Validation of single photon absorptiometry for on-farm measurement of density and mineral content of tail bone in cattle

A validation study examined the accuracy of a purpose-built single photon absorptiometry (SPA) instrument for making on-farm in vivo measurements of bone mineral density (BMD) in tail bones of cattle. In vivo measurements were made at the proximal end of the ninth coccygeal vertebra (Cy9) in steers of two age groups (each n = 10) in adequate or low phosphorus status. The tails of the steers were then resected and the BMD of the Cy9 bone was measured in the laboratory with SPA on the resected tails and then with established laboratory procedures on defleshed bone. Specific gravity and ash density were measured on the isolated Cy9 vertebrae and on 5-mm2 dorso-ventral cores of bone cut from each defleshed Cy9. Calculated BMD determined by SPA required a measure of tail bone thickness and this was estimated as a fraction of total tail thickness. Actual tail bone thickness was also measured on the isolated Cy9 vertebrae. The accuracy of measurement of BMD by SPA was evaluated by comparison with the ash density of the bone cores measured in the laboratory. In vivo SPA measurements of BMD were closely correlated with laboratory measurements of core ash density (r = 0.92). Ash density and specific gravity of cores, and all SPA measures of BMD, were affected by phosphorus status of the steers, but the effect of steer age was only significant (P < 0.05) for steers in adequate phosphorus status. The accuracy of SPA to determine BMD of tail bone may be improved by reducing error associated with in vivo estimation of tail bone thickness, and also by adjusting for displacement of soft tissue by bone mineral. In conclusion a purpose-built SPA instrument could be used to make on-farm sequential non-invasive in vivo measurements of the BMD of tailbone in cattle with accuracy acceptable for many animal studies.

Extracting DNA from ‘jaws’: high yield and quality from archived tiger shark (Galeocerdo cuvier) skeletal material

Archived specimens are highly valuable sources of DNA for retrospective genetic/genomic analysis. However, often limited effort has been made to evaluate and optimize extraction methods, which may be crucial for downstream applications. Here, we assessed and optimized the usefulness of abundant archived skeletal material from sharks as a source of DNA for temporal genomic studies. Six different methods for DNA extraction, encompassing two different commercial kits and three different protocols, were applied to material, so-called bio-swarf, from contemporary and archived jaws and vertebrae of tiger sharks (Galeocerdo cuvier). Protocols were compared for DNA yield and quality using a qPCR approach. For jaw swarf, all methods provided relatively high DNA yield and quality, while large differences in yield between protocols were observed for vertebrae. Similar results were obtained from samples of white shark (Carcharodon carcharias). Application of the optimized methods to 38 museum and private angler trophy specimens dating back to 1912 yielded sufficient DNA for downstream genomic analysis for 68% of the samples. No clear relationships between age of samples, DNA quality and quantity were observed, likely reflecting different preparation and storage methods for the trophies. Trial sequencing of DNA capture genomic libraries using 20 000 baits revealed that a significant proportion of captured sequences were derived from tiger sharks. This study demonstrates that archived shark jaws and vertebrae are potential high-yield sources of DNA for genomic-scale analysis. It also highlights that even for similar tissue types, a careful evaluation of extraction protocols can vastly improve DNA yield.

A mitochondrial species identification assay for Australian blacktip sharks (Carcharhinus tilstoni, C. limbatus and C. amblyrhynchoides) using real-time PCR and high-resolution melt analysis

Tropical Australian shark fisheries target two morphologically indistinguishable blacktip sharks, the Australian blacktip (Carcharhinus tilstoni) and the common blacktip (C. limbatus). Their relative contributions to northern and eastern Australian coastal fisheries are unclear because of species identification difficulties. The two species differ in their number of precaudal vertebrae, which is difficult and time consuming to obtain in the field. But, the two species can be distinguished genetically with diagnostic mutations in their mitochondrial DNA ND4 gene. A third closely related sister species, the graceful shark C. amblyrhynchoides, can also be distinguished by species-specific mutations in this gene. DNA sequencing is an effective diagnostic tool, but is relatively expensive and time consuming. In contrast, real-time high-resolution melt (HRM) PCR assays are rapid and relatively inexpensive. These assays amplify regions of DNA with species-specific genetic mutations that result in PCR products with unique melt profiles. A real-time HRM PCR species-diagnostic assay (RT-HRM-PCR) has been developed based on the mtDNA ND4 gene for rapid typing of C. tilstoni, C. limbatus and C. amblyrhynchoides. The assay was developed using ND4 sequences from 66 C. tilstoni, 33. C. limbatus and five C. amblyrhynchoides collected from Indonesia and Australian states and territories; Western Australia, the Northern Territory, Queensland and New South Wales. The assay was shown to be 100% accurate on 160 unknown blacktip shark tissue samples by full mtDNA ND4 sequencing.