A Pilot Study on the Effectiveness of
Hematoxylin Staining for Improved Age Determination of Skate Vertebrae
Figure 6. Longnose skate (Raja rhina) vertebral thin sections from the same individual prepared using the standard and new method. The unstained specimen (top) was aged at 22 years. The stained specimen (bottom) was aged at 16 years. Increased contrast on corpus calcareum tip of stained specimen may lead to interpretation differences between the two methods.
Figure 7. Alaska skate (Bathyraja parmifera) vertebral thin sections from the same individual. The unstained specimen (top) was aged at 9 years. The stained specimen (bottom) was aged at 9 years. Contrast has been increased with staining for the entire thin section including the intermedialia and corpus calcarea.
The Age and Growth (A&G) Program is currently evaluating a new histological preparation method for age determination using skate vertebrae. The stained-thin-section method was developed and successfully applied to Malacoraja senta, an Atlantic skate species, by Lisa Natanson at the Northeast Fisheries Science Center (NEFSC) and later adapted and applied to spiny dogfish (Squalus acanthias) by Walter Bubley at the University of New Hampshire (UNH).
The A&G Program initiated a pilot study in December 2010 to test this new method on vertebral thin sections from Alaskan skate species (big (Raja binoculata), longnose (R. rhina), and Alaska (Bathyraja parmifera) skates). This study’s main objective is to compare ageing interpretations and precision between the unstained (standard) method of thin section preparation and the new method (Figs. 6 and 7).
To date, vertebrae from 33 skates have been prepared using both methods: big (n = 10), longnose (n = 13), and Alaska (n = 10). Two thin sections per individual were removed from vertebrae at a thickness of approximately 0.30 mm using an Isomet™ 5000 linear precision saw. One untreated thin section from each skate was slide-mounted as a control. The other thin section was decalcified with a rapid decalcifier (RDO), stained with Harris hematoxylin, destained with acid-alcohol solution, soaked in glycerin, and slide mounted. Completed thin sections were examined under a dissecting microscope with transmitted light.
A small number of specimens have been aged by two people, a reader and a tester, using both methods. Within a sample of longnose skates collected in 2009 in the Gulf of Alaska, unstained vertebrae (n = 29) yielded a reader-tester percent agreement of 34.5%, with a reader-tester bias of 17:2. A subsample of vertebrae from the 2009 specimens, selected for discrepancies between the reader and tester’s age estimates using the standard approach, were stained and evaluated.
The stained subsample of longnose skates (n = 13) yielded a reader-tester percent agreement of 23.1%, with a reader-tester bias of 7:3. Observed ages ranged from 1 to 22 years for unstained thin sections and 4 to 17 years for stained thin sections. While bias appears to have improved using the new method, percent agreement decreased; however, due to the small sample size tested thus far, results should not be considered conclusive. Furthermore, the specimens selected for staining already had a high age discrepancy between the reader and tester using the standard approach.
Both reader and tester noted that general specimen clarity and growth-zone contrast improved with staining. Subsequently, this method may improve interpretation while addressing potential inter-reader bias for Alaskan skate species. The staining method may also assist in resolving current ageing criteria differences between North Pacific age determination laboratories. All Alaskan skate species will be further evaluated quantitatively for precision and bias with additional specimens. The development of an active vertebral thin section staining program is ongoing.
