dyspnea, and pallor most evident on the muzzle and ears (see table 1). Generally, those signs were transient and mild to moderate. However, in 19 of the animals from four of the five groups, after the first or second tap, symptoms were persistent and severe; five died, and the remaining 14 were euthanized (see table 1). Survival rates of the mice for tap 1 were 90–100%; for tap 2, 85–100%; and for tap 3, 35–100%. No clinical abnormalities were observed in control animals after a sham paracentesis. Jackson and others (1999a) conclude that the abnormal clinical signs observed after paracentesis were compatible with circulatory shock.

Some IACUCs have established policies (see table 2) that require tapping of abdominal fluid to be performed under anesthesia and to be followed by injection of 1–2 ml of warmed saline to minimize posttapping hypovolemic shock. It is not clear that anesthesia use does reduce overall distress (for example, anesthetic administration causes handling stress), and it is possible that hypovolemic reactions can be minimized if the fluid is drawn off slowly (as is recommended for removing ascitic fluid from human patients—generally, up to 5% of body weight over 4 hours). However, if more than one tapping is to be permitted, the clinical signs observed by Jackson and others (1999a) after tapping indicate that some level of fluid replacement might alleviate some distress.

There was some discussion at the ILAR working-group meeting of whether it was preferable for animal welfare to conduct fewer taps, which would mean that more mice would be needed to produce the required amount of antibody, or whether one should perform several taps and reduce mouse use by 50–75%. It is evident from the Jackson and others (1999a) paper that the clinical signs and evidence of animal discomfort and distress increase sharply once ascites has developed to the point where fluid can be harvested. Thus, one can reduce animal use; but if distress is high and increasing steadily in the 4–6 days from the first to the third and terminal tapping, then the extra taps are likely to be accompanied by substantial distress. If the animals are tapped just once under terminal anesthesia, the extent of distress will be minimized, but more animals will be put through (the procedure. The overriding criterion should be animal distress. Therefore, the number of taps should be limited and varied according to animal welfare and characteristics of the hybridoma being used. Some hybridomas seem to cause little distress (Jackson 1999a), and multiple taps could be allowed. Determining the number of taps depends on careful monitoring of the animal; if signs of distress appear, a terminal tap should be performed.

A number of IACUCs have adopted a 0.2 ml volume limit on the pristane injection based on recommendations made by McGuill and Rowan (1989) (table 2). Members of the committee did not object to this as a guideline, but several reported that, in some strains of mice, 0.2 ml might not be sufficient to produce ascites and that as much as 0.5 ml might be required. There are some questions about the suitability of IFA as a substitute for pristane, and the committee recommends that IACUCs determine the reaction of animals to IFA before permitting its routine use. Most IACUCs with public policies recommend that