Yeşil Kaplumbağa Humerus Kemiklerinde İskeletkronolojisi Yönteminde Döner ve Kriyostat Mikrotomun Karşılaştırılması

Year 2024, Volume: 29 Issue: 3, 823 - 829, 31.12.2024

Emre Sandık , Bektaş Sönmez , Sukran Yalçın Ozdilek

https://doi.org/10.53433/yyufbed.1483061

Abstract

İskeletkronolojisi, deniz kaplumbağalarının yaşı hakkında bilgi sağlamak için kullanılan güçlü bir araçtır. Histolojik olarak hazırlanmış humerus kemiklerinin enine kesitlerinin dijital görüntüleri yaş tayini için yaygın olarak kullanılmaktadır. Sağlıklı yaş tahmini için kritik öneme sahip olan dijital görüntülerin kalitesi, mikrotomla ilişkili histoloji tekniğine ve türüne bağlı olarak değişir. Bu çalışmanın amacı, döner ve kriyostat mikrotomlarla hazırlanan humerus kemiklerinin kesitlerinin dijital görüntülerinin kalitesini karşılaştırmaktır. Çalışma için 2021 yuvalama sezonu Samandağ kumsalında karaya ölü olarak vurmuş 11 juvenil yeşil kaplumbağanın (ortalama CCL 292,1 mm) humerus kemikleri kullanıldı. Üç okuyucu, iki farklı mikrotom ve ilgili protokollerle yapılan histolojik kesitlerin kalitesini ve gördükleri LAG'lerin okunabilirliğini 0 ile 5 arasında puanladı. İki protokolün kalite puanları arasındaki farklılıkları belirlemek için Wilcoxon işaretli eşleştirilmiş sıralama testi kullanılmıştır. Buna ek olarak, okuyucuların uyumluluğu, her protokol için en az iki aynı puana sahip numunelerin oranı belirlenerek değerlendirilmiştir. Kriyostat miktotom protokolünden elde edilen skorlar döner mikrotom protokolüne kıyasla daha geniş bir varyasyona sahipti. Döner mikrotom protokolü kriyostat mikrotom protokolünden daha yüksek medyan skor değerlerine sahiptir (p<0,001). Üç kriyostat okuyucusunun uyumluluğu (0,73) döner mikrotomunkinden (0,91) daha düşüktür. Kriyostat mikrotom tekniği daha az zaman alıcı ve daha az maliyetli olmasına rağmen, bu çalışmanın sonuçları rotary mikrotom protokolünün kriyostat mikrotom protokolüne tercih edildiğini göstermiştir. Gelecekteki çalışmalar, iskelet kronolojisi hakkında değerli bilgiler edinmek için çeşitli boyama protokolleri gibi diğer teknikleri test edebilir.

Keywords

Kriyostat, Mikrotom, Samandağ, Yaş belirleme, Yeşil kaplumbağa

Ethical Statement

The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper.

Supporting Institution

Türkiye Bilimsel ve Teknolojik Araştırma Kurumu (TÜBİTAK)

Project Number

122Z247

Thanks

This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) Grant numbers 122Z247. We would like to thank Op. Dr. Hasan Keser from Çanakkale Mehmet Akif Ersoy State Hospital, Dr. Ali Fuat Çiçek in Pathology Laboratory, and technicians Hilal Köksal Elagöz and Gönül Cömert Özkan, Op. Dr. Gökhan BAŞTÜRK from Çanakkale Provincial Health Director, 7th Regional Directorate of Nature Conservation and National Parks and Samandağ Environmental Protection and Tourism Association for their contributions.

Comparison of Rotary and Cryostat Microtomes in the Skeletochronology on Green Turtle Humerus Bones

Abstract

Skeletochronology is a powerful tool that provides information on the age of sea turtles. Digital images of histologically prepared humerus bone sections are commonly used for age determination. The quality of digital images, which is critical for robust age estimation, varies depending on the type and histological technique associated with microtome. This study aimed to compare the quality of digital image sections of humerus bones prepared by rotary and cryostat microtomes. The humerus bones of 11 juvenile stranded (dead) green turtles (mean CCL 292.1 mm) on Samandağ beach during the 2021 nesting season were used for the study. Three readers scored the quality of histologic sections prepared with two different microtomes and associated protocols, as well as the readability of the lines of arrested growth (LAGs), which they saw from 0 to 5. The Wilcoxon-signed rank-paired test was used to ascertain the disparities between the quality scores of the two protocols. In addition, the compatibility of readers was evaluated by determining the proportion of specimens with at least two identical scores for each protocol. The scores obtained from the cryostat microtome protocol had a wider variation compared to the rotary microtome protocol. The rotary microtome protocol has higher median score values (4) than the cryostat microtome protocol (3) (p<0,001). The compatibility of three cryostat microtome readers was lower than that of the rotary microtome. Although the cryostat microtome is less time-consuming and less costly, the results of this study showed that the rotary microtome protocol was preferable to the cryostat microtome protocol. Future studies may test techniques like various staining protocols to gain valuable insights into skeletochronology.

Keywords

Age determination, Cryostat, Green turtle, Microtome, Samandağ

Ethical Statement

The authors declare that they have no known competing interests or personal relationships that could have appeared to influence the work reported in this paper.

Supporting Institution

The Scientific and Technological Research Council of Turkey (TUBITAK)

Project Number

122Z247

Thanks

This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) Grant numbers 122Z247. We would like to thank Op. Dr. Hasan Keser from Çanakkale Mehmet Akif Ersoy State Hospital, Dr. Ali Fuat Çiçek in Pathology Laboratory, and technicians Hilal Köksal Elagöz and Gönül Cömert Özkan, Op. Dr. Gökhan BAŞTÜRK from Çanakkale Provincial Health Director, 7th Regional Directorate of Nature Conservation and National Parks and Samandağ Environmental Protection and Tourism Association for their contributions.

References

• Avens, L., Taylor, J. C., Goshe, L. R., Jones, T. T., & Hastings, M. (2009). Use of skeletochronological analysis to estimate the age of leatherback sea turtles Dermochelys coriacea in the western North Atlantic. Endangered Species Research, 8(3), 165-177. https://doi.org/10.3354/esr00202
• Castanet, J. (1994). Age estimation and longevity in reptiles. Gerontology, 40, 174-192. https://doi.org/10.1159/000213586
• Goshe, L. R., Avens, L., Bybee, J., & Hohn, A. A. (2009). An evaluation of histological techniques used in skeletochronological age estimation of sea turtles. Chelonian Conservation and Biology, 8, 217-222. https://doi.org/10.2744/CCB-0777.1
• Goshe, L. R., Avens, L., Scharf, F. S., & Southwood, A. L. (2010). Estimation of age at maturation and growth of Atlantic green turtles (Chelonia mydas) using skeletochronology. Marine Biology, 157(8), 1725-1740. https://doi.org/10.1007/s00227-010-1446-0
• Goshe, L. R., Avens, L., Snover, M. L., & Hohn, A. A. (2020). Protocol for processing sea turtle bones for age estimation. NOAA Technical Memorandum NMFS-SEFSC-746. https://doi.org/10.25923/gqva-9y22
• Goshe, Lisa R. et al. (2020). Protocol for processing sea turtle bones for age estimation. https://doi.org/10.25923/gqva-9y22
• Grillo, F., Bruzzone, M., Pigozzi, S., Prosapio, S., Migliora, P., Fiocca, R., & Mastracci, L. (2017). Immunohistochemistry on old archival paraffin blocks: is there an expiry date? Journal of Clinical Pathology, 70(11), 988-993. https://doi.org/10.1136/jclinpath-2017-204387
• Guarino, F. M., Di Maio, A., & Caputo, V. (2004). Age estimation by phalangeal skeletochronology of Caretta caretta from the Mediterranean Sea. Italian Journal of Zoology, 71(S2), 175-179. https://doi.org/10.1080/11250000409356630
• Klevezal, G. (1996). Recording structures of mammals. Determination of age and reconstruction of life history. Rotterdam, The Netherlands: Routledge. https://doi.org/10.1201/9780203741146
• Lenz, A. J., Avens, L., Campos Trigo, C., & Borges‐Martins, M. (2016). Skeletochronological estimation of age and growth of loggerhead sea turtles (Caretta caretta) in the western South Atlantic Ocean. Austral Ecology, 41(5), 580-590. https://doi.org/10.1111/aec.12347
• Lojda, Z., Gossrau, R., & Schiebler, T. H. (2012). Enzyme histochemistry: a laboratory manual. Springer Science & Business Media.
• Luque, P. L., Learmonth, J. A., Santos, M. B., Ieno, E., & Pierce, G. J. (2009). Comparison of two histological techniques for age determination in small cetaceans. Marine Mammal Science, 25(4), 902-919. https://doi.org/10.1111/j.1748-7692.2009.00304.x
• R Core Team (2023). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.
• Read, F. L., Hohn, A. A., & Lockyer, C. H. (2018). A review of age estimation methods in marine mammals with special reference to monodontids. NAMMCO Scientific Publications. https://doi.org/10.7557/3.4474
• Rossell, C. & R, Sheehan, J. L. (1998). Comparison of histological staining procedures for skeletochronological studies. Herpetological Review, 29, 95.
• Sinsch U., Leskovar C., Drobig, A., König, A., & Grosse, W. R. (2007). Life-history traits in green toad (Bufo viridis) populations: indicators of habitat quality. Canadian Journal of Zoology, 85, 665-673. https://doi.org/10.1139/Z07-046
• Sinsch, U. (2015). Skeletochronological assessment of demographic life-history traits in amphibians. The Herpetological Journal, 25, 5-13.
• Snover, M. L., & Hohn, A. A. (2004). Validation and interpretation of annual skeletal marks in loggerhead (Caretta caretta) and Kemp’s ridley (Lepidochelys kempii) sea turtles. Fishery Bulletin, 102, 682-692.
• Snover, M. L., Avens, L., & Hohn, A. A. (2007). Back-calculating length from skeletal growth marks in loggerhead sea turtles Caretta caretta. Endangered Species Research, 3(1), 95-104. https://doi.org/10.3354/esr003095
• Sönmez, B. (2019). Morphological variations in the green turtle (Chelonia mydas): A field study on an eastern Mediterranean nesting population. Zoological Studies, 58. https://doi.org/10.6620/ZS.2019.58-16
• Şirin, A., & Başkale, E. (2021). Age structure of stranded Loggerhead Turtles (Caretta caretta) in Turkey. Zoology in the Middle East, 67(4), 302-308. https://doi.org/10.1080/09397140.2021.1992836
• Türkozan, O., Özdilek, Ş. Y., Ergene, S., Uçar, A. H., Sönmez, B., Yılmaz, C., Kaçar, Y., & Aymak, C. (2013). Strandings of loggerhead (Caretta caretta) and green (Chelonia mydas) sea turtles along the eastern Mediterranean coast of Turkey. The Herpetological Journal, 23(1), 11-15.
• Usategui-Martín, A., Valverde, R. A., Ostiategui-Francia, P., Fariñas-Bermejo, A., Paz-Sánchez, Y., & Liria-Loza, A. (2023). First skeletochronological analysis on loggerhead yearlings (Caretta caretta) in the Canary Islands. Marine Biology, 170(8), 95. https://doi.org/10.1007/s00227-023-04239-8
• Woodward, H. N, Horner, J. R., Farlow, J. O. (2014). Quantification of intraskeletal histovariability in Alligator mississippiensis and implications for vertebrate osteohistology. PeerJ Life Environment, e422. https://doi.org/10.7717/peerj.422
• Zug, G. R., Balazs, G. H., Wetherall, J. A., Parker, D. M., & Murakawa, S. K. (2002). Age and growth of Hawaiian seaturtles (Chelonia mydas): an analysis based on skeletochronology. Fishery Bulletin, 100, 117-127.
• Zug, G. R., Wynn, A. H. & Ruckdeschel, C. (1986). Age determination of loggerhead sea turtles, Caretta caretta, by incremental growth marks in the skeleton. Smithsonian Contributions to Zoology, 427, 1-34. https://doi.org/10.5479/si.00810282.427