Handling and Tagging Techniques for Implanting Juvenile American Shad with a New Acoustic Microtransmitter.

The use of telemetry techniques to better understand the behavior and survival of juvenile American shad (Alosa sapidissima), as they migrate through hydropower systems, has been challenging because shad are widely known to be particularly sensitive to handling. The goal of this study was to develop a tagging protocol using a new, acoustic micro transmitter that minimizes the detrimental effects of the tagging process and maximizes post-tagging survival of juvenile American shad. Limiting out-of-water handling and the use of brackish saltwater (7.5 parts per thousand) before and after tagging improved survival for shad tagged using a simple pectoral implantation method. This protocol provides a detailed, step-by-step procedure for tagging juvenile shad with acoustic transmitters. Fish tagged using this procedure and held in the laboratory for 60 days had an 81.5% survival rate, compared to 70% for their untagged counterparts. The successful tagging and handling practices developed in this study could be applied to field telemetry studies of juvenile shad and other sensitive species.

Integrating computer vision algorithms and RFID system for identification and tracking of group-housed animals: an example with pigs.

Precision livestock farming aims to individually and automatically monitor animal activity to ensure their health, well-being, and productivity. Computer vision has emerged as a promising tool for this purpose. However, accurately tracking individuals using imaging remains challenging, especially in group housing where animals may have similar appearances. Close interaction or crowding among animals can lead to the loss or swapping of animal IDs, compromising tracking accuracy. To address this challenge, we implemented a framework combining a tracking-by-detection method with a radio frequency identification (RFID) system. We tested this approach using twelve pigs in a single pen as an illustrative example. Three of the pigs had distinctive natural coat markings, enabling their visual identification within the group. The remaining pigs either shared similar coat color patterns or were entirely white, making them visually indistinguishable from each other. We employed the latest version of the You Only Look Once (YOLOv8) and BoT-SORT algorithms for detection and tracking, respectively. YOLOv8 was fine-tuned with a dataset of 3,600 images to detect and classify different pig classes, achieving a mean average precision of all the classes of 99%. The fine-tuned YOLOv8 model and the tracker BoT-SORT were then applied to a 166.7-min video comprising 100,018 frames. Results showed that pigs with distinguishable coat color markings could be tracked 91% of the time on average. For pigs with similar coat color, the RFID system was used to identify individual animals when they entered the feeding station, and this RFID identification was linked to the image trajectory of each pig, both backward and forward. The two pigs with similar markings could be tracked for an average of 48.6 min, while the seven white pigs could be tracked for an average of 59.1 min. In all cases, the tracking time assigned to each pig matched the ground truth 90% of the time or more. Thus, our proposed framework enabled reliable tracking of group-housed pigs for extended periods, offering a promising alternative to the independent use of image or RFID approaches alone. This approach represents a significant step forward in combining multiple devices for animal identification, tracking, and traceability, particularly when homogeneous animals are kept in groups.

In precision livestock farming, monitoring animal activity is crucial to ensure their health, well-being, and productivity. While digital cameras and computer vision algorithms offer a promising solution for this task, tracking individual animals of similar appearance when housed in groups can be challenging. Close interaction among animals can lead to a loss of individual identity, which affects tracking accuracy. To overcome this problem, we developed a framework that combines camera images with radio frequency identification (RFID) ear tags. This methodology was applied to a pen housing 12 pigs, with an RFID reader located inside the feeder. Among the pigs, three had unique coat markings, enabling them to be tracked most of the time without losing their identity (87% of the time). The remaining pigs could not be visually distinguished from each other, so information from the RFID system was used to recover lost IDs every time pigs entered the feeder. The framework achieves 97% accuracy in tracking, offering a reliable solution for monitoring group-housed pigs.

Pinpointing pigs: performance and challenges of an ultra-wideband real-time location system for tracking growing-finishing pigs under practical conditions.

Real-Time Location Systems (RTLSs) are promising precision livestock farming tools and have been employed in behavioural studies across various farm animal species. However, their application in research with fattening pigs is so far unexplored. The implementation of these systems has great potential to gain insight into pigs' spatial behaviour such as the use of functional areas and pigs' proximity to each other as indicators for social relationships. The aim of this study was therefore to validate the accuracy, precision, and data quality of the commercial Noldus Information Technology BV TrackLab system. We conducted different measurement sets: first, we performed static measurements in 12 pens at four different locations in each pen at three heights each using a single ultra-wideband tag (UWB). We recorded unfiltered x- and y-coordinates at 1 Hz. We repeated these measurements with six tags aligned in a 2 × 3 grid with varied spacing to test interference between the tags. We also tested dynamic performance by moving the tags along the centre line of the pens. Finally, we measured the data quality with 55 growing pigs in six pens, including the identification of location 'jumps' from the inside to the outside of the pen. Each pen housed ten animals fitted with a UWB tag attached to their farm ear tag. We collected data for 10 days and analysed seven 24-h periods of raw and filtered data. The mean accuracy of the RTLS measurements was 0.53 m (precision: 0.14 m) for single and 0.46 m (precision: 0.07 m) for grouped tags. Accuracy improved with increasing measurement height for single tags but less clearly for grouped tags (P [height single] = 0.01; P [height grouped] = 0.22). When tags were fitted to animals, 63.3% of the filtered data was lost and 21.8% of the filtered location estimates were outside the pens. Altogether, the TrackLab system was capable of fairly accurate and precise assignment of the functional areas where individual animals were located, but was insufficient for the analysis of social relationships. Furthermore, the frequent occurrence of gaps in signal transmission and the overall high data loss rates presented significant limitations. Additionally, the challenging hardware requirements for attaching sensors to the animals underline the need for further technological advances in RTLS for the application with growing-finishing pigs.

Survival, growth and tag retention of juvenile European eel (Anguilla anguilla L.) with implanted 12 mm passive integrated transponder tags and acoustic tags.

To evaluate the efficiency of tagging juvenile European eels with implanted 12 mm passive integrated transponder (PIT) tags or Eel/Lamprey acoustic transmitters (ELATs), the authors studied tag retention, survival and growth of eels (7-25 g). Experimental eels were obtained from an eel farm, tagged and then released in a series of shallow dug-out ponds with a surface area of c. 200 m2 . Tagged and control eels were distributed evenly, with 50 tagged and 50 control eels in each of four ponds, giving a total of 200 tagged and 200 control eels mixed. After 76 days, the ponds were drained, and eels were sampled and measured. A total of 344 eels (86%) were recaptured, indicating high survival. Tag retention was 99% as only one of the recaptured PIT-tagged eels had lost the tag and none of the ELAT tagged. The results demonstrated that tagging juvenile eels >16 cm with these small tags is indeed feasible. The growth of tagged and control fish was differentiated but generally low in length and negative in mass but did not differ between the three groups.

Development and validation of a 1 K sika deer (Cervus nippon) SNP Chip.

BACKGROUND: China is the birthplace of the deer family and the country with the most abundant deer resources. However, at present, China's deer industry faces the problem that pure sika deer and hybrid deer cannot be easily distinguished. Therefore, the development of a SNP identification chip is urgently required. RESULTS: In this study, 250 sika deer, 206 red deer, 23 first-generation hybrid deer (F1), 20 s-generation hybrid deer (F2), and 20 third-generation hybrid deer (F3) were resequenced. Using the chromosome-level sika deer genome as the reference sequence, mutation detection was performed on all individuals, and a total of 130,306,923 SNP loci were generated. After quality control filtering was performed, the remaining 31,140,900 loci were confirmed. From molecular-level and morphological analyses, the sika deer reference population and the red deer reference population were established. The Fst values of all SNPs in the two reference populations were calculated. According to customized algorithms and strict screening principles, 1000 red deer-specific SNP sites were finally selected for chip design, and 63 hybrid individuals were determined to contain red deer-specific SNP loci. The results showed that the gene content of red deer gradually decreased in subsequent hybrid generations, and this decrease roughly conformed to the law of statistical genetics. Reaction probes were designed according to the screening sites. All candidate sites met the requirements of the Illumina chip scoring system. The average score was 0.99, and the MAF was in the range of 0.3277 to 0.3621. Furthermore, 266 deer (125 sika deer, 39 red deer, 56 F1, 29 F2,17 F3) were randomly selected for 1 K SNP chip verification. The results showed that among the 1000 SNP sites, 995 probes were synthesized, 4 of which could not be typed, while 973 loci were polymorphic. PCA, random forest and ADMIXTURE results showed that the 1 K sika deer SNP chip was able to clearly distinguish sika deer, red deer, and hybrid deer and that this 1 K SNP chip technology may provide technical support for the protection and utilization of pure sika deer species resources. CONCLUSION: We successfully developed a low-density identification chip that can quickly and accurately distinguish sika deer from their hybrid offspring, thereby providing technical support for the protection and utilization of pure sika deer germplasm resources.

Mark-release-recapture of male Aedes aegypti (Diptera: Culicidae): Use of rhodamine B to estimate movement, mating and population parameters in preparation for an incompatible male program.

Rapid advances in biological and digital support systems are revolutionizing the population control of invasive disease vectors such as Aedes aegypti. Methods such as the sterile and incompatible insect techniques (SIT/IIT) rely on modified males to seek out and successfully mate with females, and in doing so outcompete the wild male population for mates. Currently, these interventions most frequently infer mating success through area-wide population surveillance and estimates of mating competitiveness are rare. Furthermore, little is known about male Ae. aegypti behaviour and biology in field settings. In preparation for a large, community scale IIT program, we undertook a series of mark- release-recapture experiments using rhodamine B to mark male Ae. aegypti sperm and measure mating interactions with females. We also developed a Spatial and Temporally Evolving Isotropic Kernel (STEIK) framework to assist researchers to estimate the movement of individuals through space and time. Results showed that ~40% of wild females captured daily were unmated, suggesting interventions will need to release males multiple times per week to be effective at suppressing Ae. aegypti populations. Males moved rapidly through the landscape, particularly when released during the night. Although males moved further than what is typically observed in females of the species, survival was considerably lower. These unique insights improve our understanding of mating interactions in wild Ae. aegypti populations and lay the foundation for robust suppression strategies in the future.

Multi-seasonal systematic camera-trapping reveals fluctuating densities and high turnover rates of Carpathian lynx on the western edge of its native range.

Camera-trapping and capture-recapture models are the most widely used tools for estimating densities of wild felids that have unique coat patterns, such as Eurasian lynx. However, studies dealing with this species are predominantly on a short-term basis and our knowledge of temporal trends and population persistence is still scarce. By using systematic camera-trapping and spatial capture-recapture models, we estimated lynx densities and evaluated density fluctuations, apparent survival, transition rate and individual's turnover during five consecutive seasons at three different sites situated in the Czech-Slovak-Polish borderland at the periphery of the Western Carpathians. Our density estimates vary between 0.26 and 1.85 lynx/100 km2 suitable habitat and represent the lowest and the highest lynx densities reported from the Carpathians. We recorded 1.5-4.1-fold changes in asynchronous fluctuated densities among all study sites and seasons. Furthermore, we detected high individual's turnover (on average 46.3 ± 8.06% in all independent lynx and 37.6 ± 4.22% in adults) as well as low persistence of adults (only 3 out of 29 individuals detected in all seasons). The overall apparent survival rate was 0.63 ± 0.055 and overall transition rate between sites was 0.03 ± 0.019. Transition rate of males was significantly higher than in females, suggesting male-biased dispersal and female philopatry. Fluctuating densities and high turnover rates, in combination with documented lynx mortality, indicate that the population in our region faces several human-induced mortalities, such as poaching or lynx-vehicle collisions. These factors might restrict population growth and limit the dispersion of lynx to other subsequent areas, thus undermining the favourable conservation status of the Carpathian population. Moreover, our study demonstrates that long-term camera-trapping surveys are needed for evaluation of population trends and for reliable estimates of demographic parameters of wild territorial felids, and can be further used for establishing successful management and conservation measures.

Assessing DNA for fish identifications from reference collections: the good, bad and ugly shed light on formalin fixation and sequencing approaches.

Natural history collections are repositories of biodiversity and are potentially used by molecular ecologists for comparative taxonomic, phylogenetic, biogeographic and forensic purposes. Specimens in fish collections are preserved using a combination of methods with many fixed in formalin and then preserved in ethanol for long-term storage. Formalin fixation damages DNA, thereby limiting genetic analyses. In this study, the authors compared the DNA barcoding and identification success for frozen and formalin-fixed tissues obtained from specimens in the CSIRO Australian National Fish Collection. They studied 230 samples from fishes (consisting of >160 fish species). An optimized formalin-fixed, paraffin-embedded DNA extraction method resulted in usable DNA from degraded tissues. Four mini barcoding assays of the mitochondrial DNA (mtDNA) were characterized with Sanger and Illumina amplicon sequencing. In the good quality DNA (without exposure to formalin), up to 88% of the specimens were correctly matched at the species level using the cytochrome oxidase subunit 1 (COI) mini barcodes, whereas up to 58% of the specimens exposed to formalin for less than 8 weeks were correctly identified to species. In contrast, 16S primers provided higher amplification success with formalin-exposed tissues, although the COI gene was more successful for identification. Importantly, the authors found that DNA of a certain size and quality can be amplified and sequenced despite exposure to formalin, and Illumina sequencing provided them with greater power of resolution for taxa identification even when there was little DNA present. Overall, within parameter constraints, this study highlights the possibilities of recovering DNA barcodes for identification from formalin-fixed fish specimens, and the authors provide guidelines for when successful identification could be expected.

Quantification of alizarin red S uptake in coregonid eggs after mass-marking.

An easy method to measure the uptake rate of the persistent dye alizarin red S (ARS) during marking of whitefish eggs was established and used to measure the ARS content in three different whitefish species during and at the end of the marking procedure. Those values show that only 6-10% of the ARS in the marking solution will be absorbed by the eggs (0.0061-0.0119 mg per egg). Additional analyses 6, 15 and 36 months after marking showed ARS levels below the response level (<6.9 µg kg-1 ).

[A multiplex PCR method based on nuclear and cytoplasmic inheritance to identify the horse and donkey-derived components of Asini Colla Corii and the hide].

To identify the original components of Asini Colla Corii and its raw material hides provides a guarantee for authenticity of Asini Colla Corii. It is urgent for Asini Colla Corii production enterprises and market supervision departments to develop effective identification methods of Asini Colla Corii and hides derived from horses, donkeys, mules and hinnies. This study screened species-specific DNA sequences of nuclear and mitochondrial genomes as detection targets, designed horse and donkey specific primers and established multiple PCR identification methods for identifying the animal hides (including the horse, donkey, mule and hinny) and Asini Colla Corii containing horse-derived and donkey-derived components. Our method can identify the horse, donkey, mule and hinny hides and horse, donkey-derived components of Asini Colla Corii with high species specificity (no crossed amplification was observed ). The limit of detection was 0.2 ng DNA. The method developed in this study provides technical support for Asini Colla Corii production enterprises and market supervision departments.

Movement patterns of the grey field slug (Deroceras reticulatum) in an arable field.

We report the results of an experiment on radio-tracking of individual grey field slugs in an arable field and associated data modelling designed to investigate the effect of slug population density in their movement. Slugs were collected in a commercial winter wheat field in which a 5x6 trapping grid had been established with 2m distance between traps. The slugs were taken to the laboratory, radio-tagged using a recently developed procedure, and following a recovery period released into the same field. Seventeen tagged slugs were released singly (sparse release) on the same grid node on which they had been caught. Eleven tagged slugs were released as a group (dense release). Each of the slugs was radio-tracked for approximately 10 h during which their position was recorded ten times. The tracking data were analysed using the Correlated Random Walk framework. The analysis revealed that all components of slug movement (mean speed, turning angles and movement/resting times) were significantly different between the two treatments. On average, the slugs released as a group disperse more slowly than slugs released individually and their turning angle has a clear anticlockwise bias. The results clearly suggest that population density is a factor regulating slug movement.

Longer is Not Always Better: Optimizing Barcode Length for Large-Scale Species Discovery and Identification.

New techniques for the species-level sorting of millions of specimens are needed in order to accelerate species discovery, determine how many species live on earth, and develop efficient biomonitoring techniques. These sorting methods should be reliable, scalable, and cost-effective, as well as being largely insensitive to low-quality genomic DNA, given that this is usually all that can be obtained from museum specimens. Mini-barcodes seem to satisfy these criteria, but it is unclear how well they perform for species-level sorting when compared with full-length barcodes. This is here tested based on 20 empirical data sets covering ca. 30,000 specimens (5500 species) and six clade-specific data sets from GenBank covering ca. 98,000 specimens ($>$20,000 species). All specimens in these data sets had full-length barcodes and had been sorted to species-level based on morphology. Mini-barcodes of different lengths and positions were obtained in silico from full-length barcodes using a sliding window approach (three windows: 100 bp, 200 bp, and 300 bp) and by excising nine mini-barcodes with established primers (length: 94-407 bp). We then tested whether barcode length and/or position reduces species-level congruence between morphospecies and molecular operational taxonomic units (mOTUs) that were obtained using three different species delimitation techniques (Poisson Tree Process, Automatic Barcode Gap Discovery, and Objective Clustering). Surprisingly, we find no significant differences in performance for both species- or specimen-level identification between full-length and mini-barcodes as long as they are of moderate length ($>$200 bp). Only very short mini-barcodes (<200 bp) perform poorly, especially when they are located near the 5$^\prime$ end of the Folmer region. The mean congruence between morphospecies and mOTUs was ca. 75% for barcodes $>$200 bp and the congruent mOTUs contain ca. 75% of all specimens. Most conflict is caused by ca. 10% of the specimens that can be identified and should be targeted for re-examination in order to efficiently resolve conflict. Our study suggests that large-scale species discovery, identification, and metabarcoding can utilize mini-barcodes without any demonstrable loss of information compared to full-length barcodes. [DNA barcoding; metabarcoding; mini-barcodes; species discovery.].

Surgical implantation of radio tags in three eel species (Anguilla spp.) in South Africa.

Studies have reported poor survival of surgically tagged freshwater fishes in warm African waters. This study aimed to assess the applicability of using radio telemetry (and surgical implantation of tags) for Anguilla spp. Nineteen yellow eels (Anguilla bengalensis, A. marmorata and A. mossambica) were surgically implanted with radio tags between October 2018 and January 2019 in the Thukela River, South Africa. Most eels were alive 6 months after tagging, and recaptured eels displayed advanced or complete healing at the incision site. Therefore, this method appears suitable for African freshwater eels.

A new model study species: high accuracy of discrimination between individual freckled hawkfish (Paracirrhites forsteri) using natural markings.

Variations between distinct natural markings of freckled hawkfish (Paracirrhites forsteri) could allow in situ identification of individuals from underwater photography. Receiver operating characteristic analysis was used to assess the ability of the Interactive Individual Identification System (I3 S) software to assist in discriminating between images of P. forsteri individuals. This study's results show the high discriminant ability of I3 S to differentiate between unlike individuals and identify images of the same individual. The ability to use automatic computer-aided assistance in the study of this species will enable future research to explore behaviour and movements of individuals in the wild.

Differentiating three Indian shads by applying shape analysis from digital images.

Tenualosa ilisha, T. toli and Hilsa kelee are three Indian shad species of the family Clupeidae. In past, relationships among these shads have been explored in few morphological studies but still remain poorly understood. In this study, 120 specimens of three shad species were collected from the wild and a truss network system of 13 landmarks was interconnected to yield 77 size-adjusted characters. Discriminant function analysis (DFA) produced 13 discriminating variables in delineating the three species. The landmark based morphometric distance between origin of dorsal fin and posterior end of eye was the most important character in discriminating the three species. Three prominent characters extracted from DFA (distance between posterior end of operculum and posterior end of eye, distance between origin of dorsal fin and posterior end of eye and distance between insertion of pelvic fin and end of operculum) were used as traditional morphometric characters and their ratio to standard length were found significant in differentiating the three shad species. The present study strongly supports the efficiency of landmark-based morphological characters generated from digital images in discriminating the species and can be used as a rapid diagnostic tool in recognizing them.

Nail varnish helps researchers pinpoint numbers of rare bats.

Emma Huntley explains how researchers from the Zoological Society of London are using an inventive way to calculate bat populations.

A comparison of population estimation techniques for individually unidentifiable free-roaming dogs.

BACKGROUND: Measuring the size of free roaming dog populations quickly and accurately is critical in the implementation of numerous preventive health and population control interventions. However, few studies have investigated the relative performance of population size assessment tools when applied to dogs. The aim of this study was to compare the commonly used mark-resight methodology with distance sampling methods, which are less resource intensive, to estimate free-roaming dog abundance in Goa, India. Twenty-six working zones were surveyed along all roads twice by the same surveyor at the same time of day, following a vaccination campaign which included marking of vaccinated dogs with a coloured paint. The Chapman estimate was then used to evaluate the mark-resight abundance. Additionally, the number of dogs and perpendicular distance from the road for all dogs sighted was recorded. This was used to estimate dog density and abundance using distance sampling methods. The detection function was fitted based on goodness-of-fit and AIC. RESULTS: The Chapman abundance estimate for the entire study area was 5202 dogs (95%CI 4733.8-5671.0), and the distance sampling method abundance estimate was 5067 dogs (95%CI 4454.3-5764.2). For individual working zones, after taking other factors into account in a mixed effects model, the average distance sampling estimate was 35% higher (95%CI 20-53%) than the Chapman estimate. There was also evidence of a difference in estimates between surveyors of 21% (95%CI 7-37%) and between days (22% lower on day 2, 95%CI 8-38%) for individual working zones. CONCLUSION: Our study demonstrated that the distance sampling estimates were comparable overall to the Chapman method of abundance estimation of free roaming dogs across the entire study region but there was noticeable variation between the two methods when individual zones were compared. Consequently, distance sampling methods may be suitable to enumerate dogs over large areas in a more time efficient manner than the widely used mark-resight approach.

Welfare and Scientific Considerations of Tattooing and Ear Tagging for Mouse Identification.

Ear tagging is perceived as less painful or stressful than tattooing and therefore is generally considered less harmful or costly to welfare. However, ear tags are more difficult to read than tattoos and can fall out, and mice usually require restraint for the tag numbers to be read accurately. We assessed the welfare and scientific implications of tattooing by using a commercial device compared with restraint in a device versus ear tagging. Male and female BALB/c mice (n = 32) underwent procedures after 1 wk of tail or nonaversive (tunnel) handling to determine whether tunnel handling reduced anxiety. Pain was evaluated using both the Mouse Grimace Scale (MGS) and manual and automated behavior analyses; light-dark preference testing and voluntary interaction with the handler's hand were used to assess anxiety. Tail inflammation after tattooing was quantified using bioluminescent imaging, and ear tag and tattoo misidentification rates were estimated from volunteer staff records. Tunnel handling reduced anxiety compared with tail handling. According to the MGS, tattooing was not more painful than ear tagging but caused significant tail inflammation and more agitation and anxiety. However, all tattoos were read correctly without handling, whereas all ear tagged mice needed restraint, and at least 25% of the tag codes were misread. Handling stress together with identification errors at this rate represent potentially serious concerns regarding the scientific integrity of data from studies using ear tagging. These concerns are unlikely to arise with tattooing. Although tattooing was stressful, so were restraint and ear tagging. However, considering the other major advantages of tattooing, the total costs associated with tattooing were not substantially greater than for ear tagging.

Examining the viability of dorsal fin pigmentation for individual identification of poorly-marked delphinids.

Dolphin photo-identification has traditionally relied only on distinctive markings on the dorsal fin-this is problematic for delphinids whose populations exhibit a low mark ratio. We used common dolphins (genus Delphinus) as a model species to assess the viability of using pigmentation for photo-identification. Using a photo-identification catalogue of 169 adult individuals collected between 2002 and 2013, we extracted features that quantified pigmentation in a manner that was robust to lighting artefacts and dorsal fin orientation. We determined the proportion of individuals which exhibited pigmentation and examined temporal stability by (i) visually examining individuals and (ii) testing for seriation. We found 88-91% of images could be manually matched to the correct individual in the catalogue based on pigmentation patterns alone. A linear discriminant analysis classifier correctly identified the correct individual 77% of the time. We found 95% common dolphins exhibited distinctive pigmentation-all of which were temporarily stable. Our work challenges the current thinking that pigmentation is an unreliable feature for delphinid photo-identification and suggests that this feature could be applied to common dolphins and other poorly-marked delphinids.