The effect of acute cold ischemia duration on testicular tissue

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Kestel, H. Deliktas, I. Akarken, B. Sahin, Y. Dere, E. Saruhan, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7871593/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Feb, 2026 Read the published version in BMC Urology → Version 1 posted 10 You are reading this latest preprint version Abstract Purpose Testicular cancer primarily affects men aged 15–34. Partial orchiectomy has recently emerged as a viable testis-sparing surgical technique. However, prolonged spermatic cord clamping may induce ischemia-reperfusion (I/R) injury due to elevated reactive oxygen species (ROS) production. This study aimed to evaluate the protective role of testicular hypothermia against I/R injury and to determine the maximum tolerable cold ischemia duration in a rat model. Spermatogenesis was assessed using the Johnsen scoring system. Materials and Methods Thirty sexually mature Wistar albino rats were divided into four groups. Following scrotal incision and testicular cooling with ice (2–4°C), Group I (Sham, n = 6) underwent orchiectomy. Groups II, III, and IV (n = 8 each) were subjected to 10, 20, and 30 minutes of ischemia, respectively, followed by 1-hour reperfusion. Biochemical parameters (TNF-α, IL-6, TAS, TOS, OSI) and histopathological evaluations, including Johnsen scoring, were conducted. Results Inflammatory markers and oxidative stress indices increased significantly with prolonged ischemia, while antioxidant capacity and Johnsen scores declined in a time-dependent manner. The most severe biochemical and histological impairments were observed in the 30-minute group. Conclusion Cold ischemia exceeding 20 minutes leads to marked testicular damage, despite hypothermic protection. Findings emphasize the need to minimize ischemic duration during testis-sparing surgeries. ischemia-reperfusion injury hypothermia partial orchiectomy testicular torsion oxidative stress Johnsen score Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Testicular cancer predominantly affects men between the ages of 15 and 34. While radical orchiectomy has long been the standard of care, partial orchiectomy—or testis-sparing surgery—has gained increasing acceptance, particularly for small, localized, benign lesions. This conservative approach aims to preserve both the endocrine and exocrine functions of the testes. However, temporary clamping of the spermatic cord during such procedures, though necessary to control hemorrhage and minimize tumor dissemination, poses a significant risk of ischemia-reperfusion (I/R) injury, which may compromise testicular viability. I/R injury results from the paradoxical exacerbation of tissue damage following the restoration of blood supply after a period of ischemia. The associated surge in reactive oxygen species (ROS) overwhelms intrinsic antioxidant defenses, leading to oxidative stress, inflammation, germ cell apoptosis, and architectural disruption. Numerous interventions, including pharmacological agents and thermal modulation, have been investigated to mitigate I/R injury. Among them, hypothermia has emerged as a particularly promising strategy, owing to its capacity to suppress metabolic demand and reduce ROS formation. Experimental evidence has demonstrated that testicular hypothermia—often achieved through ice slush application—attenuates oxidative stress and preserves histological integrity. Nevertheless, the precise duration of cold ischemia that affords optimal protection remains ambiguous. Therefore, this study sought to determine the maximum allowable duration of cold ischemia that minimizes biochemical and histopathological damage in a controlled rat model. Materials and Methods This study was conducted with ethical approval from the Pamukkale University Rectorate Experimental Animals Ethics Committee (Approval No: 60758568-020/20966; Date: 25.10.2018). Thirty sexually mature Wistar albino rats (250–350 g) were randomly divided into four experimental groups. Group I (Sham) consisted of six rats, while Groups II (10 minutes of ischemia), III (20 minutes), and IV (30 minutes) each comprised eight rats. All animals were maintained under controlled conditions, including a 12-hour light/dark cycle at 22 ± 1°C, with ad libitum access to food and water. Anesthesia was induced using intraperitoneal ketamine (50 mg/kg) and xylazine (5 mg/kg). Through a midline scrotal incision, both testes were exposed and subjected to cooling with ice slush for 10 minutes. A digital thermometer was used to ensure maintenance of the target temperature range (2–4°C). In Group I, bilateral orchiectomy was performed immediately after cooling. In Groups II through IV, ischemia was induced via spermatic cord clamping for 10, 20, and 30 minutes, respectively, followed by 1 hour of reperfusion. All animals were sacrificed via cervical dislocation post-procedure. Testicular samples were promptly harvested and placed on ice. For biochemical analysis, approximately 100 ± 10 mg of testicular tissue per sample was homogenized in phosphate-buffered saline (pH 7.4) and centrifuged. The supernatants were analyzed for malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), total antioxidant status (TAS), and total oxidant status (TOS). For histological evaluation, tissues were fixed in 10% buffered formaldehyde, processed using an automated tissue processor, and embedded in paraffin. Sections of 4–5 µm thickness were prepared and stained with hematoxylin and eosin (H&E). Microscopic assessment included measurement of tubular diameters (20 tubules per animal) and application of the Johnsen scoring system to evaluate spermatogenesis. The Johnsen scoring system is a 10-point scale used to evaluate the degree of spermatogenesis in seminiferous tubules. A score of 10 indicates complete spermatogenesis with full germ cell maturation, while a score of 1 reflects complete absence of germ cells and only Sertoli cells present. Statistical analysis was conducted using SPSS v24 (IBM Corp., NY, USA). Non-parametric Kruskal-Wallis tests were employed, and p-values < 0.05 were considered statistically significant. Where applicable, post-hoc analyses were conducted. As the data did not meet the assumptions of normality, non-parametric Kruskal–Wallis tests were used to compare differences among groups. When statistically significant differences were observed, post-hoc pairwise comparisons were performed using the Dunn–Bonferroni method to identify the specific group differences. Results Biochemical analyses revealed a significant elevation in TNF-α and IL-6 levels in Group IV relative to the Sham group (p = 0.001 and p = 0.007, respectively). TAS levels demonstrated a progressive decline with increasing ischemia duration, particularly in Groups III and IV (p = 0.001). Conversely, TOS and OSI levels were significantly elevated in all ischemia groups compared to the Sham group. Histopathological analysis indicated that Group I exhibited well-preserved seminiferous tubules with minimal germ cell shedding. Group II showed moderate reductions in tubular diameter and increased epithelial desquamation. Group III exhibited pronounced degenerative changes, including multinucleated giant cells. The most extensive damage was observed in Group IV, characterized by marked epithelial disruption and vascular congestion. Johnsen scoring further corroborated these findings. Mean scores were as follows: Group I: 9.5 ± 0.2, Group II: 7.0 ± 0.3, Group III: 6.5 ± 0.4, Group IV: 5.5 ± 0.5 (p < 0.05), indicating a time-dependent decline in spermatogenic activity. Discussion The current findings underscore the detrimental effects of prolonged ischemia on testicular tissue, even under hypothermic conditions. The significant elevations in inflammatory cytokines (TNF-α, IL-6) and oxidative stress markers (TOS, OSI), coupled with reductions in TAS, affirm the progressive nature of I/R injury. While hypothermia mitigated the severity of damage, its protective effect diminished notably beyond 20 minutes of ischemia. These results are congruent with existing literature, which emphasizes the temporal threshold beyond which testicular damage becomes increasingly irreversible. Histological observations, including tubular atrophy and germ cell loss, were reflected in reduced Johnsen scores, thereby validating the scoring system as a reliable quantitative tool. Although these outcomes are promising, extrapolation to human physiology must be approached with caution. Differences in testicular architecture, blood flow dynamics, and thermal regulation between species may influence clinical applicability. Nevertheless, the consistency of findings across preclinical models suggests that strategic use of hypothermia—within defined time constraints—may enhance outcomes in testis-sparing surgeries. Conclusion In conclusion, this study demonstrates that while testicular cooling confers protective benefits against I/R injury, its efficacy diminishes with extended ischemia. Limiting ischemic duration to a maximum of 20 minutes under hypothermic conditions appears crucial for preserving testicular architecture and function. These insights may inform surgical protocols and stimulate further translational research aimed at optimizing fertility-preserving interventions. Limitations and Future Directions This study is subject to certain limitations. The use of a rat model, while appropriate for controlled experimental assessment, may not fully recapitulate human testicular physiology, particularly in terms of thermal regulation and vascular architecture. Additionally, the evaluation was limited to a single reperfusion duration and excluded alternative protective interventions. Future studies incorporating extended reperfusion periods, pharmacologic strategies, and translational models are warranted to validate and expand these findings, ensuring their applicability to clinical practice and enhancing testis-sparing surgical approaches. Declarations Acknowledgements Not applicable. Authors’ contributions The authors’ responsibilities were as follows—MK: designed the research and participated in data collection; MK, HD, IA, BS: conducted the statistical analyses; MK, IA: drafted the manuscript; YD, ES, HT and HS: revised the manuscript, and read and approved the final manuscript. All authors have read and approved the final version of the manuscript. Funding This study was funded by the Scientific Research Projects Coordination Unit of Muğla Sıtkı Koçman University (Project No: 19/084/01/3/4). Data availability The data that support the findings of the study are available from the corresponding author upon reasonable request. Ethics Statement ( Animal Studies : Sexually mature Wistar albino rats.) This study was approved by the Ethics Committee of the Pamukkale University Rectorate for Experimental Animals (Approval number: 60758568-020/20966, Date: 25.10.2018). Clinical trial number: not applicable. Consent to Participate declaration: not applicable. Consent to Publish declaration: not applicable. Competing interests The authors declare no competing interests. ORCID Mustafa Kestel: https://orcid.org/0000-0002-9894-1655 Hasan Deliktaş: - İlker Akarken: https://orcid.org/0000-0002-2863-3112 Barbaros Şahin: - Yelda Dere: https://orcid.org/0000-0003-0238-2236 Ercan Saruhan: https://orcid.org/0000-0001-6416-1442 Hüseyin Tarhan: https://orcid.org/0000-0003-1398-1592 Hayrettin Şahin: https://orcid.org/0000-0003-1061-1238 References Ozkisacik S, Yazici M, Gursoy H, Serter M, Culhaci N. The effects of short-interval postconditioning in preventing testicular ischemia-reperfusion injury in rats, J Pediatr Surg , vol. 46, no. 3, pp. 546–550, Mar. 2011, 10.1016/j.jpedsurg.2010.08.056 Erdem AO et al. 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Tables Table 1 The comparison of biochemical data between groups Parameters Group I (Sham) Group II (10 min) Group III (20 min) Group IV (30 min) p-value TNF–α (Median/ IQR, ng/L) 112,17/11,16 104,76/33,53 115,93/8,60 135,93/34,13 0,002 IL – 6 (Median/ IQR, ng/L) 10,35/3,25 12,33/4,54 12,89/4,17 16,31/5,59 0,008 MDA (Median/ IQR, nmol/ml) 0,37/0,12 0,35/0,11 0,42/0,11 0,36/0,17 0,290 SOD (Median/ IQR, ng/ml) 0,50/0,35 0,38/0,32 0,42/0,38 0,40/0,18 0,380 TAS (Median/ IQR, mmol TroloxEq/L) 3,20/0,56 2,78/0,40 2,47/0,69 2,36/0,40 0,001 TOS (Median/ IQR, µmol H 2 O 2 Eq/L) 4,65/0,88 5,75/1,49 5,96/1,69 6,15/2,44 0,005 OSI 1,51/0,33 2,04/0,88 2,32/1,16 2,65/0,97 0,001 TNF-α : Tumor necrosis factor-alpha, IL-6 : Interleukin-6, MDA : Malondialdehyde, SOD : Superoxide dismutase, TAS : Total antioxidant status, TOS : Total oxidant status, OSI : Oxidative stress index Table 2 Mean tubule diameters of the groups Groups Tubular diameter (µm, Mean ± SD) p-value Group I (sham) 248,02 ± 0,6 0.009 Group II 227,74 ± 2,1 Group III 233,24 ± 3,1 < 0.001 Group IV 211,97 ± 2,4 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 04 Feb, 2026 Read the published version in BMC Urology → Version 1 posted Editorial decision: Revision requested 12 Jan, 2026 Reviews received at journal 08 Jan, 2026 Reviewers agreed at journal 08 Jan, 2026 Reviews received at journal 16 Dec, 2025 Reviewers agreed at journal 07 Dec, 2025 Reviewers invited by journal 07 Dec, 2025 Editor invited by journal 20 Nov, 2025 Editor assigned by journal 18 Nov, 2025 Submission checks completed at journal 18 Nov, 2025 First submitted to journal 15 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7871593","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":556626232,"identity":"7c091349-3f0b-4579-9f23-43f097d3800e","order_by":0,"name":"M. 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12:45:34","extension":"html","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78191,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7871593/v1/3aac64cdee8f10b2c747de44.html"},{"id":97882891,"identity":"f8ab68ea-3001-4bc7-845c-c61226f941e5","added_by":"auto","created_at":"2025-12-10 12:45:34","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":242473,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of TNF-a levels according to experimental groups\u003c/p\u003e","description":"","filename":"TNFalphaDistributionbyGroup300dpi.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7871593/v1/45de7b126361904f50e6124a.jpg"},{"id":97900361,"identity":"4c271eae-eef7-48c4-81ba-4dee3cb53df7","added_by":"auto","created_at":"2025-12-10 15:45:25","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":230925,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of OSI levels according to experimental groups\u003c/p\u003e","description":"","filename":"OSIDistributionbyGroup300dpi.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7871593/v1/c5f0c4d0b78611ae0b5411ad.jpg"},{"id":97882894,"identity":"e732a305-1fbe-4711-97f0-c3e38c470f0f","added_by":"auto","created_at":"2025-12-10 12:45:34","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":198031,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical analysis of intergroup mean tubular diameters\u003c/p\u003e","description":"","filename":"MeanTubularDiameterbyGroup300dpi.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7871593/v1/2ebbeb1d3a6f71b8a1579caf.jpg"},{"id":97882889,"identity":"8b30c22f-d4a8-4a74-a7e5-064aed4ce5fc","added_by":"auto","created_at":"2025-12-10 12:45:34","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":92059,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical analysis of Johnsen Score\u003c/p\u003e","description":"","filename":"johnsenscoregraph300dpi.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7871593/v1/490f860e1e7660658eb0fc60.jpg"},{"id":102234426,"identity":"7df07c11-b9f8-4f19-a7f7-30e25a3cf609","added_by":"auto","created_at":"2026-02-09 16:11:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1337922,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7871593/v1/13a5b6f9-9b8b-4503-9ef9-c6d11a1197a9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The effect of acute cold ischemia duration on testicular tissue","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTesticular cancer predominantly affects men between the ages of 15 and 34. While radical orchiectomy has long been the standard of care, partial orchiectomy\u0026mdash;or testis-sparing surgery\u0026mdash;has gained increasing acceptance, particularly for small, localized, benign lesions. This conservative approach aims to preserve both the endocrine and exocrine functions of the testes. However, temporary clamping of the spermatic cord during such procedures, though necessary to control hemorrhage and minimize tumor dissemination, poses a significant risk of ischemia-reperfusion (I/R) injury, which may compromise testicular viability.\u003c/p\u003e\u003cp\u003eI/R injury results from the paradoxical exacerbation of tissue damage following the restoration of blood supply after a period of ischemia. The associated surge in reactive oxygen species (ROS) overwhelms intrinsic antioxidant defenses, leading to oxidative stress, inflammation, germ cell apoptosis, and architectural disruption.\u003c/p\u003e\u003cp\u003eNumerous interventions, including pharmacological agents and thermal modulation, have been investigated to mitigate I/R injury. Among them, hypothermia has emerged as a particularly promising strategy, owing to its capacity to suppress metabolic demand and reduce ROS formation. Experimental evidence has demonstrated that testicular hypothermia\u0026mdash;often achieved through ice slush application\u0026mdash;attenuates oxidative stress and preserves histological integrity.\u003c/p\u003e\u003cp\u003eNevertheless, the precise duration of cold ischemia that affords optimal protection remains ambiguous. Therefore, this study sought to determine the maximum allowable duration of cold ischemia that minimizes biochemical and histopathological damage in a controlled rat model.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e This study was conducted with ethical approval from the Pamukkale University Rectorate Experimental Animals Ethics Committee (Approval No: 60758568-020/20966; Date: 25.10.2018).\u003c/p\u003e\u003cp\u003eThirty sexually mature Wistar albino rats (250\u0026ndash;350 g) were randomly divided into four experimental groups. Group I (Sham) consisted of six rats, while Groups II (10 minutes of ischemia), III (20 minutes), and IV (30 minutes) each comprised eight rats. All animals were maintained under controlled conditions, including a 12-hour light/dark cycle at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, with ad libitum access to food and water.\u003c/p\u003e\u003cp\u003eAnesthesia was induced using intraperitoneal ketamine (50 mg/kg) and xylazine (5 mg/kg). Through a midline scrotal incision, both testes were exposed and subjected to cooling with ice slush for 10 minutes. A digital thermometer was used to ensure maintenance of the target temperature range (2\u0026ndash;4\u0026deg;C). In Group I, bilateral orchiectomy was performed immediately after cooling. In Groups II through IV, ischemia was induced via spermatic cord clamping for 10, 20, and 30 minutes, respectively, followed by 1 hour of reperfusion. All animals were sacrificed via cervical dislocation post-procedure.\u003c/p\u003e\u003cp\u003eTesticular samples were promptly harvested and placed on ice. For biochemical analysis, approximately 100\u0026thinsp;\u0026plusmn;\u0026thinsp;10 mg of testicular tissue per sample was homogenized in phosphate-buffered saline (pH 7.4) and centrifuged. The supernatants were analyzed for malondialdehyde (MDA), superoxide dismutase (SOD), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), total antioxidant status (TAS), and total oxidant status (TOS).\u003c/p\u003e\u003cp\u003eFor histological evaluation, tissues were fixed in 10% buffered formaldehyde, processed using an automated tissue processor, and embedded in paraffin. Sections of 4\u0026ndash;5 \u0026micro;m thickness were prepared and stained with hematoxylin and eosin (H\u0026amp;E). Microscopic assessment included measurement of tubular diameters (20 tubules per animal) and application of the Johnsen scoring system to evaluate spermatogenesis. The Johnsen scoring system is a 10-point scale used to evaluate the degree of spermatogenesis in seminiferous tubules. A score of 10 indicates complete spermatogenesis with full germ cell maturation, while a score of 1 reflects complete absence of germ cells and only Sertoli cells present.\u003c/p\u003e\u003cp\u003eStatistical analysis was conducted using SPSS v24 (IBM Corp., NY, USA). Non-parametric Kruskal-Wallis tests were employed, and p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant. Where applicable, post-hoc analyses were conducted. As the data did not meet the assumptions of normality, non-parametric Kruskal\u0026ndash;Wallis tests were used to compare differences among groups. When statistically significant differences were observed, post-hoc pairwise comparisons were performed using the Dunn\u0026ndash;Bonferroni method to identify the specific group differences.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eBiochemical analyses revealed a significant elevation in TNF-α and IL-6 levels in Group IV relative to the Sham group (p\u0026thinsp;=\u0026thinsp;0.001 and p\u0026thinsp;=\u0026thinsp;0.007, respectively). TAS levels demonstrated a progressive decline with increasing ischemia duration, particularly in Groups III and IV (p\u0026thinsp;=\u0026thinsp;0.001). Conversely, TOS and OSI levels were significantly elevated in all ischemia groups compared to the Sham group.\u003c/p\u003e\u003cp\u003eHistopathological analysis indicated that Group I exhibited well-preserved seminiferous tubules with minimal germ cell shedding. Group II showed moderate reductions in tubular diameter and increased epithelial desquamation. Group III exhibited pronounced degenerative changes, including multinucleated giant cells. The most extensive damage was observed in Group IV, characterized by marked epithelial disruption and vascular congestion.\u003c/p\u003e\u003cp\u003eJohnsen scoring further corroborated these findings. Mean scores were as follows: Group I: 9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2, Group II: 7.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.3, Group III: 6.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.4, Group IV: 5.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating a time-dependent decline in spermatogenic activity.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe current findings underscore the detrimental effects of prolonged ischemia on testicular tissue, even under hypothermic conditions. The significant elevations in inflammatory cytokines (TNF-α, IL-6) and oxidative stress markers (TOS, OSI), coupled with reductions in TAS, affirm the progressive nature of I/R injury.\u003c/p\u003e\u003cp\u003eWhile hypothermia mitigated the severity of damage, its protective effect diminished notably beyond 20 minutes of ischemia. These results are congruent with existing literature, which emphasizes the temporal threshold beyond which testicular damage becomes increasingly irreversible. Histological observations, including tubular atrophy and germ cell loss, were reflected in reduced Johnsen scores, thereby validating the scoring system as a reliable quantitative tool.\u003c/p\u003e\u003cp\u003eAlthough these outcomes are promising, extrapolation to human physiology must be approached with caution. Differences in testicular architecture, blood flow dynamics, and thermal regulation between species may influence clinical applicability. Nevertheless, the consistency of findings across preclinical models suggests that strategic use of hypothermia\u0026mdash;within defined time constraints\u0026mdash;may enhance outcomes in testis-sparing surgeries.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, this study demonstrates that while testicular cooling confers protective benefits against I/R injury, its efficacy diminishes with extended ischemia. Limiting ischemic duration to a maximum of 20 minutes under hypothermic conditions appears crucial for preserving testicular architecture and function. These insights may inform surgical protocols and stimulate further translational research aimed at optimizing fertility-preserving interventions.\u003c/p\u003e"},{"header":"Limitations and Future Directions","content":"\u003cp\u003eThis study is subject to certain limitations. The use of a rat model, while appropriate for controlled experimental assessment, may not fully recapitulate human testicular physiology, particularly in terms of thermal regulation and vascular architecture. Additionally, the evaluation was limited to a single reperfusion duration and excluded alternative protective interventions. Future studies incorporating extended reperfusion periods, pharmacologic strategies, and translational models are warranted to validate and expand these findings, ensuring their applicability to clinical practice and enhancing testis-sparing surgical approaches.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors\u0026rsquo; responsibilities were as follows\u0026mdash;MK: designed the research and participated in data collection; MK, HD, IA, BS: conducted the statistical analyses; MK, IA: drafted the manuscript; YD, ES, HT and HS: revised the manuscript, and read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eAll authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by the Scientific Research Projects Coordination Unit of Muğla Sıtkı Ko\u0026ccedil;man University (Project No: 19/084/01/3/4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of the study are available from the\u003c/p\u003e\n\u003cp\u003ecorresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement (\u003c/strong\u003e\u003cstrong\u003eAnimal Studies\u003c/strong\u003e:\u0026nbsp;Sexually mature Wistar albino rats.)\u003c/p\u003e\n\u003cp\u003eThis study was approved by the\u0026nbsp;Ethics Committee of the Pamukkale University Rectorate for Experimental Animals (Approval number: 60758568-020/20966, Date: 25.10.2018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u0026nbsp;\u003c/strong\u003enot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate declaration:\u003c/strong\u003e not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish declaration:\u003c/strong\u003e not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMustafa Kestel:\u003c/strong\u003e https://orcid.org/0000-0002-9894-1655\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHasan Deliktaş:\u003c/strong\u003e -\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eİlker Akarken:\u003c/strong\u003e https://orcid.org/0000-0002-2863-3112\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBarbaros Şahin:\u003c/strong\u003e -\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eYelda Dere:\u003c/strong\u003e https://orcid.org/0000-0003-0238-2236\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eErcan Saruhan:\u003c/strong\u003e https://orcid.org/0000-0001-6416-1442\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eH\u0026uuml;seyin Tarhan:\u003c/strong\u003e https://orcid.org/0000-0003-1398-1592\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHayrettin Şahin:\u003c/strong\u003e https://orcid.org/0000-0003-1061-1238\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eOzkisacik S, Yazici M, Gursoy H, Serter M, Culhaci N. 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Elsevier Ltd; Aug. 2014. pp. 593\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.jpurol.2014.06.005\u003c/span\u003e\u003cspan address=\"10.1016/j.jpurol.2014.06.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eProtective effect. of allicin on ischemia-reperfusion injury caused by testicular torsion-detorsion in rats.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMohamed MZ, Morsy MA, Mohamed HH, Hafez HM. Paeonol protects against testicular ischaemia-reperfusion injury in rats through inhibition of oxidative stress and inflammation. Andrologia. 2020;52(6):1\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/and.13599\u003c/span\u003e\u003cspan address=\"10.1111/and.13599\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOkur MH, et al. Protective Effect of Cordycepin on Experimental Testicular Ischemia/Reperfusion Injury in Rats. J Invest Surg. 2018;31(1):1\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1080/08941939.2016.1246629\u003c/span\u003e\u003cspan address=\"10.1080/08941939.2016.1246629\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eŞekerci \u0026Ccedil;A, et al. Protective Effects of Ellagic Acid on Testicular Ischemia-Reperfusion Injury in Rats. J Urol Surg. Jun. 2023;10(2):160\u0026ndash;6. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.4274/jus.galenos.2022.2022.0049\u003c/span\u003e\u003cspan address=\"10.4274/jus.galenos.2022.2022.0049\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDemir EA, et al. Arbutin abrogates testicular ischemia/reperfusion injury in rats through repression of inflammation and ER stress. Tissue Cell. Jun. 2023;82:102056. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.tice.2023.102056\u003c/span\u003e\u003cspan address=\"10.1016/j.tice.2023.102056\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTanyeli A, G\u0026uuml;ler MC, \u0026Ouml;zbek Şebin S, Bircan B, Şebin E, Ekinci FN, Akdemir. Potential Beneficial Effects of Apelin-13 on Testicular Ischemia/Reperfusion Injury. New Trends Med Sci. May 2024. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.56766/ntms.1476451\u003c/span\u003e\u003cspan address=\"10.56766/ntms.1476451\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHeidenreich A, Angerer-Shpilenya M. Organ-preserving surgery for testicular tumours. BJU Int. Feb. 2012;109(3):474\u0026ndash;90. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/j.1464-410X.2011.10913.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1464-410X.2011.10913.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAlmodhen F, He X, Loutochin O, Jednak R, Capolicchio JP, El-Sherbiny MT. Protective role of hypothermia on ischemia of prepubertal rodent testicle. Urology. 2011;77(3). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1016/j.urology.2010.09.050\u003c/span\u003e\u003cspan address=\"10.1016/j.urology.2010.09.050\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. 762.e8-762.e12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBo X, Wang P, Nie Y, Li R, Lu J, Wang H. Protective effect of hypothermia and vitamin E on spermatogenic function after reduction of testicular torsion in rats. Exp Ther Med. May 2020;20(2):796\u0026ndash;801. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3892/etm.2020.8800\u003c/span\u003e\u003cspan address=\"10.3892/etm.2020.8800\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei SM, Huang YM. Effect of sulforaphane on testicular ischemia-reperfusion injury induced by testicular torsion-detorsion in rats. Sci Rep. Dec. 2024;14(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-024-74756-z\u003c/span\u003e\u003cspan address=\"10.1038/s41598-024-74756-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei SM, Huang YM. Safranal ameliorates testicular ischemia-reperfusion injury in testicular torsion-detorsion rat modelSafranal mejora la lesi\u0026oacute;n por isquemia-reperfusi\u0026oacute;n testicular en un modelo de rata con detorsi\u0026oacute;n de torsi\u0026oacute;n testicular. Rev Int Androl. 2024;22(4):33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.22514/j.androl.2024.028\u003c/span\u003e\u003cspan address=\"10.22514/j.androl.2024.028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDemir EA et al. Jun., Syringic acid ameliorates ischemia/reperfusion-induced testicular injury in rats via suppressing of HMGB1/NF-κB axis and endoplasmic reticulum stress, \u003cem\u003eEuropean Journal of Trauma and Emergency Surgery\u003c/em\u003e, vol. 49, no. 3, pp. 1595\u0026ndash;1602, 2023, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1007/s00068-023-02227-7\u003c/span\u003e\u003cspan address=\"10.1007/s00068-023-02227-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRezakhaniha B, et al. The evaluation of citral effects on experimental unilateral testicular ischemia/reperfusion injury. Andrologia. Dec. 2022;54(11). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1111/and.14605\u003c/span\u003e\u003cspan address=\"10.1111/and.14605\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZhou J, Peng C, Zhu X, Yao W, Luo Y, Yang L. Application value of strain elastography in testicular injury assessment after torsion. Front Pediatr. 2024;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.3389/fped.2024.1477821\u003c/span\u003e\u003cspan address=\"10.3389/fped.2024.1477821\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWei SM, Huang YM. Effect of sulforaphane on testicular ischemia-reperfusion injury induced by testicular torsion-detorsion in rats. Sci Rep. Dec. 2024;14(1). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41598-024-74756-z\u003c/span\u003e\u003cspan address=\"10.1038/s41598-024-74756-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cdiv class=\"gridtable\"\u003e\n \u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eThe comparison of biochemical data between groups\u003c/div\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eParameters\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup I\u003c/div\u003e\n \u003cdiv class=\"SimplePara\"\u003e(Sham)\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup II\u003c/div\u003e\n \u003cdiv class=\"SimplePara\"\u003e(10 min)\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup III\u003c/div\u003e\n \u003cdiv class=\"SimplePara\"\u003e(20 min)\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup IV\u003c/div\u003e\n \u003cdiv class=\"SimplePara\"\u003e(30 min)\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003ep-value\u003c/div\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eTNF\u0026ndash;\u0026alpha; (Median/ IQR, ng/L)\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e112,17/11,16\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e104,76/33,53\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e115,93/8,60\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e135,93/34,13\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e0,002\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eIL \u0026ndash; 6 (Median/ IQR, ng/L)\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e10,35/3,25\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e12,33/4,54\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e12,89/4,17\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e16,31/5,59\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e0,008\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eMDA (Median/ IQR, nmol/ml)\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0,37/0,12\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0,35/0,11\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0,42/0,11\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0,36/0,17\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0,290\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eSOD (Median/ IQR, ng/ml)\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0,50/0,35\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0,38/0,32\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0,42/0,38\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0,40/0,18\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0,380\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eTAS (Median/ IQR, mmol TroloxEq/L)\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e3,20/0,56\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e2,78/0,40\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e2,47/0,69\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e2,36/0,40\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e0,001\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eTOS (Median/ IQR, \u0026micro;mol H\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eO\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eEq/L)\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e4,65/0,88\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e5,75/1,49\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e5,96/1,69\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e6,15/2,44\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e0,005\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eOSI\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e1,51/0,33\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e2,04/0,88\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e2,32/1,16\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e2,65/0,97\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003e0,001\u003c/span\u003e\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"6\"\u003e\u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eTNF-\u0026alpha;\u003c/span\u003e: Tumor necrosis factor-alpha, \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eIL-6\u003c/span\u003e: Interleukin-6, \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eMDA\u003c/span\u003e: Malondialdehyde, \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eSOD\u003c/span\u003e: Superoxide dismutase, \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eTAS\u003c/span\u003e: Total antioxidant status, \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eTOS\u003c/span\u003e: Total oxidant status, \u003cspan type=\"Bold\" class=\"Bold\" name=\"Emphasis\"\u003eOSI\u003c/span\u003e: Oxidative stress index\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"gridtable\"\u003e\n \u003cdiv id=\"13\" class=\"btn-xs-small Annotation tooltipped\" data-position=\"top\" data-tooltip=\"\"\u003e\u003cbr\u003e\u003c/div\u003e\n \u003ctable id=\"Tab2\" border=\"1\" class=\"fr-table-selection-hover\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eMean tubule diameters of the groups\u003c/div\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroups\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eTubular diameter (\u0026micro;m, Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/div\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003ep-value\u003c/div\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup I (sham)\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e248,02\u0026thinsp;\u0026plusmn;\u0026thinsp;0,6\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e0.009\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup II\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e227,74\u0026thinsp;\u0026plusmn;\u0026thinsp;2,1\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup III\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e233,24\u0026thinsp;\u0026plusmn;\u0026thinsp;3,1\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\" rowspan=\"2\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e\u0026lt;\u0026thinsp;0.001\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cdiv class=\"SimplePara\"\u003eGroup IV\u003c/div\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cdiv class=\"SimplePara\"\u003e211,97\u0026thinsp;\u0026plusmn;\u0026thinsp;2,4\u003c/div\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-urology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"buro","sideBox":"Learn more about [BMC Urology](http://bmcurol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/buro/default.aspx","title":"BMC Urology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"ischemia-reperfusion injury, hypothermia, partial orchiectomy, testicular torsion, oxidative stress, Johnsen score","lastPublishedDoi":"10.21203/rs.3.rs-7871593/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7871593/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e\u003cp\u003eTesticular cancer primarily affects men aged 15\u0026ndash;34. Partial orchiectomy has recently emerged as a viable testis-sparing surgical technique. However, prolonged spermatic cord clamping may induce ischemia-reperfusion (I/R) injury due to elevated reactive oxygen species (ROS) production. This study aimed to evaluate the protective role of testicular hypothermia against I/R injury and to determine the maximum tolerable cold ischemia duration in a rat model. Spermatogenesis was assessed using the Johnsen scoring system.\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e\u003cp\u003eThirty sexually mature Wistar albino rats were divided into four groups. Following scrotal incision and testicular cooling with ice (2\u0026ndash;4\u0026deg;C), Group I (Sham, n\u0026thinsp;=\u0026thinsp;6) underwent orchiectomy. Groups II, III, and IV (n\u0026thinsp;=\u0026thinsp;8 each) were subjected to 10, 20, and 30 minutes of ischemia, respectively, followed by 1-hour reperfusion. Biochemical parameters (TNF-α, IL-6, TAS, TOS, OSI) and histopathological evaluations, including Johnsen scoring, were conducted.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eInflammatory markers and oxidative stress indices increased significantly with prolonged ischemia, while antioxidant capacity and Johnsen scores declined in a time-dependent manner. The most severe biochemical and histological impairments were observed in the 30-minute group.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eCold ischemia exceeding 20 minutes leads to marked testicular damage, despite hypothermic protection. Findings emphasize the need to minimize ischemic duration during testis-sparing surgeries.\u003c/p\u003e","manuscriptTitle":"The effect of acute cold ischemia duration on testicular tissue","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-10 12:45:27","doi":"10.21203/rs.3.rs-7871593/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-12T09:54:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-08T23:47:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"307381243762210227997938634253611102906","date":"2026-01-08T16:06:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-16T16:22:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98114771411826733830530889105091337669","date":"2025-12-08T00:57:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-08T00:50:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-11-20T10:21:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-18T07:08:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-11-18T07:06:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Urology","date":"2025-10-15T20:45:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-urology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"buro","sideBox":"Learn more about [BMC Urology](http://bmcurol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/buro/default.aspx","title":"BMC Urology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc1de380-fecb-4632-861b-b95d9c7b7783","owner":[],"postedDate":"December 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:07:20+00:00","versionOfRecord":{"articleIdentity":"rs-7871593","link":"https://doi.org/10.1186/s12894-026-02071-y","journal":{"identity":"bmc-urology","isVorOnly":false,"title":"BMC Urology"},"publishedOn":"2026-02-04 15:57:38","publishedOnDateReadable":"February 4th, 2026"},"versionCreatedAt":"2025-12-10 12:45:27","video":"","vorDoi":"10.1186/s12894-026-02071-y","vorDoiUrl":"https://doi.org/10.1186/s12894-026-02071-y","workflowStages":[]},"version":"v1","identity":"rs-7871593","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7871593","identity":"rs-7871593","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}