inspector into surgical log.
?. [Daily for 1 week after surgery]
Grounds for early euthanasia: mice will be euthanized if the body score becomes less than 2, if animal movement is compromised, or if animal becomes unresponsive to mild stimulus. If mouse has these symptoms, (1) euthanize mouse, (2) record the euthanasia in the surgical log, (3) alert RARC staff that an adverse event occurred (email svmvets@rarc.wisc.edu and explain what happened).
?. [Daily for 1 week after surgery]
If an infection develops (1) email svmvets@rarc.wisc.eduv and request antibiotics (2) record the event in the surgical log.