Age Estimation from Teeth in Longman’s Beaked Whales (Indopacetus Pacificus) Stranded in New Caledonia (South Pacific)
At a Glance
Section titled “At a Glance”| Metadata | Details |
|---|---|
| Publication Date | 2021-05-01 |
| Journal | Archimer (Ifremer) |
| Authors | Christina Lockyer, Claire Garrigue |
| Institutions | Écologie Marine Tropicale des Océans Pacifique et Indien |
| Citations | 1 |
| Analysis | Full AI Review Included |
Executive Summary
Section titled “Executive Summary”This study details the development and comparison of two distinct material preparation protocols for the age estimation of Longman’s beaked whale teeth, focusing on optimizing the visualization of Growth Layer Groups (GLGs) in dentine and cementum for engineering and material science applications.
- Material Focus: Calcified tissues (dentine and cementum) from cetacean teeth, used as chronological biomarkers.
- Methodology Comparison: Two protocols were tested: 1) Untreated, precision-cut thin sections (150 µm) using a diamond saw; and 2) Decalcified, stained ultra-thin sections (10-25 µm) prepared via wafering, chemical treatment (RDO™), and freezing microtome.
- Optimal Tissue Matrix: Cementum was identified as the preferred tissue for age estimation, consistently providing clearer and higher GLG counts than dentine.
- Processing Enhancement: Decalcification and histological staining (Ehrlich’s Acid Haematoxylin) significantly improved GLG resolution, yielding higher counts (up to 26 GLGs observed) compared to untreated sections.
- Dentine Limitations: Dentine analysis was compromised by material anomalies, including extensive mineral resorption and the presence of pulp stones, making GLG counting unreliable in this tissue for the species studied.
- Precision Equipment: The methodology relies on high-precision cutting tools (Isomet low-speed diamond saw) and specialized histological equipment (freezing microtome) for preparing ultra-thin, high-quality sections.
Technical Specifications
Section titled “Technical Specifications”| Parameter | Value | Unit | Context |
|---|---|---|---|
| Thin Section Thickness (Method 1) | 150 | µm | Cut centrally through crown and root using Isomet diamond saw. |
| Thick Wafer Thickness (Method 2) | 2.0-2.5 | mm | Central wafer removed for decalcification process. |
| Ultra-Thin Section Thickness (Method 2) | 10-25 | µm | Cut from decalcified wafer using freezing microtome. |
| Freezing Stage Temperature | ~ -12 | °C | Operating temperature of the benchtop freezing microtome. |
| Rapid Decalcification Time (RDO™) | Up to 15 | h | Duration for decalcification of the 2.5 mm wafer. |
| EAH Staining Time (Optimal Uptake) | Up to 45 | min | Duration for stain uptake in decalcified sections. |
| Dentine GLG Width | 25 | µm | Estimated width of Growth Layer Groups in dentine. |
| Cementum GLG Width | 10-15 | µm | Estimated width of Growth Layer Groups in cementum. |
| Maximum Age Estimate (Male #2) | 20-26 | GLGs | Based on stained cementum sections (assumed equivalent to years). |
| Microscope Magnification Range | x7-x40 | N/A | Used for examining untreated thin sections. |
Key Methodologies
Section titled “Key Methodologies”The study employed two primary protocols for preparing tooth sections for microscopic analysis:
Protocol 1: Untreated Thin Sectioning (Mechanical Preparation)
Section titled “Protocol 1: Untreated Thin Sectioning (Mechanical Preparation)”- Cleaning: Teeth were thoroughly cleaned of all residual gum tissue.
- Mounting: Teeth were affixed to a wood block using standard hobby glue sticks.
- Precision Cutting: 2-3 sections, approximately 150 µm thick, were cut centrally through the crown and root using a Buehler Isomet low-speed circular diamond saw.
- Analysis: Sections were examined under a binocular dissecting microscope using alternating transmitted plain light and transmitted polarized light to identify GLGs in dentine and cementum.
Protocol 2: Decalcified and Stained Ultra-Thin Sectioning (Histological Preparation)
Section titled “Protocol 2: Decalcified and Stained Ultra-Thin Sectioning (Histological Preparation)”- Wafering: A thicker central section (wafer, 2.0-2.5 mm) was removed adjacent to the first thin section.
- Decalcification: The wafer was soaked in RDO™ (a rapid decalcifying solution) for up to 15 hours.
- Microtome Slicing: Ultra-thin sections (10-25 µm) were cut from the decalcified wafer using a freezing microtome (stage temperature approximately -12 °C).
- Staining: Sections were placed in an agitated, ripe solution of Ehrlich’s Acid Haematoxylin (EAH) for up to 45 minutes.
- Rinsing and Differentiation: Sections were rinsed in water, then soaked in a weak ammonia solution (approx. 3 min) until the color uniformly shifted from deep red to purple-blue.
- Mounting: Sections were floated onto 5% gelatin-coated slides, air-dried, and permanently mounted under glass cover slips using DPX (a fast-drying mixture of distyrine and xylene).
- Analysis: Sections were examined using transmitted plain light microscopy.
Commercial Applications
Section titled “Commercial Applications”The specialized material processing and analysis techniques developed in this research are highly relevant to industries requiring high-resolution structural analysis of hard, brittle, or composite materials.
- Biomaterials and Dental Research:
- Failure analysis and wear studies of dental composites, ceramics, and prosthetic materials.
- Developing standardized preparation protocols for analyzing bone and enamel interfaces.
- Forensic and Archaeological Science:
- Establishing reliable chronological markers (like GLGs) in calcified tissues for age determination in human and animal remains.
- Optimizing decalcification and staining methods for degraded or mineralized samples.
- Precision Machining and Metrology:
- Application of low-speed diamond saws for preparing ultra-flat, damage-free cross-sections of brittle engineering materials (e.g., semiconductors, geological samples, advanced ceramics) for SEM/TEM analysis.
- Environmental and Toxicological Monitoring:
- Using calcified tissues as chronological archives to map the deposition history of heavy metals (e.g., Cadmium, as discussed in the paper) in long-lived organisms, requiring precise sectioning and elemental mapping.
- Histopathology and Medical Device Integration:
- Specialized preparation of tissue samples containing hard inclusions or medical implants, where decalcification and microtome sectioning are necessary to study tissue-device integration without damaging the surrounding matrix.
View Original Abstract
Seven Longman’s beaked whales mass stranded in New Caledonia in November 2013, of which 4 ultimately died, in a first worldwide event reported for this poorly known Ziphioid species. Teeth were extracted, collected and thoroughly cleaned of gum tissue from 3 females ranging from juvenile to adult and one adult male. These were sectioned (crown-root) and prepared using two different methods and examined under microscope magnification when Growth Layer Groups (GLGs) in both dentine and cement were successfully identified. The methods employed for aging included 1) sectioning centrally at approx. 150 µm through crown and root on an Isomet circular diamond saw and examining under a microscope using both transmitted polarised light and plain light; and, 2) thick sectioning (wafering) at approx. 2.5 mm and subsequent decalcifiation in RDO™ (a proprietory brand, Illinois, USA) and then thin sectioning the wafer at 10-25 µm and staining with Ehrlich’s acid haematoxylin. GLGs were investigated in both dentine (25 micron) and cementum (10-15 µm). Layering was evident in both tissues but higher counts were more evident in thin stained sections of cementum. Although dentinal GLGs in untreated tooth sections have been used successfully for aging in Ziphioid species Hyperoodon ampullatus (Christensen 1973, Feyrer et al., 2020), it is believed this is the first time that teeth have been used for estimating age from GLGs in this tropical species.\n