WHAT’S YOUR DIAGNOSIS Thomas M. Donnelly, DVM, Column Editor

Fecal bulking in a frequently mated female rat Genaro A. Coria-Avila, DVM, MSc, Shann Menard, BA, Brigitte Boulard, BSc, Richard Ryan, BSc, Ivana Lemme, BA & James G. Pfaus, PhD One month into a study examining the effects of flupenthixol on sexual behavior and conditioned partner preference, we noticed that one female Long-Evans rat started to lose weight. The animal was one of forty 200–250 g Long-Evans rats housed in groups of four in wire-bottomed mesh cage. Each rat had free access to laboratory-rat pelleted feed and water. The animal room was maintained at 21 ± 2˚ C and 30–80% humidity on a reverse 12:12-h light/dark cycle. Half the rats in the study received intraperitoneal (i.p.) injections of 0.25 mg/kg every four days, 30 minutes before they engaged in sexual behavior. The other half received 1 ml/kg of 0.9% saline and served as a control group. The affected rat was in the flupenthixol treatment group, and the technician responsible for administering the IP injections reported the presence of a hard mass in the rat’s abdominal cavity. We examined the rat; palpation of the abdominal cavity indicated the presence of a firm fecal mass distending the colon. On neurologic examination the rat displayed normal postural reactions, spinal reflexes, and nociception. Its gait, however, was slightly abnormal, but because flupenthixol is a dopamine antagonist, we considered the uncharacteristic movement to be a consequence of altered motor activity1. However, as constipation is another reported side effect of flupenthixol1, we decided not to start any treatment but to monitor and observe the rat daily in its cage. Two days later, the rat died unexpectedly. We performed an immediate necropsy. In the abdominal cavity, we saw a prominent distended bladder containing 2.5 ml of dark urine (Fig. 1). Urinalysis revealed a pH of 5.7 (normal pH 7.3–8.5) and the presence of blood. Following centrifuga-

tion, we found that solids (largely erythrocytes) comprised 30% of the total urine. The urine supernatant remained red after centrifugation, indicating the likely presence of hemoglobin. Further gross observation of the abdominal cavity revealed flecks of blood scattered throughout the mesentery and gastrointestinal tract. The colon was enlarged and turgid with a 9- x 1.5-cm fecal mass (Fig. 2a). The anus appeared blocked with a firm plug of dry feces and surrounding hair. Yet surprisingly, the rectum was not distended with feces. The vagina, which was ventrally adjacent to the empty segment of the rectum, was severely distended to 3 x 2 cm (Fig. 2b). Thorough dissection of the vagina revealed the presence of white caseous contents. Microscopic examination of the caseous mass indicated it was made up of numerous spermatozoa. No bacteria or other microorganisms were seen within the mass. We postulated that the rat’s death might be a consequence of our experimental protocol and reviewed our procedures for possible explanations. Flupenthixol is an antipsychotic agent, but we were interested in the drug’s dopamine receptor antagonist effect on sexual behavior and conditioned partner preference. We required the female rats to mate frequently. To prevent pregnancy or pseudopregnancy (which would stop the females accepting a male), we ovariectomized all the rats two weeks before starting the study. For the surgery, we anesthetized the animals with a 4:3 mixture of ketamine hydrochloride (50 mg/ml) and xylazine hydrochloride (4 mg/ml) administered by i.p. injection at a dose of 1 ml/kg of body weight. For one week following the surgery, we treated all the rats with the analgesic flunixin meglu-

FIGURE 1 | Exposed abdominal cavity of a female adult Long-Evans rat. Notice the enlarged bladder (white arrow) filled with blood-tinged urine and the fecal mass in the colon (black arrow).

mine (2.5 mg/kg once a day) and the antibiotic enrofloxacin (5 mg/kg once a day). The rats all recovered uneventfully. Two weeks after ovariectomy, we induced sexual receptivity in the rats with subcutaneous estradiol benzoate (10 µg) followed by progesterone (500 µg) 44 hours later. Four hours after progesterone administration, we examined the female rats’ sexual receptivity and male partner preference in copulatory trials. We continued to induce sexual receptivity by administering this hormonal treatment every four days for 10 trials (i.e., 40 days). Thirty minutes before each copulatory trial, half of the ovariectomized rats received flupenthixol and the

Center for Studies in Behavioral Neurobiology, Department of Psychology, Concordia University, 7141 Sherbrooke West, Montréal, QC H4B 1R6, Canada. Correspondence should be addressed to G.A.C.-A. ([email protected]).

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FIGURE 2 | (a) Distended colon from the rat seen in Figure 1. Notice that the fecal bulk starts at the level of the anterior border of the vagina (black arrow). On the right, the two uterine horns and the empty bladder (white arrows) can be seen. (b) Enlarged view of colon and vagina in Figure 2a. Notice that the rectum is empty (black arrow) and the vagina is severely distended (white arrow).

other half received saline. The affected rat had received flupenthixol and died during the seventh conditioning trial, about one month after ovariectomy. Based on the rat’s weight loss, clinical presentation, and necropsy results, what do you think was the cause of death? How common is this problem?

What’s your diagnosis?

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Diagnosis | Colonic obstruction due to severe vaginal distension The female rat’s colonic obstruction was a consequence of the pressure induced by a distended vagina packed with caseous copulatory-like plugs of spermatozoa. We believe the rat died of multiple organ dysfunction syndrome (MODS), previously referred to as multiple organ failure. This is a progressive condition characterized by initiation of some clotting mechanisms and combined failure of several key organs, usually due to sepsis following injury or surgery. We considered that bacteremia and sepsis might have developed from the colonic obstruction2. In the progression from sepsis to MODS, uncontrolled inflammatory response and hemolysis lead to acute failure of the heart, lungs, liver, and kidneys. Heart failure can lead to hypotension, reducing the blood supply to the kidneys and instigating renal failure. Hemolysis due to the breakdown of red blood cells increases the level of free hemoglobin, potentially inducing acute renal failure from renal tubular damage. Kidney failure in turn leads to azotemia; depending on the severity and duration of renal dysfunction, metabolic acidosis, hyperkalemia, and abnormal fluid balance may also develop, leading to cardiac damage and failure. Although this sequence of events is speculative, the findings at gross necropsy are similar to findings described in humans with MODS and consistent with this diagnosis. We propose two possible explanations for the origin of colon obstruction. The first is straightforward: in rats, ejaculation is usually accompanied by the formation and placement of a copulatory plug in the vagina, which under normal circumstances is dislodged by intromissions during subsequent copulatory activity by the same or a different male3. Rodent copulatory plugs are typically hard, rubbery, or waxy in consistency, the exclusive product of male secretions4. LAB ANIMAL

The plug forms as the result of two successive reactions between proteins secreted by the enlarged vesicular glands (seminal vesicles) and anterior prostate. The male rat first secretes procoagulase (vesicular glands), which reacts with vesiculase (anterior prostate) to form coagulase. The newly formed coagulase then reacts with another vesicular gland secretion, coagulinogen, to form the coagulated protein of the plug4. In intact (non-ovariectomized) female rats, mating and copulatory plug deposition are followed by either pseudopregnancy or pregnancy5; female rats will not accept copulation with any male until they are again sexually receptive (after 10–22 days, depending on reproductive state). This is sufficient time to allow vaginal peristalsis to disintegrate and evacuate the remnants of the copulatory plug, a process triggered by the presence of the vaginal plug itself6. In ovariectomized rats, however, sexual receptivity can be artificially induced by injections of estradiol benzoate and progesterone, allowing copulation every four days (or more frequently). Such frequent mating can lead to subsequent development of the caseous sperm plug we observed, rather than the more typical copulatory plug (Fig. 3). Mating every four days may leave little time for the female to eliminate the sperm plug. This may have allowed sperm plugs to accumulate before disintegration and expulsion. In addition, vaginal distension induces a vagino-uterine reflex that causes normal caudocranial reverse peristaltic movements, helping sperm transport in the female reproductive tract7,8. The cranially directed peristalsis may have worsened the compaction of the sperm plugs, swelling the vagina and obstructing the rectum. While ovariectomy and hormone replacement for sexual receptivity are common procedures in our and other laboratories, to our knowledge, this is the first case

FIGURE 3 | Close-up view of multiple caseous sperm plugs found in the opened distended vagina of the frequently mated female adult Long-Evans female rat seen in Figures 1 and 2.

reported in which sperm plugs have had such disastrous consequences. A second possible explanation is related to the effects of flupenthixol. In humans, the drug is used as an antipsychotic1, with reports of adverse side effects that include dry mouth, urinary retention, paralytic ileus, and constipation1,9,10. We wonder if constipation could have occurred in the female rat as a direct consequence of flupenthixol injections during the conditioning trials. If this were true, then the constipation most likely was the result of digestive tract paralysis caused by flupenthixol’s anticholinergic properties11. We hypothesize that a functional interrelationship between the rectum and vagina may participate in the expulsion of sperm plugs. As the rectum and vagina are Volume 35, No. 10 | NOVEMBER 2006

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in close proximity, craniocaudal peristalsis during defecation may assist in vaginal evacuation, thereby facilitating spermplug removal. In this case, we speculate that a paralyzed rectum may have allowed the build-up of multiple sperm plugs during the copulatory trials. This would create a vicious cycle, inducing sperm-plug accumulation, which in turn would provoke obstruction of the colon, resulting in the clinical features described above. Constipation in rats can be easily diagnosed with abdominal palpation. In cases where female rats have been ovariectomized and allowed to copulate frequently, sperm plug-induced obstruction may cause constipation. Other causes of constipation in rats12 can include insufficient fiber intake, dehydration, hypothyroidism, hypokalemia, injuries to the anal sphincter 13, strictures 14, diverticula, tumors 15, and medications such as antipsychotics9,16, antidepressants 17 , and some analgesics (such as morphine18 and apomorphine19). In dogs and cats, the treatment for constipation often includes suppository laxatives, colonic evacuation with enemas, or manual extraction under general anesthesia20. In laboratory rats, these treatments have yet to be proven effective. ACKNOWLEDGMENTS The authors thank Paul Widden (microscopy) and Franc Rogan (urinalysis) for their contributions to this case.

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Canadian Pharmacists Association. Compendium of Pharmaceuticals and Specialties (Ottawa, Ontario, Canada, 2005). Sharma, S. & Mink, S. Septic shock. http://www.emedicine.com/MED/topic2101.htm. Wallach, S.J. & Hart, B.L. The role of the striated penile muscles of the male rat in seminal plug dislodgement and deposition. Physiol. Behav. 31(6), 815–821 (1983). Voss, R. Male accessory glands and the evolution of copulatory plugs in rodents. Occas. Papers Mus. Zool. Univ. Michigan 689, 1–27 (1979). Frye, C.A. & Erskine, M.S. Influence of time of mating and paced copulation on induction of pseudopregnancy in cyclic female rats. J. Reprod. Fertil. 90(2), 375–385 (1990).

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Crane, L.H. & Martin, L. Postcopulatory myometrial activity in the rat as seen by videolaparoscopy. Reprod. Fertil. Dev. 3(6), 685–698 (1991). Toner, J.P. & Adler, N.T. Influence of mating and vaginocervical stimulation on rat uterine activity. J. Reprod. Fertil. 78(1), 239–249 (1986). Shafik, A. Study of the uterine response to vaginal distension: the ‘vagino-uterine reflex’. Gynecol. Obstet. Invest. 44(4), 265–269 (1997). Jokinen, K., Koskinen, T. & Selonen, R. Flupenthixol versus diazepam in the treatment of psychosomatic disorders: a doubleblind, multi-centre trial in general practice. Pharmatherapeutica 3(9), 573–581 (1984). Lloyd-Williams, M. Treatment of depression with flupenthixol in terminally ill patients. Eur. J. Cancer Care (Engl.) 3(3), 133–134 (1994). Pharmacorama. Dopamine receptor antagonists. http://www.pharmacorama.com/en/Sections/ Catecholamines_7_4.php. Caldarella, M. et al. Visceral sensitivity and symptoms in patients with constipation- or diarrhea-predominant irritable bowel syndrome (IBS): effect of a low-fat intraduodenal infusion. Am. J. Gastroenterol. 100(2), 383– 389 (2005). Sanmiguel, C.P. & Soffer, E.E. Constipation caused by functional outlet obstruction. Curr. Gastroenterol. Rep. 5(5), 414–418 (2003). Holschneider, A.M., Amano, S., Urban, A. & Donhauser, G. Animal experimental studies on the free transplantation of smooth colon muscles as an artificial sphincter in the rat. [German]. Z. Kinderchir. 39(3), 182–190 (1984). Colecchia, G. & Nardi, M. Colorectal cancer in pregnancy. A case report. [Italian]. G. Chir. 20(4), 159–161 (1999). Kovacs, G. Prescription of psychotropic drugs for schizophrenic outpatients in Hungary. [Hungarian]. Neuropsychopharmacol. Hung. 7(1), 4–10 (2005). Wilde, M.I. & Benfield, P. Tianeptine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy in depression and coexisting anxiety and depression. Drugs 49(3), 411–439 (1995). Tamayo, A.C. & Diaz-Zuluaga, P.A. Management of opioid-induced bowel dysfunction in cancer patients. Support. Care Cancer 12(9), 613–618 (2004). Gowing, L., Ali, R. & White, J. Buprenorphine for the management of opioid withdrawal. (Cochrane Review). Cochrane Database of Systematic Reviews 2(CD002025). DOI: 10.1002/14651858.CD002025. (2006). Merck Veterinary Manual. Constipation and obstipation. http://merckvetmanual.com/mvm/ index.jsp?cfile=htm/bc/23303.htm.

LAB ANIMAL welcomes reader contributions to “What’s Your Diagnosis” in case history/ diagnosis format. Submissions should include two to five illustrations or photos. Please email manuscripts to [email protected]. Selections are made on the basis of relevance and interest to readers. Please refer to the Guide to Authors for information on submission of digital figures.

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