Analysis of Gross and Histopathologic Changes from Repeated Celiotomies in African Clawed Frogs (Xenopus laevis) - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Official websites use .gov A .gov website belongs to an official government organization in the United States. Secure .gov websites use HTTPS A lock ( Lock Locked padlock icon ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites. Search Log in Dashboard Publications Account settings Log out Search… Search NCBI Primary site navigation Search Logged in as: Dashboard Publications Account settings Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide PERMALINK Copy As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice J Am Assoc Lab Anim Sci . 2026 Mar;65(2):279–285. doi: 10.30802/AALAS-JAALAS-24-097 Search in PMC Search in PubMed View in NLM Catalog Add to search Analysis of Gross and Histopathologic Changes from Repeated Celiotomies in African Clawed Frogs ( Xenopus laevis ) Darya Mailhiot Darya Mailhiot , DVM, DACLAM 1 Animal Resources Center and Department of Surgery, University of Chicago, Chicago, Illinois; Find articles by Darya Mailhiot 1, *, † , Rebecca Turcios Rebecca Turcios , DVM 1 Animal Resources Center and Department of Surgery, University of Chicago, Chicago, Illinois; Find articles by Rebecca Turcios 1, † , Genevieve Remmers Genevieve Remmers , DVM, DACVP 2 IDEXX BioAnalytics, Colombia, Missouri; and Find articles by Genevieve Remmers 2 , Jennifer Brazzell Jennifer Brazzell , DVM, DACVP 2 IDEXX BioAnalytics, Colombia, Missouri; and Find articles by Jennifer Brazzell 2 , George P Langan George P Langan , DVM, DACLAM 1 Animal Resources Center and Department of Surgery, University of Chicago, Chicago, Illinois; Find articles by George P Langan 1 , Bridget M Clancy Bridget M Clancy , DVM, DACLAM 1 Animal Resources Center and Department of Surgery, University of Chicago, Chicago, Illinois; Find articles by Bridget M Clancy 1 , Kelly Kries Kelly Kries , DVM 3 University of Illinois College of Veterinary Medicine, University of Illinois at Urbana–Champaign, Urbana, Illinois Find articles by Kelly Kries 3 , Kerith R Luchins Kerith R Luchins , DVM, DACLAM, DACAW 1 Animal Resources Center and Department of Surgery, University of Chicago, Chicago, Illinois; Find articles by Kerith R Luchins 1 Author information Article notes Copyright and License information 1 Animal Resources Center and Department of Surgery, University of Chicago, Chicago, Illinois; 2 IDEXX BioAnalytics, Colombia, Missouri; and 3 University of Illinois College of Veterinary Medicine, University of Illinois at Urbana–Champaign, Urbana, Illinois * Corresponding author. Email: [email protected] † These authors contributed equally to this study. Received 2024 Sep 10; Revised 2024 Oct 2; Accepted 2026 Jan 12; Issue date 2026 Mar. © American Association for Laboratory Animal Science PMC Copyright notice PMCID: PMC13086188 PMID: 41692417 Abstract Repeated survival surgical oocyte collection in African clawed frogs ( Xenopus laevis ) is commonly performed to access high-quality, viable oocytes for research procedures and to reduce the number of animals used in research. Despite this common practice, there is no evidence-based limit for the total number of survival celiotomies that can be performed. To provide an improved reference, a retired colony of experimental frogs (n = 31) was euthanized for gross and histopathologic evaluation and then compared with 4 experimentally naive age-matched controls (n = 4). Experimental animals underwent 4-11 celiotomies (average 6 ± 2) and were between 180 and 1,646 days (average 448 ± 245.2 days) from their last surgery. Body weight, residual skin, and coelomic wall sutures were counted. Surgically incised skin and coelomic wall musculature were collected for histopathology. A cumulative skin score and a coelomic wall score were developed to reflect all histopathologic evidence of acute and chronic inflammation. The cumulative skin score was significantly predicted by the number of surgeries ( P = 0.003), but not predicted by the number of skin sutures remaining ( P = 0.14). The coelomic wall score was significantly predicted by the number of coelomic sutures placed ( P < 0.001) and the number of surgeries ( P < 0.001). Neither one of the histopathology scores correlated to the number of days since the last surgery ( P > 0.2). As one would expect, increasing the number of surgeries and coelomic sutures correlated with the histopathologic finding of a chronic inflammatory process in the skin. Although a specific recommendation for a maximum number of surgeries could not be discerned, this study provides an improved reference for Xenopus survival surgical oocyte collection underscoring the recommendation to use absorbable sutures within the body cavity and to remove all external nonabsorbable sutures 10-14 days postoperatively. Abbreviations and Acronyms: CSS, cumulative skin score; CWS, coelomic wall score; OM, osseous metaplasia Introduction For decades, African clawed frogs ( Xenopus laevis ) have been used as animal models in toxicology, vertebrate embryology, cellular biology, and biomedical research. In addition to their adaptability and longevity, one of the main advantages of Xenopus frogs as a research model is the reliable and precisely staged development of oocytes. 1 At our institution, investigators have performed surgical oocyte collection for experimental purposes. Despite the same surgical approach among Xenopus frogs, there can be differences in the quality and quantity of oocytes collected. Multiple factors such as temperature, age, and experimental techniques may contribute to this change, but individual variations also occur. 2 To reduce the number of animals used in research and capitalize on the quality of biologic materials collected, it is common for a single frog to undergo multiple major survival surgeries. Most institutions have IACUC policies in place to limit the number and frequency of surgeries for Xenopus frogs, but the policies are varied and largely unsupported by adequate data. To our knowledge, there have been no published, evidence-based recommendations on the maximum number of major survival surgeries for Xenopus frogs, and no literature providing insight on the effect multiple major survival surgeries can have on their anatomy and the development of pathology. The Guide for the Care and Use of Laboratory Animals emphasizes the continued monitoring and assessment of animals undergoing multiple survival surgeries. 3 However, observation of clinical signs indicative of pain and distress in amphibians is difficult, as pain experienced by amphibians is understudied and, by nature, difficult to define. 4 Furthermore, identifying objective markers of amphibian stress or overall welfare is notoriously difficult due to their stoic nature and their evolutionarily driven need to hide signs of pain or discomfort as a prey species. 5 There have been physiologic and neuroanatomical studies that attempt to describe pain response in nonmammalian vertebrates, and these studies suggest that the response is comparable to that of mammals. 4 – 7 Although amphibian pain and nociceptive capacity have been understudied, there still exists a fundamental understanding that unless the contrary is known or established, it should be considered that procedures that cause pain in humans may also cause pain in other animals. 3 While Xenopus frogs have been an invaluable and extremely popular source of biologic material in research for decades, very little evidence-based research exists that examines their ideal care, husbandry, and recovery from invasive surgical procedures. At our institution, surgical extraction of oocytes had been performed for years, and a colony was retired after the principal investigator opted to purchase oocytes from a vendor rather than maintaining a colony long-term. Using this opportunity, we examined histopathologic changes to the skin and coelomic muscle layers of X. laevis frogs that had undergone repeated celiotomies to develop a better understanding of histopathologic changes of skin and coelomic wall after repeated surgeries. In this study, we hypothesize that animals that receive repeated celiotomies will have greater histopathologic and gross anatomic changes with increased number of celiotomies performed. Materials and Methods Animals. The University of Chicago animal care program is accredited by AAALAC International, and all procedures performed for this study were approved by The University of Chicago IACUC. Housing was consistent with the Guide for the Care and Use of Laboratory Animals 3 ; therefore, animals were maintained in a room with controlled temperature (17-23 °C) and humidity (30%-70%), with a 12-hour light/12-hour dark cycle. The frogs were maintained single-housed or in pairs in 4 L of water in an automated continuous flow recirculating system (Marine Biotech, Beverly, MA) with 10% water changes daily. Whole PVC pipes (McMaster-Carr, Elmhurst, IL) were cut into half-PVC pipes and given to each 4-L housing cage. They were leeched for 7 days in reverse osmosis water prior to being used for enrichment. Water was filtered through a water purification system, adjusted to 400-1,200 μS, maintained at pH 7.0-7.75 and a temperature of 62-65 °F (17-22 °C). Conductivity, pH, and temperature were monitored daily and adjusted manually to maintain these parameters. Water underwent nitrogenous waste biofiltration, mechanical filtration of particulates, chemical charcoal filtration, and UV irradiation. Frogs were fed exclusively a diet of Nasco frog brittle 3 times a week (Nasco, Fort Atkinson, WI). Healthy, adult, research-bred female X. laevis (Nasco, Fort Atkinson, WI), between 3 and 7 years of age, were used for this study. Of the 35 animals included in this study, 31 were experimental frogs previously used for oocyte collection only. The remaining 4 were age-matched, experimentally naive control animals obtained from the same vendor as the experimental animals (Xenopus 1, Dexter, MI). Surgical oocyte extraction. All surgical procedures were performed by members of the investigator’s laboratory who were trained by senior members and veterinary staff. For oocyte collection, Xenopus frogs were anesthetized using 0.1% MS-222 (Syndel, Ferndale, WA). Depth of anesthesia was assessed by loss of righting reflex and negative response to stimuli. Once anesthetized, animals were moved to a separate 4-L container with a shallow amount of 0.1% MS-222, and a moistened, clean paper towel was placed over the pelvis and hindlimbs to prevent desiccation. The animals were placed in dorsal recumbency and the ventral aspect of the frog was rinsed for 5 seconds with a steady stream of 0.9% sterile saline aimed directly over the incision site. The surgeon used powder-free latex gloves and sterile surgical instruments throughout the procedure. Using a no. 11 blade and forceps, a 1- to 2-cm skin incision was made off-midline along the ventral abdomen in a caudomedial to a craniolateral orientation. A second, corresponding incision was made through the body wall running parallel with the coelomic musculature. Within the coelom, the ovary was identified, gently extracted, and the oocyte mass was excised. The body wall musculature and the skin were closed in 2 separate layers with 5-0 nonabsorbable nylon suture in a simple interrupted pattern. Historically, using absorbable suture material for the body wall closure along with nonabsorbable suture material for the skin closure resulted in an increased incidence of surgical site infections and dehiscence for this laboratory. For this reason, the animals in this study received nonabsorbable suture for body wall and skin closure. Animals were recovered in clean system water and observed until they were responsive to stimuli, after which they were monitored every other day for evidence of surgical site infection or inflammation. Postoperative analgesics were not used and omission was approved by the IACUC due to historical concern of interference with the research. Skin sutures were left to fall out with the animal’s natural shedding and not proactively removed. Of the 35 animals used for this study, 4 were unoperated controls and the remaining 31 animals underwent 4-11 celiotomies based on the experimental necessities and quality of the oocyte collection. Animals were grouped according to the number of surgeries they underwent ( Table 1 ). Animals were given at least 3 months of recovery before undergoing an additional celiotomy, and the contralateral side of the body was accessed for the following surgery. Table 1. Description of Study Groups Group No. animals in group No. surgeries Days since last surgery No. skin sutures present No. coelomic sutures present Control 4 0 N/A N/A N/A 1 9 4 407-857 0-5 7-13 2 8 5-6 340-527 0-2 11-19 3 11 7 352-1,646 0-2 10-19 4 3 10-11 180-325 0-2 18-24 Open in a new tab Experimental and control animals were organized into groups based on the number of celiotomies they underwent. Average days since their last surgery, number of external skin sutures and coelomic wall sutures at the time of necropsy were also recorded. Necropsy. All of the animals in this study were euthanized and examined over a period of 2 years. Most data were collected within a 3-month period due to the dissolution of an established research colony. Necropsies were performed and data were collected by individuals (R.T., K.K., B.M.C.) not blind to the experimental procedures performed on each frog. For their scheduled necropsy, animals were anesthetized with an immersion bath in buffered MS-222 (Syndel, Ferndale, WA) at 10 g/L until movement, response to stimuli, and pedal withdrawal had ceased. 8 External skin sutures were counted if they remained intact and grossly visible at the time of necropsy ( Figure 1A ). Body weights were recorded to the nearest 10th of a gram, after which animals were decapitated, under deep plane of anesthesia, using scissors as a method of euthanasia. Three animals underwent heart removal as their physical method of euthanasia prior to any analysis; therefore, their body weights were excluded from data analysis (n = 32 for body weights). Figure 1. Open in a new tab Images of Gross Anatomy Taken at Necropsy. (A) An experimental frog that received 11 surgeries and had 2 residual skin sutures was observed. Healed surgical incisions and scars are visible from previous surgeries. (B) The same animal has the skin reflected so individual coelomic wall sutures could be counted. After these measurements, the skin was incised along midline from caudal to cranial and gently reflected laterally. Body wall sutures were counted if they remained intact and grossly visible ( Figure 1B ). Skin samples containing all surgical incisions were collected from both right and left sides of the ventrum and placed in 10% neutral buffered formalin (Leica Biosystems, Richmond, IL). A midline incision was made into the linea alba, and samples of body wall were collected to include all previous surgical incisions. The coelomic cavity was inspected for the presence of any adhesions, and any tissues that appeared grossly abnormal (eg, discoloration, distension) were collected for histopathologic analysis. Once all measurements and tissues were collected, samples were submitted to IDEXX BioAnalytics (Columbia, MO) for analysis and scoring. Histopathology. Groups were assigned based on the number of surgeries performed ( Table 1 ). For each animal that was submitted, 2 representative skin and coelomic wall sections were trimmed, paraffin-embedded, stained with hematoxylin and eosin, and evaluated by light microscopy by a board-certified anatomic pathologist (G.R.). Histopathological changes in the skin and coelomic wall were independently microscopically evaluated and graded for each animal. All animals were examined for the following microscopic changes in the skin: epidermal hyperplasia, epidermal and dermal histiocytic and/or lymphocytic infiltrates, dermal edema, dermal granulation tissue formation, decreased and/or dilated mucus glands, decreased granular glands, mineral deposition, and osseous metaplasia. Similarly, each animal was examined for the following microscopic changes in the coelomic wall: histiocytic infiltrates, fibrosis, and granulation tissue. Each of these microscopic changes were graded individually, as to severity, using a grading system whereby 0 = no significant change, 1 = minimal change, 2 = mild change, 3 = moderate change, and 4 = severe change ( Table 2 ). International Harmonization of Nomenclature and Diagnostic Criteria 9 were used to guide for nomenclature of the microscopic changes described. Table 2. Histopathology Scoring System Score Grade Histologic features 0 No significant change • Normal skin and/or coelomic wall architecture; no histologic abnormalities observed 1 Minimal • Lesions focal, rarely multifocal • Very slight edema • Minimal/low-density granulation tissue or fibrosis • Minimal epidermal hyperplasia • Rare/subtle reduction in granular/mucus glands • Sparse mononuclear inflammation • Rare mineral deposition or osseous metaplasia 2 Mild • Lesions focal to multifocal • Low-grade edema • Mild/minor granulation tissue or fibrosis • Mild epidermal hyperplasia • Slight reduction in granular/mucus glands • Scattered/low-density mononuclear inflammation • Occasional mineral deposition or osseous metaplasia 3 Moderate • Lesions focally extensive to multifocal • Mild to moderate edema • Prominent/noticeable granulation tissue or fibrosis • Moderate epidermal hyperplasia • Clear reduction in granular/mucus glands • Moderate/pronounced mononuclear inflammation • Mineral deposition and osseous metaplasia more frequent 4 Severe • Lesions multifocal, coalescing • Moderate to marked edema • Extensive/advanced granulation tissue or fibrosis • Severe/high-grade epidermal hyperplasia • Marked/near-complete loss of granular/mucus glands • Dense/intense mononuclear inflammation • Widespread mineral deposition and osseous metaplasia Open in a new tab The grading system depicts how each microscopic change was individually graded as to severity. International Harmonization of Nomenclature and Diagnostic Criteria were used to guide for nomenclature of the microscopic changes described. Each animal was given a cumulative skin score (CSS) and coelomic wall score (CWS). The CSS is the sum of all the microscopic changes assessed and graded in the skin. A CWS is the sum of all the graded microscopic changes observed in the coelomic wall. Statistical analysis. Data were analyzed statistically using R 4.3.2 software (R Core Team, Vienna, Austria). Kruskal-Wallis tests were used to examine statistical differences between experimental groups and the control groups. Subsequent Wilcoxon rank sum tests were performed to further assess statistical significance. A P value of <0.05 was considered statistically significant. A linear regression analysis was performed to identify correlates between histopathology scores and the number of surgeries, histopathology scores and the days since surgeries, and histopathology scores and the number of residual sutures. Results Necropsy. Experimental animals underwent anywhere from 4 to 11 celiotomies (average 6 ± 2) and were between 180 and 1,646 days (average 448 ± 245.2 days) from their last surgery. Average body weight was 155.8 ± 23.82 g for the animals that were weighed prior to a secondary method of euthanasia. Experimental animals averaged 0.6 ± 1.12 residual skin sutures at the time of necropsy, and the average number of residual coelomic sutures was far greater, with 13.6 ± 3.61 counted at necropsy. The average number of residual skin sutures in all experimental groups (control group = 0, group 1 = 0.8, group 2 = 0.6, group 3 = 0.4, group 4 = 1.3) was less than the number of residual coelomic sutures (control group = 0, group 1 = 10.8, group 2 = 14.4, group 3 = 13.6, group 4 = 20.0) ( Table 1 ). Upon inspection of the coelomic cavity, only one animal that had undergone 11 surgeries had an adhesion between the small intestine and the right ovary; no abnormalities were noted on histopathology. The remaining animals had normal gross anatomy. Histopathology. Epidermis. Microscopic examination of the epidermis of experimental animals revealed variable minimal to mild hyperplasia and minimal lymphocytic infiltrate. The epidermal hyperplasia and lymphocytic infiltrate were not correlated to the number of celiotomies. Dermis. Microscopic examination of the dermis and subcutis of experimental animals revealed histiocytic infiltrates accompanied by granulation tissue and fibrosis surrounding suture material (suture granulomas), mild to moderate dermal edema most prominent near suture granulomas and areas of epidermal hyperplasia, and minimal to mild decreases in mucus glands and granular glands. The magnitude of the decrease in granular glands was greater than the magnitude of decrease in mucosal glands. The decrease in mucus and granular glands was not evenly distributed in the dermis of the experimental animals and was often associated with areas of increased edema, suture granulomas, and granulation tissue/fibrosis, interpreted as samples of skin closely adjacent and/or at the site of celiotomies. Osseous metaplasia was observed more frequently in the dermis of experimental animals but was present in the skin of one animal of the control group. The osseous metaplasia was randomly distributed in the dermis and did not appear to be associated with suture granulomas or other pathologic findings. Dermal mineralization (Eberth-Katschenko layer) was evaluated to determine whether the experimental animals had an increase or decrease in mineralization given the presence and potential increase in osseous metaplasia. There was no difference in dermal mineralization between the experimental and control animals. A representative photomicrograph of osseous metaplasia is shown in Figure 2 . Figure 2. Open in a new tab Representative Osseous Metaplasia. The figure depicts osseous metaplasia (OM) identified in the dermis. The lesion was observed more frequently in the dermis of experimental animals, but was present in the skin of one animal of the control group. Hematoxylin and eosin staining; original magnification, ×10; scale bar, 300 μm. Photomicrographs of normal control epidermis and dermis and cutaneous pathologic findings in experimental animals are presented in Figure 3A-C . Figure 3. Open in a new tab Xenopus laevis . The figures depict ventral coelomic skin (A-C) and abdominal wall (D-F). Images depict histologically unremarkable control animal tissues (A, D), mild changes in celiotomy animal tissues (B, E), and moderate to marked changes (C, F) in celiotomy animal tissues. (A) Normal ventral coelomic skin of X. laevis . (B) There is a minimal decrease in mucus glands (mg) and mild decrease in granular glands (gg). There is mild dermal fibrosis (f). (C) There is moderate epidermal hyperplasia with low numbers of apoptotic keratinocytes and mild mononuclear inflammation (in). There is a decrease in mucus glands, and granular glands are absent. There is moderate dermal edema (ed). (D) Normal abdominal wall skeletal muscle of X. laevis . (E) There is minimal to mild fibrosis (f) surrounding suture residua (s). (F) There is marked mononuclear to heterophilic inflammation (in) surrounding suture residua (s). Hematoxylin and eosin staining: (A-C) original magnification, ×20; scale bar, 20 μm; (D-F) original magnification, ×40; scale bar, 40 μm. de, dermis; ep, epidermis; gg, granular gland; mg, mucus gland; sm, subcutaneous muscle. Coelomic wall. Microscopic examination of the coelomic wall revealed histiocytic infiltrates associated with skeletal muscle degeneration/necrosis, granulation tissue, and fibrosis. Histiocytic infiltrates were often associated with clear space or suture residua and were interpreted to be suture granulomas. The number of suture granulomas seen on histopathology was dependent on the number of sutures present in the selected sections examined. The histiocytic infiltrate, fibrosis, and granulation tissue were minimal to mild in groups 1, 2, and 3, and moderate in group 4. As depicted in Table 3 , all experimental groups had higher histopathology scores compared to the control group. However, there were no statistically significant differences between the experimental groups. Photomicrographs of normal control coelomic wall and coelomic wall pathologic findings in experimental animals are presented in Figure 3D-F . Table 3. Summary of Histopathology Scores per Experimental Group Group No. surgeries Cumulative skin pathology score Coelomic wall pathology score Control 0 1.5 (0.75, 2.5) 0 (0,0) 1 4 14 (12, 16) 5 (4, 5) 2 5-6 11.5 (11, 15) 3.5 (3, 6) 3 7 12 (10, 14) 6 (5, 8) 4 10-11 13 (13, 14) 7 (6.5, 9.5) Open in a new tab Data for skin and coelomic wall histopathology scores are presented as: median score (25th percentile, 75th percentile). Cumulative Skin Score (CSS). Cumulative Skin Scores are presented in Table 3 . Linear regression results are displayed in Table 4 . As modeled by linear regression, the results indicate that each additional surgery led to a 0.86 (95% CI [0.32, 1.40]; P value 0.001) increase in CSS. Each additional skin suture increases CSS by 1.10 (95% CI [−0.38, 2.60]; P value 0.14), but this increase was not significant. CSS decreased by 3.0 points (95% CI [−11, 4.6]; P value 0.43) with the increasing number of days since the last surgery, but this was also not significant. Each experimental group differed significantly from the control group, and no statistically significant differences were found between the 4 experimental groups ( Table 5 ). Table 4. Results of Linear Regression Analysis Characteristic No. surgeries No. sutures in tissue layer Days since last surgery β 95% CI P value β 95% CI P value β 95% CI P value CSS 0.86 0.32, 1.3 0.003 1.1 −0.38, 2.6 0.14 −3.0 −11, 4.6 0.43 CWS 0.76 0.51, 1.0 <0.001 0.31 0.17, 0.45 <0.001 −3.9 −9.5, 1.6 0.2 Open in a new tab The analysis modeled associations between Cumulative Skin Score and Coelomic Wall Score and number of surgeries, number of residual sutures and days since last surgery.Abbreviations: CSS, cumulative skin score; CWS, coelomic wall score. Table 5. Results of Kruskal-Wallis Test for Cumulative Skin Score and Coelomic Wall Score Characteristic Overall K-W P value 0 compared with 1 P value 0 compared with 2 P value 0 compared with 3 P value 0 compared with 4 P value K-W 1-4 P value CSS 0.008 0.007 0.008 0.005 0.050 0.246 CWS 0.001 0.006 0.007 0.004 0.032 0.026 Open in a new tab Each experimental group was compared against the control group. All experimental groups differ significantly from the control group. No significance is seen between experimental groups. Abbreviations: CSS, cumulative skin score; CWS, coelomic wall score; K-W, Kruskal-Wallis test. Coelomic Wall Score. Coelomic wall histopathology scores are presented in Table 3 . Linear regression results are displayed in Table 4 . As modeled by linear regression analysis, the coelomic wall histopathology score increased by 0.31 points (95% CI [0.17, 0.45]; P value <0.001) as the number of coelomic sutures increased and increased by 0.76 (95% CI [0.51, 1.0]; P value <0.001] with the number of surgeries. The coelomic wall histopathology score decreased by 3.9 points (95% CI [−9.5, 1.6], P value 0.2) and was not predicted by the number of days since the last surgery. All experimental groups had higher histopathology scores relative to the control group. However, there was no statistically significant difference between the experimental groups. Discussion Amphibian skin performs many critical homeostatic functions, including osmoregulation, respiration, and protection from microbial pathogens and predation. 10 – 12 Research procedures that significantly affect the skin and underlying coelomic wall musculature may result in systemic stressors that could affect animal welfare. Similar to mammalian species, amphibian tissues subjected to repeated trauma or injury can undergo inflammation, tissue proliferation, and remodeling of tissue architecture. 11 , 13 , 14 While early life stages of X. laevis frogs have been demonstrated to have better skin regenerative properties than that of mammals, postmetamorphic X. laevis adults are still capable of experiencing the same pathologic changes seen in mammals, and with age these regenerative properties decline. 13 , 14 Despite this, it remains unclear just how much repeated surgical insult can affect tissue architecture. In this study the dissolution of a colony of experimental X. laevis frogs was used as an opportunity to study these changes. At the time of necropsy, experimental animals were at least 5 months past their last surgery, and animals had undergone between 4 and 11 surgeries. Despite being months past their last surgery, some animals had visible skin sutures, and all animals in this study had grossly visible coelomic wall sutures ( Table 1 ). Only one animal that had 11 celiotomies had an adhesion of coelomic viscera, which is a potential sequela associated with coelomic surgeries. 15 The severity of the CSS did not correlate to the number of days since the last surgery. This suggests a different primary driver for the development of pathology. Histopathologic findings were observed in the skin and coelomic wall musculature of all experimental animals. Thus, it was not surprising that there was a strong correlation between increasing CSS and the number of surgeries. From this, we can conclude that repeated surgeries result in a greater likelihood of developing cutaneous and coelomic wall pathology. The CSS of all treatment groups was significantly different from the control group; however, no significant difference in CSS was identified between treatment groups. The number of residual skin sutures counted did not correlate with CSS. This was not altogether surprising, as skin sutures that were placed were left to naturally dislodge via periodic shedding of skin. Turnover of the stratum corneum of the amphibian epidermis takes anywhere from 1 to 14 days. 11 , 12 At the time of this study, enough time had passed to allow for some skin sutures to dislodge, but there were some individual animals that still possessed skin sutures over a year after their last surgery. The persistence of suture could be attributed to partial suture placement into the dermis, which does not naturally turn over and slough like epidermis. The epidermis of X. laevis in health is 5-6 cell layers thick, 11 making strictly epidermal suture placement difficult and the likelihood of dermal suture placement likely. It is recommended that skin sutures placed be removed at 10-14 days postsurgery to ensure that all sutures—even those accidentally left in the dermis—are removed in a timely fashion. 16 In this study, the number of skin sutures placed at the time of surgery was not known; however, future studies could examine whether the number of skin sutures initially placed could contribute to the degree or severity of skin histopathology changes observed. There has only been one study that has examined the effect that different suture materials have on amphibian skin healing and pathology. 16 From this study, all 5 suture materials (silk, monofilament nylon, polydioxanone, polyglactin, and chromic gut) caused greater inflammation than allowing a stab incision to heal by second intention. Of the 5 used, nonabsorbable monofilament nylon appears to be the best suture material to use on X. laevis skin, as it resulted in the least histologic tissue reaction and dehiscence. Similar inflammatory responses to suture materials have been reported in numerous species of reptiles, which suggests that further studies on optimizing suture material preference for amphibians and reptiles are needed. 16 , 17 In amphibians, physical evidence of inflammation seen following celiotomies includes dermal, subcutaneous, and epidermal edema, as well as hyperplasia. 16 Animals appeared to show decreases in mucosal and granular glands, which is a reported sequelae to skin insult in amphibians. 12 , 18 Granular glands are integral to the frogs’ innate immunity, as they are primarily responsible for developing antimicrobial peptides, and mucosal glands assist in maintaining a physical mucosal barrier to protect the skin from desiccation and pathogens. 11 Therefore, a reduction in their numbers could impact a frog’s overall ability to maintain homeostasis. In this study, the granular glands appear to be much more greatly impacted than the mucosal glands. This may suggest that either the mucus glands are more resistant to the dermal necrosis caused by celiotomy, or the granular glands may be more sensitive to these changes. Epidermal hyperplasia was not observed in the control group, but it was present in experimental groups, and it did not appear to be correlated to the number of celiotomies. The presence of dermal mineralization did not appear to be correlated to celiotomies, as there was one animal in the control group expressing the lesion as well. It is possible that dermal mineralization is due to an age-related or incidental background finding. In addition, the increase in osseous metaplasia could be the result of dermal necrosis secondary to celiotomy, while the osseous metaplasia seen in the control animal may have been incidental. Therefore, the correlation between osseous metaplasia and experimental procedures is unclear. Osseous metaplasia has been recorded in reptiles, fish, and mammals, including humans, following chronic inflammation or repeated trauma. 19 – 23 Few medical interventions necessitate multiple incisions over the same area, so case studies describing subsequent pathology are limited. Humans who have had multiple laparotomies in this fashion report painful, swollen scars, with occasional mention of ectopic calcification and osseocartilaginous metaplasia. 24 , 25 Further research is required to help interpret the significance of this finding. Analysis of the coelomic wall histopathology scores revealed increasing scores with both the number of residual sutures placed and the number of surgeries. Evidence of chronic inflammatory processes (fibrosis and granulation tissue) suggest frogs were either subjected to a constant low-level of foreign material, exposed to recurrent episodes of acute inflammation secondary to surgical incision, or both. Most of the changes in coelomic wall musculature were aggregated around the nonabsorbable sutures and interpreted to be suture granulomas. Chronically implanted nonabsorbable nylon suture in mammalian abdominal wall musculature has been associated with histiocytic and inflammatory cell infiltrate up to 12 months after implantation, with eventual fibrotic entrapment of the suture material within 2 years. 26 When comparing nylon, catgut, polydioxanone (PDS), and polygalactic sutures in mammalian skeletal muscle, nylon elicits the strongest inflammatory response postimplantation with eventual attempts for fibrous encapsulation by tissue. 27 Nylon sutures were found in the coelomic musculature of every experimental animal at the time of necropsy. In fact, one animal had coelomic sutures intact 1,646 days (4.5 years) after the last surgery. The use of nonabsorbable sutures for coelomic wall musculature appears to illicit a chronic foreign-body presence; therefore, the use of nonabsorbable suture for muscle closure is not recommended. We recognize that with an opportunistic study that received animals at the dissolution of a laboratory, the low n was dictated by the availability of the situation. This led to the use of nonparametric statistical methods. Thus, the nonsignificant result of this project may be due to it being underpowered. Therefore, we cannot definitively claim the absence of an effect, as the lack of significance might be due to the study’s inability to detect a real effect. We did, however, attempt to compensate for this limitation by using appropriate scoring systems and optimizing the precision of data collection (such as careful tissue sectioning and analysis) to improve the statistical power of nonparametric testing and help mitigate the risk of false negatives (type II errors) in this study with negative findings. Future studies could examine animals that underwent similar surgical procedures between 1 and 4 survival surgeries or add additional animals in each experimental group to improve statistical significance. Given their ubiquity and utility in laboratory animal medicine, it is imperative to develop an understanding of the effects that repeated celiotomies may have on the health and welfare of these animals. With this foundational information, institutions can better develop evidence-based guidelines for amphibian celiotomies. This study determined that increasing the number of survival celiotomies does affect tissue architecture and leads to histopathologic changes. For institutions with similar methodology for surgical oocyte collection, areas of refinements for this procedure include the use of absorbable sutures to close coelomic wall musculature and removal of skin sutures 10-14 days after celiotomy. Future areas of study could focus on identifying optimal suture preferences for amphibian surgeries, as most suture materials available are designed to be dissolved within a mammalian body. Although this study identified histopathologic patterns of change associated with repeated surgeries, a greater understanding of amphibian skin biology is needed to better elucidate the findings observed, specifically, to identify what significance osseous metaplasia has on X. laevis skin and what significance a change in granular or mucosal glands has on animal health. 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