Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido: 04/06/2025 Aceptado:19/08/2025 Publicado: 24/09/2025 hps://doi.org/10.52973/rcfcv-e35650 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 4 Revista Cienfica, FCV-LUZ / Vol. XXXV hps://doi.org/10.52973/rcfcv-e35717 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Preparaon and clinical applicaon of thrombocyte-leukocyte rich plasma in green turtles (Chelonia mydas). Technical note Preparación y aplicación clínica de plasma rico en trombocitos y leucocitos en la tortuga verde (Chelonia mydas). Nota técnica Agustina Algorta 1 , Fernando Fumagalli 2 , Virginia Ferrando 3 , Victoria Sorriba 4 , Adrián Carzoli 4 , Kevin Yaneselli 1* ¹Unidad de Inmunología e Inmunoterapia, Departamento de Patobiología, Facultad de Veterinaria, Universidad de la República (Udelar), PC:13000, Montevideo, Uruguay. ²Unidad de Clínica Semiológica, Departamento de Clínicas y Hospital Veterinario, Facultad de Veterinaria, Universidad de la República (Udelar), Montevideo, Uruguay. ³ONG Karumbé, Conservación de tortugas Marinas del Uruguay, Uruguay. ⁴Unidad de Imagenología, Análisis Clínicos y LEMA, Departamento de Clínicas y Hospital Veterinario, Facultad de Veterinaria, Universidad de la República (Udelar), Montevideo, Uruguay. *Autor correspondencia: kevin.yaneselli@fvet.edu.uy ABSTRACT Traumac injuries in turtles pose a significant challenge for veterinarians, requiring extended follow-up and rehabilitaon periods. With convenonal healing methods oſten yielding limited results, the realm of regenerave medicine in reples and amphibians has offered promising alternaves. Among these, the ulizaon of thrombocyte-leukocyte rich plasma and analogous hemocomponents akin to platelet-rich plasma in mammals has garnered aenon. Hence, the objecve of this study was to explore therapeuc hemocomponents in reples, specifically thrombocyte-leukocyte rich plasma, and its applicaon in a clinical case involving Chelonia mydas. Autologous thrombocyte-leukocyte rich plasma was prepared via a density gradient centrifugaon process to isolate the buffy coat and concentrate thrombocytes and leukocytes in the final formulaon. The selected clinical case involved a green turtle (Chelonia mydas) undergoing rehabilitaon at the Karumbé center, exhibing lesions and abscesses in the jaw and flipper regions. Treatment entailed abscess removal, anbioc administraon, and the applicaon of gelified thrombocyte-leukocyte rich plasma in the affected areas. Results demonstrated a recovery rate of 20.8% for thrombocytes and leukocytesin the thrombocyte- leukocyte rich plasma preparaon, which effecvely gelled upon applicaon. Regarding the clinical case evoluon, aſter 2 months of follow-up, an improvement in wound healing was observed. In conclusion, this is the first report of the preparaon of thrombocyte-leukocyte rich plasma in Chelonia mydas and its applicaon in a clinical case with favorable progression. However, more cases are needed in this species and type of lesion to obtain conclusive results on its beneficial effects in turtles. Key words: Marine turtle; thrombocytes; blood plasma; wound; abscess RESUMEN Las lesiones traumácas en tortugas pueden ser frustrantes para los veterinarios y requerir largos empos de seguimiento y rehabilitación. Ante la limitante de cicatrización han surgido novedosas opciones desde la medicina regenerava en reples y anfibios como el uso de plasma rico en trombocitos y leucocitos y otros hemocomponentes comparables con el plasma rico en plaquetas en mamíferos. Por ello, el objevo del presente trabajo fue desarrollar hemocomponentes terapéucos en réples como el plasma rico en trombocitos y leucocitos y su aplicación en un caso clínico de tortuga verde (Chelonia mydas). La preparación de plasma rico en trombocitos y leucocitos autólogo se preparó mediante un procedimiento de centrifugación en gradiente de densidad para la separación de la capa flogísca y concentración de trombocitos y leucocitos en la formulación final. El caso clínico seleccionado fue una tortuga verde (Chelonia mydas) que se encontraba en rehabilitación en el centro Karumbé. Presentaba lesiones y abscesos en la zona de la mandíbula y en las aletas pectorales. El tratamiento consisó en la remoción de los abscesos y uso de anbiócos, a lo que se agregó depósito del plasma rico en trombocitos y leucocitos gelificado en las zonas tratadas. Los resultados mostraron que se pudo recuperar un 20,8 % de los trombocitos y leucocitos para el preparado de plasma rico en trombocitos y leucocitos y gelificar al momento de aplicación. En cuanto a la evolución del caso clínico, a los 2 meses de seguimiento, se observó una mejoría en la cicatrización de las heridas. En conclusión, fue posible por primera vez reportar la preparación de plasma rico en trombocitos y leucocitos en Chelonia mydas y su aplicación en un caso clínico que tuvo una evolución favorable. Asimismo, es necesaria mayor casuísca en esta especie y po de lesión para obtener resultados concluyentes sobre su efecto beneficioso en tortugas. Palabras clave: Tortuga marina; trombocitos; plasma sanguíneo; herida; absceso.

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION The green turtle (Chelonia mydas) is a marine species that feeds and develops in tropical and subtropical oceans, with adults predominantly residing in the South Atlanc Ocean [1 , 2 ,[3]. It is listed as an endangered species on the Internaonal Union for Conservaon of Nature (IUCN) Red List [4]. In Uruguay, the non- governmental organizaon Karumbé has operated a Sea Turtle Rehabilitaon Program connuously since 2000 [5]. The capture, rehabilitaon, and reintroducon of wild animals are common pracces in marine wildlife rescue centers. Reducing the length of stay in these facilies is crucial to ensure animal welfare and opmize resources. However, traumac injuries—caused by predaon or fishing gear—are common in both capve and wild turtles. These injuries, affecng soſt ssues or bone, oſten require prolonged recovery, monitoring, and treatment, delaying rehabilitaon and complicang reintroducon into the wild [6 , 7 ,8]. Therefore, it is necessary to advance therapeuc methods that accelerate healing and shorten recovery periods. One emerging opon is the use of hemocomponents to promote wound healing in turtles. This approach is based on studies in mammals demonstrang the benefits of platelet-rich plasma (PRP) and platelet-rich fibrin, both of which concentrate growth factors and cytokines to enhance ssue regeneraon. These blood derivaves have gained popularity in both human and veterinary medicine as cost-effecve and safe therapeuc opons [9 , 10 , 11]. In turtles, the applicaon of thrombocyte-leukocyte rich plasma (TLRP) was first reported in 2015 for treang skin and bone injuries in Testudo spp., showing favorable clinical outcomes [12]. Similarly, Chen and Deem [7] reported encouraging results using hemocomponents to treat a nasal p wound in a terrestrial tortoise (Indotestudo elongata) aſter convenonal anbioc therapy and debridement failed. In reples, TLRP has also been used to treat chronic oral cavity disorders in a python and a chameleon, with evidence suggesng potenal regenerave benefits [10]. However, therapeuc use of hemocomponents in reples and amphibians remains limited, though inial findings support further exploraon in herpetological medicine. Thus, the objecve of this study was to develop therapeuc hemocomponents, specifically TLRP, in turtles sea and evaluate their clinical applicaon in a case involving a green turtle (Chelonia mydas). MATERIALS AND METHODS TLRP preparaon Autologous TLRP was prepared following the density- gradient centrifugaon protocol described by Di Ianni et al. [12]. Briefly, 13 mL of blood were drawn from the external jugular vein (cervical venous sinus) using a sterile syringe preloaded with 3.8% sodium citrate as an ancoagulant. The sample was centrifuged at 250 g (Thermo IEL CL30R centrifuge, USA) for 15 min to separate the plasma, and the cellular fracon was resuspended in an equal volume of phosphate-buffered saline (PBS). This diluted fracon was layered onto a 1:1 lymphocyte separaon medium (density: 1077 g/mL) and centrifuged at 150 g for 20 min. The thrombocyte- leukocyte (T-L) fracon was collected and washed with PBS. Finally, T-L cells were resuspended in 20–25% of the original plasma volume (FIG. 1). T-L counts were performed using a Neubauer chamber (Marienfeld, Neubauer bright-line, Germany) on both whole blood and TLRP samples. The recovery percentage of T-L was calculated using the formula: Cell recovery (%) = (100 × T-Lr) / T-Lt. Where T-Lt is the total number of T-L cells in whole blood and T-Lr is the number recovered in the final product. A representave blood smear from the treated individual is shown in FIG. 2. The procedure yielding a T-L count of 22,400 cells/μL in whole blood and 27,060 cells/μL in the TLRP, corresponding to a 20.8% recovery of T-L cells and represented a 1.2-fold concentraon (120.8%) relave the baseline. The prepared TLRP was stored frozen at −20 °C unl use. FIGURE 1. Schemac representaon of the preparaon of thrombocyte-leukocyte rich plasma (TLRP). The process includes blood collecon, inial centrifugaon, plasma sepa- raon, cellular fracon collecon, and separaon by density gradient to concentrate the inflammatory layer (buffy coat), ulmately resulng in TLRP formulaon FIGURE 2. Microscopic image (1000×) of thrombocytes, leukocytes, and erythrocytes from the blood smear of the treated specimen used for the preparaon of thrombocyte-leuko- cyte rich plasma (TLRP). Black arrow: lymphocyte; light blue arrow: thrombocyte; green arrow: monocyte; red arrow: erythrocyte 2 of 4

Thrombocyte-leukocyte rich plasma in Chelonia mydas / Algorta et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Therapeuc applicaon of TLRP A clinical case was selected involving cutaneous lesions and abscesses in the neck and forelimb regions of a green turtle undergoing rehabilitaon at the Karumbé civil associaon (collecon and capture permits: DINAMA-MVOTMA 4/2018; DINARA-MGAP 195/2018 and 50/2019). The most likely cause of the lesions was a skin wound that became secondarily infected. Such injuries are oſten the result of trauma caused by contact with rocks or abrasions sustained during stranding events. The turtle weighed 3.35 kg and measured 31 cm in standard curved length. Under general anesthesia, surgical debridement was performed to remove the abscesses, with each wound site cleaned to healthy margins. Anesthesia was induced with intravenous propofol (3–5 mg/kg) unl the desired anesthec depth was achieved. A 4.5 mm endotracheal tube was placed, and anesthesia was maintained with 3% isoflurane in a low- flow oxygen open-circuit system. Tramadol (3–5 mg/kg) was administered for analgesia. To enable gelaon and enhance retenon at the applicaon site, 10% (W/V) calcium chloride was added to the TLRP ten min prior to administraon. The hemocomponent was injected perilesionally and intralesionally (FIG. 3). In larger defects created by cureage, gelified TLRP was applied directly by allowing it to solidify within the syringe and then placing it into the wound bed, thus forming a three- dimensional matrix. Nylon film was used to wrap and retain the gelified TLRP at the applicaon sites. Clinical follow-up included a one-month anbioc therapy regimen with gentamicin at 6 mg/kg every 72 h, based on results from intralesional cultures. All procedures were conducted in accordance with the experimental protocol approved by the Ethics Commiee (CEUA) of the Facultad de Veterinaria, Universidad de la República, Uruguay (Protocol No. 1421). FIGURE 3. Therapeuc applicaon of thrombocyte-leukocyte rich plasma (TLRP). A: TLRP prepared for applicaon. B: Applicaon of TLRP aſter acvaon with calcium chloride to achieve gelificaon, improving retenon at the treatment site RESULTS AND DISCUSSION The recovery rates obtained in this study (20.8%) were lower than those reported by Di Ianni et al. [12], who documented a 48.9% recovery rate. This discrepancy could be aributed to differences in the resuspension volume used in the final formulaon, which in their case achieved a 3–4× concentraon of T-L cells relave to baseline. Despite this, using the same protocol, we previously obtained a 4× concentraon in another clinical case involving Chelonia mydas (unpublished data), supporng the protocol’s effecveness in this species. As highlighted in both mammalian and herpetological literature [10 , 12 , 13], variaons in T-L recovery are influenced by individual biological variability and methodological nuances, such as sample handling and centrifugaon parameters. However, the true therapeuc value of hemocomponents such as PRP and TLRP does not solely depend on cell concentraon but also on the quality and viability of bioacve factors released during applicaon. Although some studies in mammals suggest a possible dose-dependent effect of PRP, the opmal therapeuc concentraon has not yet been clearly established in either mammals or reples [12 , 13 , 14]. Therefore, further studies are necessary to determine effecve concentraon thresholds and to correlate them with measurable regenerave outcomes in turtles and other reple species. Clinical case progression Two days’ post-treatment, improvement was observed in the ventral mandibular wounds. Aſter two weeks, significant improvement was noted on the right side upon visual inspecon, while the leſt side showed moderate improvement. Lesions on the forelimbs also improved, with the right-side lesion reappearing in smaller size compared to pre-treatment, and only minor abrasions remaining on the leſt side. A two-month follow-up confirmed connued healing progression with no signs of infecon or adverse reacons (FIG. 4). FIGURE 4. Clinical case: Presence and progression of cutaneous lesions. A–C: Mandibular lesions; D–F: Abscessed lesions in the leſt forelimb. A and D: Before treatment. B and E: 2 days post-treatment. C and F: 21 days post-treatment Traumac injuries in turtles are among the most challenging and prolonged condions to treat, oſten resulng in frustrang outcomes when healing fails. This reality has driven the search for alternave therapies, such as hemocomponents, which are emerging as safe and cost-effecve treatment opons. In this context, this study represents one of the first documented clinical applicaons of TLRP in a green turtle (Chelonia mydas). However, literature on TLRP and related hemocomponents in herpetological medicine remains scarce [10 , 12]. Nonetheless, the clinical case presented here showed a posive response to treatment, with enhanced wound healing. This aligns with 3 of 4

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico findings reported by other authors who have observed favorable outcomes following TLRP or similar hemocomponent therapy in turtles [7],[12] and in other reple species [10 , 15 , 16]. However, despite these encouraging results, the study has some limitaons, as it involved only a single clinical case and lacked precise quantave assessment of wound healing during follow-up. CONCLUSION For the first me, the preparaon of TLRP in Chelonia mydas was successfully reported. TLRP was applied effecvely in a clinical case without adverse reacons and was associated with appropriate wound healing. To further elucidate its therapeuc effects, future studies should include a larger number of clinical cases and incorporate complete hematological and biochemical evaluaons to beer correlate physical recovery with the animal’s physiological status Conflict of Interest The authors declare that there are no conflicts of interest associated with this work. Authors’ contribuons All authors contributed equally to the concepon and wring of the manuscript. All authors crically revised the manuscript and approved the final version BIBLIOGRAPHIC REFERENCES [1] López-Mendilaharsu M, Gardner SC, Seminoff JA, Riosmena-Rodriguez R. Idenfying crical foraging habitats of the green turtle (Chelonia mydas) along the Pacific coast of the Baja California peninsula, Mexico. Aquat. Conserv.: Mar. Freshw. Ecosyst. [Internet]. 2005; 15(3):259–269. doi: hps://doi.org/cpzqgt [2] Naro-Maciel E, Becker JH, Lima EHSM, Marcovaldi MA, DeSalle R. 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