Use of X-ray computed microtomography (micro-CT) to improve the precision of age assessment in the spiny butterfly ray, Gymnura altavela

Age information is fundamental to a better understanding of species life cycles, as it factors in reproductive maturity, mortality and somatic growth. Age assessments are particularly important for threatened groups, such as rays, since the demersal behaviour of most species makes them vulnerable to fishing and pollution. The age of a ray can be determined by counting annuli present along the centre of its vertebrae. These are distinct pairs of bands that form during the concentric growth of vertebrae due to differential calcification throughout an individual's lifetime. However, annulus reading can be affected by sample processing methods, image resolution and reader biases. In this study, we compared two vertebrae processing methods: the traditional transversal cut using a low speed saw with a diamond disk and the X-ray computed microtomography (micro-CT). Vertebrae from ten Gymnura altavela sampled of the southeastern cost of Brazil were manually cleaned of organic tissue and air-dried in a room with a dehumidifier for 48 hours. For the traditional method, vertebrae were embedded in resin, cut and photographed using a stereomicroscope. Concurrently, uncut vertebrae from the same individuals were scanned using a Bruker SKYSCAN 1273 micro-CT. Two distinct settings were applied depending on vertebral width: 50 kV voltage and 220 mA current when up to 3mm and 60 kV voltage and 230 mA when larger. A 0.5 mm aluminium filter was applied in all cases. For both methods, two independent annuli reading were performed, in random order and without size information. Reproducibility between the readings was assessed using the mean percent error index (IAPE), the mean coefficient of variation (ACV) and the percent agreement (PA). Results for the traditional cutting method were: IAPE = 13.71%, ACV = 19.39 and PA = 60%. Despite the small sample size, the IAPE value was only slightly larger than the value normally used for elasmobranchs (up to 10%). In turn, the micro-CT method improved reading precision, especially for older individuals, with IAPE = 2%, ACV = 2.83 and PA = 90%. Therefore, our results highlight the potential to use micro-CT to reduce reading biases and facilitate elasmobranch age assessments.