Genetic rescue-an increase in population fitness following the introduction of new alleles-has been proven to ameliorate inbreeding depression in small, isolated populations, yet is rarely applied as a conservation tool. A lingering question regarding genetic rescue in wildlife conservation is how long beneficial effects persist in admixed populations. Using data collected over 40 years from 1192 endangered Florida panthers (Puma concolor coryi) across nine generations, we show that the experimental genetic rescue implemented in 1995-via the release of eight female pumas from Texas-alleviated morphological, genetic, and demographic correlates of inbreeding depression, subsequently preventing extirpation of the population. We present unequivocal evidence, for the first time in any terrestrial vertebrate, that genetic and phenotypic benefits of genetic rescue remain in this population after five generations of admixture, which helped increase panther abundance (> fivefold) and genetic effective population size (> 20-fold). Additionally, even with extensive admixture, microsatellite allele frequencies in the population continue to support the distinctness of Florida panthers from other North American puma populations, including Texas. Although threats including habitat loss, human-wildlife conflict, and infectious diseases are challenges to many imperiled populations, our results suggest genetic rescue can serve as an effective, multi-generational tool for conservation of small, isolated populations facing extinction from inbreeding.

The Seychelles magpie-robin\'s (SMR) five island populations exhibit some of the lowest recorded levels of genetic diversity among endangered birds, and high levels of inbreeding. These populations collapsed during the 20th century, and the species was listed as Critically Endangered in the IUCN Red List in 1994. An assisted translocation-for-recovery program initiated in the 1990s increased the number of mature individuals, resulting in its downlisting to Endangered in 2005. Here, we explore the temporal genomic erosion of the SMR based on a dataset of 201 re-sequenced whole genomes that span the past ~150 years. Our sample set includes individuals that predate the bottleneck by up to 100 years, as well as individuals from contemporary populations established during the species recovery program. Despite the SMR\'s recent demographic recovery, our data reveal a marked increase in both the genetic load and realized load in the extant populations when compared to the historical samples. Conservation management may have reduced the intensity of selection by increasing juvenile survival and relaxing intraspecific competition between individuals, resulting in the accumulation of loss-of-function mutations (i.e. severely deleterious variants) in the rapidly recovering population. In addition, we found a 3-fold decrease in genetic diversity between temporal samples. While the low genetic diversity in modern populations may limit the species\' adaptability to future environmental changes, future conservation efforts (including IUCN assessments) may also need to assess the threats posed by their high genetic load. Our computer simulations highlight the value of translocations for genetic rescue and show how this could halt genomic erosion in threatened species such as the SMR.

Small, fragmented, or isolated populations are at risk of population decline due to fitness costs associated with inbreeding and genetic drift. The King Island scrubtit Acanthornis magna greeniana is a critically endangered subspecies of the nominate Tasmanian scrubtit A. m. magna, with an estimated population of <100 individuals persisting in three patches of swamp forest. The Tasmanian scrubtit is widespread in wet forests on mainland Tasmania. We sequenced the scrubtit genome using PacBio HiFi and undertook a population genomic study of the King Island and Tasmanian scrubtits using a double-digest restriction site-associated DNA (ddRAD) dataset of 5,239 SNP loci. The genome was 1.48 Gb long, comprising 1,518 contigs with an N50 of 7.715 Mb. King Island scrubtits formed one of four overall genetic clusters, but separated into three distinct subpopulations when analyzed independently of the Tasmanian scrubtit. Pairwise FST values were greater among the King Island scrubtit subpopulations than among most Tasmanian scrubtit subpopulations. Genetic diversity was lower and inbreeding coefficients were higher in the King Island scrubtit than all except one of the Tasmanian scrubtit subpopulations. We observed crown baldness in 8/15 King Island scrubtits, but 0/55 Tasmanian scrubtits. Six loci were significantly associated with baldness, including one within the DOCK11 gene which is linked to early feather development. Contemporary gene flow between King Island scrubtit subpopulations is unlikely, with further field monitoring required to quantify the fitness consequences of its small population size, low genetic diversity, and high inbreeding. Evidence-based conservation actions can then be implemented before the taxon goes extinct.

Genetic diversity underpins evolutionary potential that is essential for the long-term viability of wildlife populations. Captive populations harbor genetic diversity potentially lost in the wild, which could be valuable for release programs and genetic rescue. The Critically Endangered Arabian leopard (Panthera pardus nimr) has disappeared from most of its former range across the Arabian Peninsula, with fewer than 120 individuals left in the wild, and an additional 64 leopards in captivity. We (i) examine genetic diversity in the wild and captive populations to identify global patterns of genetic diversity and structure; (ii) estimate the size of the remaining leopard population across the Dhofar mountains of Oman using spatially explicit capture-recapture models on DNA and camera trap data, and (iii) explore the impact of genetic rescue using three complementary computer modeling approaches. We estimated a population size of 51 (95% CI 32-79) in the Dhofar mountains and found that 8 out of 25 microsatellite alleles present in eight loci in captive leopards were undetected in the wild. This includes two alleles present only in captive founders known to have been wild-sourced from Yemen, which suggests that this captive population represents an important source for genetic rescue. We then assessed the benefits of reintroducing novel genetic diversity into the wild population as well as the risks of elevating the genetic load through the release of captive-bred individuals. Simulations indicate that genetic rescue can improve the long-term viability of the wild population by reducing its genetic load and realized load. The model also suggests that the genetic load has been partly purged in the captive population, potentially making it a valuable source population for genetic rescue. However, the greater loss of its genetic diversity could exacerbate genomic erosion of the wild population during a rescue program, and these risks and benefits should be carefully evaluated. An important next step in the recovery of the Arabian leopard is to empirically validate these conclusions, implement and monitor a genomics-informed management plan, and optimize a strategy for genetic rescue as a tool to recover Arabia\'s last big cat.

Conservation is prioritized based on accepted taxa. As a consequence, a conservation incentive exists to emphasize inter-population differences to define taxa, potentially leading to taxonomic inflation. But stressing the uniqueness of threatened populations has the side effect of hindering conservation actions that promote inter-population gene flow, such as genetic rescue. These actions may be of critical importance for severely inbred populations involved in extinction vortices, for which an inflated taxonomy can become a conservation trap. Here, we exemplify this scenario with the western capercaillie (Tetrao urogallus, Phasianidae) population in the Cantabrian Mountains, described and legally listed as a subspecies not supported by recent molecular data. The Cantabrian capercaillie population is Critically Endangered after a long-lasting decline and a recent demographic collapse. It shows clear signs of inbreeding depression, including striking clutch size decreases as well as reduced hatchability and chick survival. This critical situation could be alleviated through a genetic rescue, but this possibility is hindered by inertias rooted in the putative uniqueness of the Cantabrian capercaillie. It had been previously argued that poor taxonomy could hamper conservation, through the oblivion of populations deserving, but not having, a taxonomic status. We show that taxonomic inflation can also become an obstacle for effective conservation.

Small, isolated populations are often characterised by low levels of genetic diversity. This can result in inbreeding depression and reduced capacity to adapt to changes in the environment, and therefore higher risk of extinction. However, sometimes these populations can be rescued if allowed to increase in size or if migrants enter, bringing in new allelic variation and thus increasing genetic diversity. This study uses experimental manipulation of population size and migration to quantify their effects on fitness in a challenging environment to better understand genetic rescue. Using small, replicated populations of Tribolium castaneum experimentally evolved to different temperature regimes we tested genetic and demographic rescue, by performing large-scale manipulations of population size and migration and examining fitness consequences over multiple generations. We measured fitness in high temperature (38°C) thermal lines maintained at their usual \'small\' population size of N = 100 individuals, and with \'large\' scaled up duplicates containing N≈10,000 individuals. We compared these large lines with and without migration (m = 0.1) for 10 generations. Additionally, we assessed the effects of outcrossing at an individual level, by comparing fitness of hybrid (thermal line × stock) offspring with within-line crosses. We found that, at the population level, a rapid increase in the number of individuals in the population resulted in reduced fitness (represented by reproductive output and survival through heatwave conditions), regardless of migration. However, at an individual level, the hybrid offspring of migrants with native individuals generally demonstrated increased longevity in high temperature conditions compared with individuals from thermal selection lines. Overall, these populations showed no evidence that demographic manipulations led to genetic or evolutionary rescue. Following the effects of migration in individuals over several generations may be the next step in unravelling these conflicting results. We discuss these findings in the context of conservation intervention.

Drug dependence is characterized by a switch in motivation wherein a positively reinforcing substance can become negatively reinforcing. Put differently, drug use can transform from a form of pleasure-seeking to a form of relief-seeking. Ventral tegmental area (VTA) GABA neurons form an anatomical point of divergence between two double dissociable pathways that have been shown to be functionally implicated and necessary for these respective motivations to seek drugs. The tegmental pedunculopontine nucleus (TPP) is necessary for opiate conditioned place preferences (CPP) in previously drug-naïve rats and mice, whereas dopaminergic (DA) transmission in the nucleus accumbens (NAc) is necessary for opiate CPP in opiate-dependent and withdrawn (ODW) rats and mice. Here, we show that this switch in functional anatomy is contingent upon the gap junction-forming protein, connexin-36 (Cx36), in VTA GABA neurons. Intra-VTA infusions of the Cx36 blocker, mefloquine, in ODW rats resulted in a reversion to a drug-naïve-like state wherein the TPP was necessary for opiate CPP and where opiate withdrawal aversions were lost. Consistent with these data, conditional knockout mice lacking Cx36 in GABA neurons (GAD65-Cre;Cx36 fl(CFP)/fl(CFP)) exhibited a perpetual drug-naïve-like state wherein opiate CPP was always DA independent, and opiate withdrawal aversions were absent even in mice subjected to an opiate dependence and withdrawal induction protocol. Further, viral-mediated rescue of Cx36 in VTA GABA neurons was sufficient to restore their susceptibility to an ODW state wherein opiate CPP was DA dependent. Our findings reveal a functional role for VTA gap junctions that has eluded prevailing circuit models of addiction.

There is an urgent need to find a way to improve the genetic diversity of captive South China tiger (SCT, Panthera tigris amoyensis), the most critically endangered taxon of living tigers, facing inbreeding depression. The genomes showed that 13 hybrid SCTs from Meihuashan were divided into two groups; one group included three individuals who had a closer relationship with pureblood SCTs than another group. The three individuals shared more that 40% of their genome with pureblood SCTs and might be potential individuals for genetic rescuing in SCTs. A large-scale genetic survey based on 319 pureblood SCTs showed that the mean microsatellite inbreeding coefficient of pureblood SCTs decreased significantly from 0.1789 to 0.0600 (p = 0.000009) and the ratio of heterozygous loci increased significantly from 38.5% to 43.2% (p = 0.02) after one individual of the Chongqing line joined the Suzhou line and began to breed in the mid-1980s, which is a reason why the current SCTs keep a moderate level of microsatellite heterozygosity and nucleotide diversity. However, it is important to establish a back-up population based on the three individuals through introducing one pureblood SCT into the back-up population every year. The back-up population should be an important reserve in case the pureblood SCTs are in danger in the future.

Augmenting depleted genetic diversity can improve the fitness and evolutionary potential of wildlife populations, but developing effective management approaches requires genetically monitored test cases. One such case is the small, isolated and inbred Cotter River population of an endangered Australian freshwater fish, the Macquarie perch Macquaria australasica, which over 3 years (2017-2019) received 71 translocated migrants from a closely related, genetically more diverse population. We used genetic monitoring to test whether immigrants bred, interbred with local fish and augmented population genetic diversity. We also investigated whether levels of river flow affected recruitment, inbreeding depression and juvenile dispersal. Fish length was used to estimate the age, birth year cohort and growth of 524 individuals born between 2016 and 2020 under variable flow conditions. DArT genome-wide genotypes were used to assess individual ancestry, heterozygosity, short-term effective population size and identify parent-offspring and full-sibling families. Of 442 individuals born after translocations commenced, only two (0.45%) were of mixed ancestry; these were half-sibs with one translocated parent in common. Numbers of breeders and genetic diversity for five birth year cohorts of the Cotter River fish were low, especially in low-flow years. Additionally, individuals born in the year of lowest flow evidently suffered from inbreeding depression for juvenile growth. The year of highest flow was associated with the largest number of breeders, lowest inbreeding in the offspring and greatest juvenile dispersal distances. Genetic diversity decreased in the upstream direction, flagging restricted access of breeders to the most upstream breeding sites, exacerbated by low river flow. Our results suggest that the effectiveness of translocations could be increased by focussing on upstream sites and moving more individuals per year; using riverine sources should be considered. Our results indicate that river flow sufficient to facilitate fish movement through the system would increase the number of breeders, promote individuals\' growth, reduce inbreeding depression and promote genetic rescue.

UNASSIGNED: Epididymal sperm preservation is a simple conservation approach that can help prevent the loss of high genetic quality of farm animals. The chance of loss increases, especially during disease outbreaks or other interruptions to normal reproduction function.
UNASSIGNED: This study looked into the ability of preserved ram epididymal sperm to fertilize oocytes. Due to motility becoming an issue following sperm storage for fertilization, the sperm microinjection known as intracytoplasmic sperm injection approach was employed.
UNASSIGNED: The study was divided into two parts. First, involved the preservation of epididymal sperm at 5°C for 12 days. During preservation, sperm quality parameters namely motility, viability, intact membrane, acrosome, and Deoxyribonucleic acid (DNA) are evaluated every three days. For the fertility test in the second experiment, matured oocytes were injected with immotile sperm discovered in the last days of preservation. The presence of pronucleus development following in vitro culture is used as an indicator of sperm\'s ability to activate and fertilize oocytes.
UNASSIGNED: All sperm quality parameters significantly (p < 0.05) declined during preservation time. On day 12, motility was discovered to be 0%, but viable sperm, sperm with intact membrane, acrosome, and DNA remained at 41.86% ± 9.30%, 31.18% ± 5.15%, 21.88% ± 1.93%, and 33.35% ± 8.74%, respectively. On the fertility test, we inject immotile sperm from day 12 of preservation, which has the lowest motility found, into matured oocytes. Those sperms are able to activate (52.05% ± 7.15%) and fertilize (31.37% ± 1.75%) the injected oocytes, but their fertilizing ability is significantly lower (p < 0.05) when compared to the sperm derived from the ejaculate.
UNASSIGNED: In this study, simple preservation of epididymal sperm reduces all sperm quality criteria, particularly motility. Using the microinjection approach preserved sperm which had no motility, still demonstrated its ability to activate and fertilize the oocytes. According to that, this study provides potential approaches and tools for using genetically superior animals that have lost their ability to execute regular fertilization, and also prolong reproduction function.