Comparative Medicine Vol 50, No 2 Copyright 2000 April 2000 by the American Association for Laboratory Animal Science Poor Quality of Oocytes from Xenopus laevis Used in Laboratory Experiments: Prevention by Use of Antiseptic Surgical Technique and Antibiotic Supplementation Holger-Andreas Elsner,1,2 Hans-Hinrich Hönck,3 Frank Willmann,4 Hans-Jürgen Kreienkamp,3 and Franz Iglauer4 Background and Purpose: Episodic phases of continuous poor-quality oocytes obtained from South American Clawed Frogs (Xenopus laevis) often are observed. In publications dealing with the surgical technique of oocyte removal, the frogs’ robust constitution and resistance against infections provided by magainins are pointed out. For this reason, clean rather than sterile conditions for the surgical procedure are mostly recommended. However, in most instances, antibiotics are added to the buffer medium when in vitro experiments are performed using oocytes. Methods: After a long phase of poor oocyte quality at our facility, involving oocytes that had been obtained by use of a “clean” surgical procedure, we subsequently cultured oocytes in a buffer medium containing the three antibi- otics: penicillin G, gentamicin, and streptomycin. Results: During DNA injection experiments, the oocytes developed black spots on their surface by postoperative day two. Pure culture of the gram-negative non-fermentative rod Pseudomonas fluorescens was obtained from the impaired oocytes; the isolate was resistant to the three antibiotics. By contrast, after aseptic surgical removal and culture of oocytes in buffer medium containing the antibiotics tetracycline and gentamicin, perfect oocytes with- out bacterial contamination were obtained. Conclusion: Whenever impaired oocyte quality is observed, microbial contamination should be considered as a possible cause. In former times, South African clawed frogs (Xenopus laevis) Despite great experience in rearing X. laevis and in oocyte were mostly used for diagnosis of gravidity in obstetrics, for si- removal from the living animal, we observed a period of poor tus preparations, or for the study of developmental processes in oocyte quality from December 1997 until January 1998. During practices/exercises of physiology. Currently, its most frequent RNA injection experiments, postoperatively healthy-appearing biomedical use is to obtain oocytes for research in molecular bi- oocytes developed marbled spots on the surface (Figure 1). ology, biophysics, and ontogenesis. These changes are reminiscent of the apoptotic stage of normal At the Laboratory Animal Facility, University Hospital Ham- cell aging (4). After only one or two days, the electric resting po- burg, South African clawed frogs have been successfully bred for tential was very low (–10 to –20 mV), wheras healthy oocytes many years. These frogs were maintained in accordance with the (Figure 2) usually remain optically unchanged after one week most recent accomodation recommendations (1), and in compliance and maintain a rather high resisting potential (–30 to –60 mV). with the governmental supervising authority (Hamburg, Ger- In most institutes, researchers complain about the disastrous many), in particular with §11, Tierschutzgesetz. The adult females episodes of continuous (weeks or months) poor oocyte quality had been received from various resources: NASCO (Fort Atkinson, from all donor frogs that belong to the same colony (5, 6). Rear- Wisconsin) or Fa. Dipl. Biol. H. Kähler (Schramsweg 13, 20249 ing conditions, parasitic or other diseases, origin of the frogs, or Hamburg, Germany), or had been bred in our facility (stock: simply the “warm season” are suspected to cause this phenom- Uhh:Xenopus). According to the categories of the National Re- enon (4, 7–10). However, during this period of poor oocyte qual- search Council (2), the hygienic status was “laboratory-reared ity and during former periods, we never found evidence for any standard” or “laboratory-bred standard.” Feeding and husbandry of these suppositions to be causative. Frogs were kept at 20 Ϯ conditions were those described elsewhere (3). Use of these labora- 2ЊC in flow-through or static tanks. As oocytes from different tory animals was indicated and licensed (§6 Abs.1 Satz 2 Nr.4 frogs were involved, genetic reasons, including inbreeding, also TierSchG) by the government office in Hamburg for laboratory were unlikely. At our facility, each X. laevis is used six times at animal experiments (16.07.1996/Lo: G 8151/591-00.33). a maximum. Body weight is monitored before and after surgery. The interval between two surgeries is at least three months. 1Department of Transfusion Medicine, Hannover Medical School, Carl-Neuberg- Frogs were not used if there was any reduction in body weight Str. 1, D-30625 Hannover; 2Institute of Medical Microbiology and Immunology, University Hospital Eppendorf, Martinistr. 52, D-20246 Hamburg; 3Institute prior to the next scheduled surgery. Each frog from our own for Cell Biochemistry and Clinical Neurobiology, University Hospital Eppendorf, stock was at least two years old and weighed Ն 90 g. Those from Martinistr. 52, D-20246 Hamburg; and 4Laboratory Animal Facility, University extramural sources had body weight of at least 120 g. The frogs Hospital Eppendorf, Martinistr. 52, D-20246 Hamburg, Germany. Dr. Iglauer’s present address is Tierschutzbeauftragter, Universität Tübingen, had good appetite, activity, and general appearance. There were Calwer Straße 7/4, 72076 Tübingen, Germany. no signs of skin discoloration or other disorders. The frog colony 206 Prevention of Poor Quality of Xenopus Oocytes Figure 1. Impaired oocytes that were useless for electrophysiologic Figure 2. Oocytes immediately after removal (no signs of impairment). experiments. mens. A short time after surgical removal, all oocytes had been was monitored for skin parasites as described (10). Since 1997, optically unchanged, with distinct black and white hemispheres our own breeding colony has been free of skin nematodes. Those (Figure 2), indicating that impairment was most likely not of extramural sources had been treated by use of methods suit- caused by disease of the donor frog, but developed later. After able for eradication of skin nematodes (10). This was done three defolliculation, marbled spots could be visualized at a seven-fold months before surgery, when the frogs arrived at our facility. magnification after 24 hours (Figure 1). Oocytes that were incu- In publications dealing with surgical oocyte removal tech- bated over 24 hours within the mesovarium remained un- niques, the frogs’ robust constitution has been discussed and changed, suggesting a normal protective effect of this structure. wound infections are rarely described. The resistance against To explain the bacterial growth despite antibiotic supplemen- infection of the surgical wound is mainly prevented by antimi- tation, minimal inhibitory concentrations (MICs) of penicillin G, crobial peptides (magainin) located in the frog’s skin (4, 7, 11). gentamicin, streptomycin, tetracycline, and ciprofloxacin (single For this reason, normal sterile conditions for surgery are re- garded unnecessary, but a “clean surgical technique” is ap- agents) against all Pseudomonas fluorescens isolates were de- praised as adequate (7), which means avoidance of coarse termined on Mueller-Hinton agar (Difco), using E-test strips contamination of the surgical area. However, in most instances, (AB Biodisk, Solna, Sweden). These test strips, which are antibiotics (streptomycin, gentamicin, penicillin G) are added to charged with an increasing concentration of each antibiotic and the buffer medium when in vitro experiments are performed are put on the agar plate, render easy determination of the MIC, which corresponds to the number printed on the strip where the with oocytes (12). At the time of the impaired oocyte quality outline of the growth inhibition zone touches the scale on the event, a “clean surgical technique” was our standard, and the strip. Incubation times were 24 hours at 30ЊC, 24 hours at 35ЊC, combination of streptomycin (100 ␮g/ml), gentamicin (100 ␮g/ and 6 days at 35ЊC. Test were performed at AB Biodisk. Entero- ml) and penicillin G (59 ␮g/ ml) was added to the Barth me- coccus faecalis (ATCC 29212), Staphylococcus aureus (ATCC dium, in which oocytes were kept. 29213), Escherichia coli (ATCC 25922), and P. aeruginosa To prove or exclude microbial contamination of the oocytes, (ATCC 27853) were used for quality control. nine oocyte specimens obtained from four frogs during the pe- Results of antibiotic susceptibility tests are shown in Table 2. riod between December 15, 1997 (approx. two weeks after the After incubation for 24 hours at 35ЊC, there were no visible colo- beginning of the episode) and February 2, 1998 (Table 1) were nies on the agar. However, there was light or very light growth examined microbiologically. The four frogs belonged to a colony after prolonged incubation at 6 days. At these conditions MICs of approximately 40 animals and were randomly chosen. The were lower than those at incubation for 24 hours at 30ЊC, re- oocyte specimens were streaked on 10% sheep blood agar with flecting weaker growth. After incubation at 30ЊC for 24 hours Columbia Blood Agar Base (Difco, Detroit), MacConkey agar and at 35ЊC for 6 days, high MICs that exceeded concentrations (Oxoid, Basingstoke, UK), and for detection of fungi on Kimmig in the Barth medium were found for penicillin G, gentamicin, agar (Merck, Darmstadt, Germany) supplemented with