Genetic diversity and individual identification of reintroduced otters (<Emphasis Type="Italic">Lutra lutra</Emphasis>) in north-eastern Spain by DNA genotyping of spraints

During the last century, otter populations in the Mediterranean area of the Iberian Peninsula were dramatically reduced and disappeared in many localities. A reintroduction programme was established in north-eastern Spain (Muga and Fluvià basins and the “Aiguamolls de l’Empordà” wetlands), by releasing 42 otters from four different Iberian populations, between 1995 and 2002. In order to evaluate the success of the reintroduction programme, we investigated microsatellite variation in the native populations of released otters as well as in the population present in the release area in 2004. We used non-invasively collected samples as the DNA source to avoid disturbing the animals in the wild. Laboratory procedures included the screening of samples and a multiple-tubes approach to detect and correct genotyping errors. Our results show that founders have been replaced by descendants in the release area and the population is in Hardy-Weinberg equilibrium, with a 1:1 sex ratio. Western Iberian otters—representing two-thirds of the founder group and released earlier—have greatly contributed to the genetic composition of the current population. The genetic patterns of otters found in a basin north of the studied area suggest a common origin with the released population. We also detected in a few otters the presence of alleles not identified in the original founder group, and we shall discuss the possible origin of these alleles. This paper is dedicated to the memory of Dr. Xavier Domingo-Roura.     

Eurasian otter (<Emphasis Type="Italic">Lutra lutra</Emphasis>) density estimate based on radio tracking and other data sources

Estimating animal population size is a critical task in both wildlife management and conservation biology. Precise and unbiased estimates are nonetheless mostly difficult to obtain, as estimates based on abundance over unit area are frequently inflated due to the “edge effect” bias. This may lead to the implementation of inappropriate management and conservation decisions. In an attempt to obtain an as accurate and conservative as possible picture of Eurasian otter (Lutra lutra) numbers, we combined radio tracking data from a subset of tracked individuals from an extensive project on otter ecology performed in Southern Portugal with information stemming from other data sources, including trapping, carcasses, direct observation of tagged and untagged individuals, relatedness estimates among genotyped individuals, and a minor contribution from non-invasive genetic sampling. In 158 km of water network, which covers a sampling area of 161 km² and corresponds to the minimum convex polygon constructed around the locations of five radio-tracked females, 21 animals were estimated to exist. They included the five radio-tracked, reproducing females and six adult males. Density estimates varied from one otter per 3.71–7.80 km of river length (one adult otter per 7.09–14.36 km) to one otter per 7.67–7.93 km² of range, depending on the method and scale of analysis. Possible biases and implications of methods used for estimating density of otters and other organisms living in linear habitats are highlighted, providing recommendations on the issue.     

Individual identification and sex determination of Eurasian otters (Lutra lutra) in Daegu city based on genetic analysis of otter spraint

The Eurasian otter, Lutra lutra, is a well known endangered species in South Korea that experienced rapid population decreases through poaching and industrialization until the 1980’s. To evaluate the genetic diversity and the existing number of otters recently found in Daegu City, 81 fecal samples collected from the Gumho River and Shincheon stream were subjected to DNA extraction for sex determination and genetic analysis using nuclear genetic markers. Individual identification and relatedness between individuals were detemined by genotypic data using twelve microsatellite loci, and sex identification was also determined based on sequence variation of the zinc finger protein gene on sex chromosomes. Our results showed that at least seven otter individuals were identified and the kinship relationships of seven individual pairs were determined. It was concluded that otters distributed widely in Daegu City have moderate levels of genetic diversity, and close monitoring of the small-sized otter population is necessary to promote successful settlement of the otters in the area.     

Evaluating otter reintroduction outcomes using genetic spatial capture–recapture modified for dendritic networks

Monitoring the demographics and genetics of reintroduced populations is critical to evaluating reintroduction success, but species ecology and the landscapes that they inhabit often present challenges for accurate assessments. If suitable habitats are restricted to hierarchical dendritic networks, such as river systems, animal movements are typically constrained and may violate assumptions of methods commonly used to estimate demographic parameters. Using genetic detection data collected via fecal sampling at latrines, we demonstrate applicability of the spatial capture–recapture (SCR) network distance function for estimating the size and density of a recently reintroduced North American river otter (Lontra canadensis) population in the Upper Rio Grande River dendritic network in the southwestern United States, and we also evaluated the genetic outcomes of using a small founder group (n = 33 otters) for reintroduction. Estimated population density was 0.23–0.28 otter/km, or 1 otter/3.57–4.35 km, with weak evidence of density increasing with northerly latitude (β = 0.33). Estimated population size was 83–104 total otters in 359 km of riverine dendritic network, which corresponded to average annual exponential population growth of 1.12–1.15/year since reintroduction. Growth was ≥40% lower than most reintroduced river otter populations and strong evidence of a founder effect existed 8–10 years post‐reintroduction, including 13–21% genetic diversity loss, 84%–87% genetic effective population size decline, and rapid divergence from the source population (F _ST accumulation = 0.06/generation). Consequently, genetic restoration via translocation of additional otters from other populations may be necessary to mitigate deleterious genetic effects in this small, isolated population. Combined with non‐invasive genetic sampling, the SCR network distance approach is likely widely applicable to demogenetic assessments of both reintroduced and established populations of multiple mustelid species that inhabit aquatic dendritic networks, many of which are regionally or globally imperiled and may warrant reintroduction or augmentation efforts.     

Genetic evaluation of an otter translocation program

The translocation of individuals from onepopulation to another is a common technique inwildlife conservation. However, the outcome oftranslocation programs is not always properlyevaluated and the relative contribution ofreleased individuals to the resident populationoften remains unknown. We used mitochondrialDNA and autosomal genetic markers to evaluatethe success of a translocation program ofEurasian otters (Lutra lutra) in Sweden.The program is regarded as successful becauseof subsequent population growths. Norwegianotters used for the restocking program could begenetically differentiated from Swedish otters.The releases took place at two sites. In anarea south of the first site, where 47 otterswere released, no genetic contribution of theintroduced animals to the population could beobserved and the genetic diversity was lowerthan before the releases. At the second site,the release of seven otters led to a change ingenetic composition of the resident population.The results of this study suggest that thegrowth of the otter population after therestocking may not be as dependent on thereleases as initially suspected. The geneticeffects of the translocations appear to berestricted to areas in the immediate vicinityof the release sites.     

Non-Invasive Genetic Mark-Recapture as a Means to Study Population Sizes and Marking Behaviour of the Elusive Eurasian Otter (Lutra lutra)

Quantifying population status is a key objective in many ecological studies, but is often difficult to achieve for cryptic or elusive species. Here, non-invasive genetic capture-mark-recapture (CMR) methods have become a very important tool to estimate population parameters, such as population size and sex ratio. The Eurasian otter (Lutra lutra) is such an elusive species of management concern and is increasingly studied using faecal-based genetic sampling. For unbiased sex ratios or population size estimates, the marking behaviour of otters has to be taken into account. Using 2132 otter faeces of a wild otter population in Upper Lusatia (Saxony, Germany) collected over six years (2006–2012), we studied the marking behaviour and applied closed population CMR models accounting for genetic misidentification to estimate population sizes and sex ratios. We detected a sex difference in the marking behaviour of otters with jelly samples being more often defecated by males and placed actively exposed on frequently used marking sites. Since jelly samples are of higher DNA quality, it is important to not only concentrate on this kind of samples or marking sites and to invest in sufficiently high numbers of repetitions of non-jelly samples to ensure an unbiased sex ratio. Furthermore, otters seemed to increase marking intensity due to the handling of their spraints, hence accounting for this behavioural response could be important. We provided the first precise population size estimate with confidence intervals for Upper Lusatia (for 2012: N^ = 20 ± 2.1, 95% CI = 16–25) and showed that spraint densities are not a reliable index for abundances. We further demonstrated that when minks live in sympatry with otters and have comparably high densities, a non-negligible number of supposed otter samples are actually of mink origin. This could severely bias results of otter monitoring if samples are not genetically identified.     

Similar estimates of population genetic composition and sex ratio derived from carcasses and faeces of Eurasian otter Lutra lutra

Collecting faeces is viewed as a potentially efficient way to sample elusive animals. Nonetheless, any biases in estimates of population composition associated with such sampling remain uncharacterized. The goal of this study was to compare estimates of genetic composition and sex ratio derived from Eurasian otter Lutra lutra spraints (faeces) with estimates derived from carcasses. Twenty per cent of 426 wild-collected spraints from SW England yielded composite genotypes for 7-9 microsatellites and the SRY gene. The expected number of incorrect spraint genotypes was negligible, given the proportions of allele dropout and false allele detection estimated using paired blood and spraint samples of three captive otters. Fifty-two different spraint genotypes were detected and compared with genotypes of 70 otter carcasses from the same area. Carcass and spraint genotypes did not differ significantly in mean number of alleles, mean unbiased heterozygosity or sex ratio, although statistical power to detect all but large differences in sex ratio was low. The genetic compositions of carcass and spraint genotypes were very similar according to confidence intervals of theta and two methods for assigning composite genotypes to groups. A distinct group of approximately 11 carcass and spraint genotypes was detected using the latter methods. The results suggest that spraints can yield unbiased estimates of population genetic composition and sex ratio.     

AGE- AND SEX-SPECIFIC MORTALITY AND POPULATION STRUCTURE IN SEA OTTERS

We used 742 beach-cast carcasses to characterize age- and sex-specific sea otter mortality during the winter of 1990-1991 at Bering Island, Russia. We also examined 363 carcasses recovered after the 1989 grounding of the T/V Exxon Valdez , to characterize age and sex composition in the living western Prince William Sound (WPWS) sea otter population. At Bering Island, mortality was male-biased (81%), and 75% were adults. The WPWS population was female-biased (59%) and most animals were subadult (79% of the males and 45% of the females). In the decade prior to 1990-1991 we found increasing sea otter densities (particularly among males), declining prey resources, and declining weights in adult male sea otters at Bering Island. Our findings suggest the increased mortality at Bering Island in 1990-1991 was a density-dependent population response. We propose male-maintained breeding territories and exclusion of juvenile females by adult females, providing a mechanism for maintaining densities in female areas below densities in male areas and for potentially moderating the effects of prey reductions on the female population. Increased adult male mortality at Bering Island in 1990-1991 likely modified the sex and age class structure there toward that observed in Prince William Sound.     

Ancient DNA reveals genotypic relationships among Oregon populations of the sea otter (<Emphasis Type="Italic">Enhydra lutris</Emphasis>)

The sea otter has experienced a dramatic population decline caused by intense human harvesting, followed by a century of recovery including relocation efforts to reestablish the species across its former range in the eastern Pacific. Although the otter was historically present along the coast in Oregon, there are currently no populations in this region and reintroduction efforts have failed. We examined the mtDNA genotypes of 16 pre-harvest otter samples from two Oregon locations in an attempt to determine the best genotypic match with extant populations. Our amplifications of a 222 base-pair portion of the control region from otters ranging in age from approximately 175–2000 years revealed four genotypes. The genotypic composition of pre-harvest otter populations appears to match best with those of contemporary populations from California and not from Alaska, where reintroduction stocks are typically derived.     

The reintroduction of the Eurasian otter (Lutra lutra) into the Netherlands: hidden life revealed by noninvasive genetic monitoring

The last recorded presence of the Eurasian otter (Lutra lutra) in the Netherlands dates from 1989 and concerned a dead individual. In 2002 a reintroduction programme was started, and between June 2002 and April 2008 a total of 30 individuals (10 males and 20 females) were released into a lowland peat marsh in the north of the Netherlands. Noninvasive genetic monitoring based on the genetic profiles obtained from DNA extracted from otter faeces (spraints) was chosen for the post-release monitoring of the population. To this end, the founding individuals were genotyped before release and spraints were collected in the release area each winter from 2002 to 2008. From June 2002 to April 2008 we analysed the genetic profile of 1,265 spraints on the basis of 7–15 microsatellite loci, 582 of which (46%) were successfully assigned to either released or newly identified genotypes. We identified 54 offspring (23 females and 31 males): the females started to reproduce after 2 years and the males after 4 years. The mating and reproductive success among males was strongly skewed, with a few dominant males fathering two-thirds of the offspring, but the females had a more even distribution. The effective population size (Ne) was only about 30% of the observed density (N), mainly because of the large variance in reproductive success among males. Most juvenile males dispersed to surrounding areas on maturity, whereas juvenile females stayed inside the area next to the mother’s territory. The main cause of mortality was traffic accidents. Males had a higher mortality rate (22 out of 41 males (54%) vs. 9 out of 43 females (21%)). During winter 2007/08 we identified 47 individuals, 41 of which originated from mating within the release area. This study demonstrates that noninvasive molecular methods can be used efficiently in post-release monitoring studies of elusive species to reveal a comprehensive picture of the state of the population.     

Prey selection in coastal Eurasian otters Lutra Iutra

Prey preferences and dietary differences between sex and age categories of Eurasian otters were studied in coastal Norwegian habitats Relative to their trapping frequency potential prey species with hard, spiny exoskeletons (crabs and sea urchins) or otherwise tough, spiny integuments (Labridae) were much less frequently found in spraints than fish species with soft integuments Spines did not protect fish with otherwise soft integuments from otter predation The number of non-fish taxa per otter stomach did not vary significantly between otter age categories despite presumed differences in hunting abilities (small cubs large cubs and subadults, adults) Relative frequency of occurrence of crabs and sea urchins was < 5% in the stomachs in each of these otter categories Anadromous, katadromous and freshwater fish species were infrequently eaten The coastal otter population during the study period probably had access to an adequate, and preferred, supply of marine fish prey
At the otter population level no prey size selection was conclusively demonstrated within the range of fish sizes sampled However, fish sizes eaten differed significantly between otter sex and age categories The fish sizes per stomach were on average larger in males than in females, regardless of age Adult males tended to eat the largest fishes Among the self provisioning age categories (subadult and adult otters) fish lengths differed significantly between otter males and females, but not between the otter age categories, and did not covary significantly with otter body length Fish eaten by females with old placental scars (potential mothers of fisheating cubs) were significantly smaller than those eaten by small cubs, provisioned by their mothers     

Procedures to genotype problematic non-invasive otter (Lutra lutra) samples

Non-invasive genetics is a powerful tool in wildlife research and monitoring, especially when dealing with elusive and rare species such as the Eurasian otter (Lutra lutra). Nevertheless, otter DNA obtained from scats and anal secretions appears to be exposed to very quick degradation processes, and the success rate in DNA amplification is lower than in other carnivores. We collected 191 samples from April to September 2011 along the river Sangro basin (Italy) which was recently re-colonized by the Eurasian otter. Using two sets of microsatellite loci (six Lut and seven OT loci), we investigated the influence of sample type and age, collection time, storage time, temperature and humidity on genotyping success and amplification success. We also tested the efficacy of different DNA extraction kits and storage buffer mediums. Finally, we compared amplification success rate, allelic dropout and false allele rates for each locus. We obtained a mean amplification success rate of 79.0 % and a genotyping success rate of 35.1 %. Fresh pure jellies yielded the highest amplification success and genotyping rate. Six microsatellite loci should be theoretically sufficient to distinguish the individual unrelated otters (PID?=?0.001), while 13 loci were needed to distinguish sibling otters (PID_sibs?=?0.002) in our population. We identified 11 otters, and molecular sexing ascertained the presence of five males, four females and two uncertain individuals. Generalized linear models highlighted a significant influence of sample type and age, temperature and humidity both on genotyping and amplification success.     

Influence of occupation history and habitat on Washington sea otter diet

Habitat characteristics are primary determinants of nearshore marine communities. However, biological drivers like predation can also be important for community composition. Sea otters (Enhydra lutris ssp.) are a salient example of a keystone species exerting top‐down control on ecosystem community structure. The translocation and subsequent population growth and range expansion of the northern sea otter (Enhydra lutris kenyoni) in Washington State over the last five decades has created a spatio‐temporal gradient in sea otter occupation time and density, and acts as a natural experiment to quantify how sea otter population status and habitat type influence sea otter diet. We collected focal observations of sea otters foraging at sites across the gradient in varying habitat types between 2010 and 2017. We quantified sea otter diet composition and diversity, and long‐term rates of energy gain across the gradient. We found that sea otter diet diversity was positively correlated with cumulative sea otter density, while rate of energy gain was negatively correlated with cumulative density. Additionally, we found that habitat type explained 1.77 times more variance in sea otter diet composition than sea otter cumulative density. Long‐term diet studies can provide a broader picture of sea otter population status in Washington State.     

Characteristics of sex-biased dispersal and gene flow in coastal river otters: implications for natural recolonization of extirpated populations

River otters (Lontra canadensis) were extirpated from much of their historic distribution because of exposure to pollution and urbanization, resulting in expansive reintroduction programmes that continue today for this and other species of otters worldwide. Bioaccumulation of toxins negatively affects fecundity among mustelids, but high vagility and different dispersal distances between genders may permit otter populations to recover from extirpation caused by localized environmental pollution. Without understanding the influence of factors such as social structure and sex-biased dispersal on genetic variation and gene flow among populations, effects of local extirpation and the potential for natural recolonization (i.e. the need for translocations) cannot be assessed. We studied gene flow among seven study areas for river otters (n = 110 otters) inhabiting marine environments in Prince William Sound, Alaska, USA. Using nine DNA microsatellite markers and assignment tests, we calculated immigration rates and dispersal distances and tested for isolation by distance. In addition, we radiotracked 55 individuals in three areas to determine characteristics of dispersal. Gender differences in sociality and spatial relationships resulted in different dispersal distances. Male river otters had greater gene flow among close populations (within 16-30 km) mostly via breeding dispersal, but both genders exhibited an equal, low probability of natal dispersal; and some females dispersed 60-90 km. These data, obtained in a coastal environment without anthropogenic barriers to dispersal (e.g. habitat fragmentation or urbanization), may serve as baseline data for predicting dispersal under optimal conditions. Our data may indicate that natural recolonization of coastal river otters following local extirpation could be a slow process because of low dispersal among females, and recolonization may be substantially delayed unless viable populations occurred nearby. Because of significant isolation by distance for male otters and low gene flow for females, translocations should be undertaken with caution to help preserve genetic diversity in this species.     

Asian small-clawed otter (Aonyx cinerea) urolithiasis prevalence in North America

The prevalence of urolithiasis in the North American Asian small-clawed otter (Aonyx cinerea) population was determined through a retrospective survey. A questionnaire regarding diet and radiographic or necropsy evidence of urolithiasis was sent to the 16 North American institutions or individuals listed in the Asian Small-Clawed Otter North American Regional Studbook. Completed forms were returned by 75% of questionnaire recipients. Individual surveys or necropsy reports were received for 79.8% of the living or dead animals listed as comprising the North American population. Renal calculi were detected in 66.1%, and cystic calculi in 23.2%, of the captive adult population that had been radiographed or necropsied. All otters with cystic calculi also had renal calculi. Bilateral renal calculi occurred in 83.8% of affected otters. Both males (61.2%) and females (72%) were frequently affected. The prevalence of urolithiasis in wild-born otters was 76.7%, and in adult (≥1 year) captive-born otters it was 53.8%. The higher prevalence in wild-born otters may be a reflection of the older mean age at which they were first evaluated. In the overwhelming majority of otters with renal calculi, the calculi were multiple and diffusely distributed throughout the renal parenchyma. Renal and cystic calculi analyzed were primarily composed of calcium oxalate or urates. Glucosuria was infrequently reported. Necropsies of dependent neonates (≤ 2 months old) that died revealed cystic calculi in one animal. Renal disease was the cause of, or a contributing factor in, the deaths of all older animals whose necropsy reports were reviewed. The captive diet may be a contributing factor to urolith formation and progression.     

Isolation and characterization of tetramicrosatellite DNA markers in the Eurasian otter (Lutra lutra)

Eight new tetramicrosatellite loci for Eurasian otter (Lutra lutra) were designed. Polymorphism of these eight loci in 29 otter individuals was tested. The results indicated that the allele numbers of each of loci ranged from three to five and the observed heterozygosity from 0.483 to 0.828. These new loci can be useful for population genetic research on otters and help improve the resolution of individual identification using noninvasive method.     

Individual identification and distribution assessment of otters (Lutra lutra) through non-invasive genetic sampling: Recovery of an endangered species in the Basque Country (Northern Spain)

Non-invasive genetic techniques have proven to be cost-effective for monitoring and studying otter populations, largely due to the linearity of otter territories and the marking behavior of the species. After a severe decline, in the 60, the Eurasian otter is recovering in the Iberian Peninsula. However, the recovery pattern is not homogeneous and the species is still considered “Threatened” in many regions. During 2007–2010 a systematic non-invasive genetic sampling effort was carried out to determine the spatial distribution and to estimate the population size of an endangered otter population in northern Iberian Peninsula (Basque Country). Samples were identified to species level by sequencing a 226 bp mtDNA fragment prior to genotyping. Among the 132 obtained samples, 127 (98.4%) belonged to the study species, one sample was genetically identified as European mink (Mustela lutreola) and one as American mink (Neovison vison) while genetic species confirmation was not possible in the three remaining samples. These results provided novel and accurate data on species distribution, highlighting an overall increase of 25% in 10 × 10 UTM grids occupied by otter and a clear pattern of re-colonization upstream of the main rivers. All samples corresponding to otter were subsequently individually genotyped using a novel multiplex panel of 11 microsatellite markers and sexed by typing the sex-chromosome-related gene ZFX/ZFY. We obtained a complete individual genetic profile for 55 samples (genotyping success 43%), corresponding to 20 different individuals (11 females, 6 males, and 3 individuals of unknown gender). The mean otter density in occupied areas estimated to be 0.09 (0.06–0.12) individuals per river kilometer. The present study enabled us to obtain updated and relevant information about this elusive species’ distribution and population size, essential to define population status and to design successful and effective management and conservation programs.     

Use of non-invasive techniques to determine population size of the marine otter in two regions of Peru

The marine otter (Lontra felina) can be found on rocky shores from the northern coast of Peru (9°S) to the extreme south of Argentina (56°S). This species is currently classified as endangered but there is little information on population size because marine otters are very difficult to observe and count. Between June and August 2012 we collected 240 samples of marine otter feces from seven localities in Peru. All locations were visited four times. One-hundred and thirty-three samples (55%) were successfully amplified at five to seven microsatellite loci and a sex-linked marker. We identified a minimum of 80 individuals (41 males and 39 females) across all locations for a density estimate of 4.4 otters per km, a value about 2X higher than estimates based on previous visual counts. Estimates using the program CAPWIRE averaged 12.6 otters/km, a value six times higher than estimates based on previous visual counts, although confidence limits were large due to the low number of recaptures. There was a strong positive relationship between the number of fresh scats and the number of unique genotypes, suggesting scat counts might be used to estimate the minimum number of otters at a site. Non-invasive genotyping of marine otter feces and scat counts will be valuable tools for estimating population sizes and monitoring movements of this secretive species.     

Preliminary Findings of Fecal Gonadal Hormone Concentrations in Six Captive Sea Otters (Enhydra lutris) after Deslorelin Implantation

The sea otter (Enhydra lutris) is a popular exhibit animal in many zoos and aquariums worldwide. Captive sea otters from these populations are owned by the United States Fish and Wildlife Service (USFWS). The USFWS has requested that these sea otters be prevented from breeding in order to save captive space for wild rescued animals. Sea otters are often housed in mixed sex groups, therefore a chemical contraceptive method or surgical removal of gonads must be used to prevent potential pregnancy. The contraceptive, Suprelorin® or deslorelin, has been used in many different species to effectively suppress reproduction but duration of effect may vary not only between species but also individuals. Here, we report the effects of one to several consecutive deslorelin implants on gonadal reproductive hormones found in fecal samples from six captive sea otters (two males and four females) compared to two control otters (one male and one female) housed at three zoological institutions. We documented the longitudinal hormone signatures of many stages of the contraceptive cycle including pretreatment (PT), stimulatory phase (S), effective contraception (EC), and hormone reversal (HR) that was characterized by a return to normal hormone levels. Deslorelin was found to be an effective contraceptive in sea otters and was found to be reversible documented by a live birth following treatment, however the duration of suppression in females was much longer than expected with a 6‐month and a 1‐year implant lasting between 3 and 4 years in females. Zoo Biol. 32:307–315, 2013. © 2012 Wiley Periodicals, Inc.     

Sex identification of Eurasian otter (<Emphasis Type="Italic">Lutra lutra</Emphasis>) non-invasive DNA samples using ZFX/ZFY sequences

We developed a new PCR/RFLP system targeted to amplify and cut a segment of the ZFX/ZFY gene in non-invasive otter (Lutra lutra) samples. This assay produced one sex-specific fragment in females (XX genotypes) and two fragments in males (XY genotypes), and is intrinsically more reliable than alternative systems (e.g., SRY genes) that are based on the amplification of a single Y-linked fragment. The new primer pair correctly identified the sex of 23 DNA extracted from sexed otter tissues. This procedure was used to sex unknown DNA extracted from otter scats. A multi-tube approach led to identify the sex of 72/91 (79%) samples, using a minimum of two PCR replicates. This procedure is currently used in non-invasive genetic monitoring of Italian otter populations.