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Richard B Harris - One of the best experts on this subject based on the ideXlab platform.
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High connectivity among Argali sheep from Afghanistan and adjacent countries: Inferences from neutral and candidate gene microsatellites
Conservation Genetics, 2011Co-Authors: Gordon Luikart, Stephen J. Amish, Albano Beja-pereira, Raquel Godinho, J. Winnie, Fred W. Allendorf, Richard B HarrisAbstract:We quantified population connectivity and genetic variation in the Marco Polo subspecies of Argali mountain sheep (Ovis ammon polii) by genotyping 9 neutral and 8 candidate gene microsatellite loci in 172 individuals noninvasively sampled across five study areas in Afghani- stan, China, and Tajikistan. Heterozygosity and allelic richness were generally high (mean H = 0.67, mean A = 6.1), but were significantly lower in the China study area (H = 0.61, P \ 0.001; A = 4.9, P \ 0.01). One mar- ker in an immune system gene (TCRG4) showed an excess of rare alleles compared to neutral expectations. Another immune system gene (GLYCAM-1) showed excessive dif- ferentiation (high FST) between study areas. Estimates of genetic differentiation were similar (FST = 0.035 vs. 0.033) with and without the two loci deviating from neutrality, suggesting that selection is not a primary driver of overall molecular variation, and that candidate gene loci can be used for connectivity monitoring, as long as selection tests are conducted to avoid biased gene flow estimates. Adequate protection of Argali and maintenance of inter-population connectivity will require monitoring and international cooperation because Argali exhibit high gene flow across international borders.
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Argali food habits and dietary overlap with domestic livestock in ikh nart nature reserve mongolia
Journal of Arid Environments, 2011Co-Authors: G J Wingard, Sukhiin Amgalanbaatar, Richard B Harris, D H Pletscher, Donald J Bedunah, B Mandakh, Richard P. ReadingAbstract:The Argali (Ovis ammon ) is a species of great conservation concern throughout its range in central Asia, and is particularly sensitive to human disturbance and habitat degradation. Recent increases in livestock numbers have potentially reduced the capacity of habitats to sustain Argali because of forage or interference competition. We studied the potential for forage competition between Argali and domestic livestock in Ikh Nart Nature Reserve, Mongolia by quantifying overlap among seasonal diets using microhistological analysis of fecal samples. We collected 100 fecal samples from Argali and 100 fecal samples from livestock during June 2002, August 2002, January 2003, and April 2003. Argali and livestock used the same plant species with the exception of five used only by livestock. We also quantified forage availability in both summer (2002) and winter (2003) seasons. Total mean biomass in summer (x¯=19.0g/m2) declined ∼82% to a winter mean of 3.5 g/m2(t = −10.00, P < 0.0001). Dietary overlap between Argali and livestock was high, varying from 72% in summer to 95% in winter. We concluded that high degree of overlap for forage coupled with the low available vegetative biomass in winter suggests the potential for competition between Argali and livestock.
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Argali food habits and dietary overlap with domestic livestock in Ikh Nart Nature Reserve, Mongolia
Journal of Arid Environments, 2011Co-Authors: G J Wingard, Sukhiin Amgalanbaatar, Richard B Harris, D H Pletscher, Donald J Bedunah, B Mandakh, Richard P. ReadingAbstract:The Argali (Ovis ammon ) is a species of great conservation concern throughout its range in central Asia, and is particularly sensitive to human disturbance and habitat degradation. Recent increases in livestock numbers have potentially reduced the capacity of habitats to sustain Argali because of forage or interference competition. We studied the potential for forage competition between Argali and domestic livestock in Ikh Nart Nature Reserve, Mongolia by quantifying overlap among seasonal diets using microhistological analysis of fecal samples. We collected 100 fecal samples from Argali and 100 fecal samples from livestock during June 2002, August 2002, January 2003, and April 2003. Argali and livestock used the same plant species with the exception of five used only by livestock. We also quantified forage availability in both summer (2002) and winter (2003) seasons. Total mean biomass in summer (x¯=19.0g/m2) declined ∼82% to a winter mean of 3.5 g/m2(t = −10.00, P
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Estimating abundance of mountain ungulates incorporating imperfect detection: Argali Ovis ammon in the Gobi Desert, Mongolia
Wildlife Biology, 2011Co-Authors: Ganchimeg Wingard, Richard B Harris, Sukh Amgalanbaatar, Richard P. ReadingAbstract:Abstract Estimating the density or abundance of mountain ungulates is difficult and rarely conducted in a statistically valid manner. The rough terrain they inhabit, their group-living habits, their relatively low density, and the difficulty of marking individuals all contribute to making rigorous estimates of abundance logistically difficult. Raw (uncalibrated) counts are usually reported, and although their drawbacks are often acknowledged, biases are rarely quantified. In September 2009, we took advantage of the presence of a radio-marked sample of Argali Ovis ammon in the Ikh Nart Nature Reserve in south-central Mongolia, as well as the area's comparatively forgiving topography to estimate abundance simultaneously using two independent methods: distance sampling and mark-resight sampling. Distance sampling produced an abundance estimate of 539 (95% CI: 196-1,081) Argali within a ∼ 330 km2 study area on the same day that we visually tallied 189 animals. Mark-resight sampling using the Poisson log-norma...
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Argali abundance in the afghan pamir using capture recapture modeling from fecal dna
Journal of Wildlife Management, 2010Co-Authors: Richard B Harris, Stephen J. Amish, Raquel Godinho, J John R Winnie, Albano Bejapereira, Vǎnia Costa, Gordon LuikartAbstract:Abstract Estimating population size in a mark–recapture framework using DNA obtained from remotely collected genetic samples (e.g., feces) has become common in recent years but rarely has been used for ungulates. Using DNA extracted from fecal pellets, we estimated the size of an Argali (Ovis ammon) population that was believed to be isolated from others within the Big Pamir Mountains, Afghanistan, an area where access was difficult and expensive. We used closed-capture models to estimate abundance, and Pradel models to examine closure assumptions, both as implemented in Program MARK. We also made visual counts of Argali in the Big Pamirs, allowing comparison of count indices of abundance with modeled estimates. Our model-averaged estimate for female Argali in the Big Pamir was 172 (95% CI = 117–232), which was about 23% higher than our best assessment using uncorrected visual counts. However, mark–recapture models suggested that males were not a closed population; thus, we were unable to provide a mean...
Eric Ariel L. Salas - One of the best experts on this subject based on the ideXlab platform.
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Habitat assessment of Marco Polo sheep (Ovis ammon polii) in Eastern Tajikistan: Modeling the effects of climate change.
Ecology and evolution, 2018Co-Authors: Eric Ariel L. Salas, Raul Valdez, Stefan Michel, Kenneth G. BoykinAbstract:Identifying the factors predicting the high-elevation suitable habitats of Central Asian Argali wild sheep and how these suitable habitats are affected by the changing climate regimes could help address conservation and management efforts and identify future critical habitat for the species in eastern Tajikistan. This study used environmental niche models (ENMs) to map and compare potential present and future distributions of suitable environmental conditions for Marco Polo Argali. Argali occurrence points were collected during field surveys conducted from 2009 to 2016. Our models showed that terrain ruggedness and annual mean temperature had strong correlations on Argali distribution. We then used two greenhouse gas concentration trajectories (RCP 4.5 and RCP 8.5) for two future time periods (2050 and 2070) to model the impacts of climate change on Marco Polo Argali habitat. Results indicated a decline of suitable habitat with majority of losses observed at lower elevations (3,300-4,300 m). Models that considered all variables (climatic and nonclimatic) predicted losses of present suitable areas of 60.6% (6,928 km2) and 63.2% (7,219 km2) by 2050 and 2070, respectively. Results also showed averaged habitat gains of 46.2% (6,106 km2) at much higher elevations (4,500-6,900 m) and that elevational shifts of habitat use could occur in the future. Our results could provide information for conservation planning for this near threatened species in the region.
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Summer and winter habitat suitability of Marco Polo Argali in southeastern Tajikistan: A modeling approach.
Heliyon, 2017Co-Authors: Eric Ariel L. Salas, Raul Valdez, Stefan MichelAbstract:Abstract We modeled summer and winter habitat suitability of Marco Polo Argali in the Pamir Mountains in southeastern Tajikistan using these statistical algorithms: Generalized Linear Model, Random Forest, Boosted Regression Tree, Maxent, and Multivariate Adaptive Regression Splines. Using sheep occurrence data collected from 2009 to 2015 and a set of selected habitat predictors, we produced summer and winter habitat suitability maps and determined the important habitat suitability predictors for both seasons. Our results demonstrated that Argali selected proximity to riparian areas and greenness as the two most relevant variables for summer, and the degree of slope (gentler slopes between 0° to 20°) and Landsat temperature band for winter. The terrain roughness was also among the most important variables in summer and winter models. Aspect was only significant for winter habitat, with Argali preferring south-facing mountain slopes. We evaluated various measures of model performance such as the Area Under the Curve (AUC) and the True Skill Statistic (TSS). Comparing the five algorithms, the AUC scored highest for Boosted Regression Tree in summer (AUC = 0.94) and winter model runs (AUC = 0.94). In contrast, Random Forest underperformed in both model runs.
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Summer and winter habitat suitability of Marco Polo Argali in southeastern Tajikistan: A modeling approach
Elsevier, 2017Co-Authors: Eric Ariel L. Salas, Raul Valdez, Stefan MichelAbstract:We modeled summer and winter habitat suitability of Marco Polo Argali in the Pamir Mountains in southeastern Tajikistan using these statistical algorithms: Generalized Linear Model, Random Forest, Boosted Regression Tree, Maxent, and Multivariate Adaptive Regression Splines. Using sheep occurrence data collected from 2009 to 2015 and a set of selected habitat predictors, we produced summer and winter habitat suitability maps and determined the important habitat suitability predictors for both seasons. Our results demonstrated that Argali selected proximity to riparian areas and greenness as the two most relevant variables for summer, and the degree of slope (gentler slopes between 0° to 20°) and Landsat temperature band for winter. The terrain roughness was also among the most important variables in summer and winter models. Aspect was only significant for winter habitat, with Argali preferring south-facing mountain slopes. We evaluated various measures of model performance such as the Area Under the Curve (AUC) and the True Skill Statistic (TSS). Comparing the five algorithms, the AUC scored highest for Boosted Regression Tree in summer (AUC = 0.94) and winter model runs (AUC = 0.94). In contrast, Random Forest underperformed in both model runs. Keywords: Ecology, Evolution, Zoology, Environmental science, Geography, Biological science
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Open-Access Geographic Data for the Argali Habitat in the Southeastern Tajik Pamirs
Data, 2016Co-Authors: Eric Ariel L. Salas, Raul Valdez, Kenneth G. BoykinAbstract:Seven Geographic Information System (GIS) layers comprise this dataset intended for understanding the Marco Polo Argali habitat in the southeastern Tajikistan Pamirs (37°33′ N, 74°09′ E). Extensive remote sensing habitat data processing and field data analysis of the Marco Polo sheep study area have yielded these layers that are now available online to download and for use by other researchers interested in studying the Argali patterns and habitat suitability in the southeastern Tajik Pamirs. It is important to note that the layers were generated using a 30-m Landsat ETM image and field data from 2012.
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Geographic Layers as Landscape Drivers for the Marco Polo Argali Habitat in the Southeastern Pamir Mountains of Tajikistan
ISPRS International Journal of Geo-Information, 2015Co-Authors: Eric Ariel L. Salas, Raul Valdez, Kenneth G. BoykinAbstract:We described in this report the essential geographic layers used as landscape drivers for the Marco Polo Argali habitat in the eastern Tajik Pamirs. Using remote sensing techniques and geographic information systems (GIS), individual layers were generated in order to acquire more information on Argali patterns and habitat suitability and to make the dataset available online. We introduced an improved object-based image analysis in our mapping of the vegetation cover by utilizing spectral, topographic, and texture variables. We exhausted every Landsat image band and texture feature combination to select the best pairing of band-texture components. For vegetation class alone, the producer’s accuracy was 90.8% and the user’s accuracy was 91.6%.
Richard P. Reading - One of the best experts on this subject based on the ideXlab platform.
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Seasonal Daytime Activity Budget of Mongolian Gobi Argali Sheep (Ovis ammon)
Mongolian Journal Of Biological Sciences, 2021Co-Authors: Barry Rosenbaum, Richard P. Reading, Sukh Amgalanbaatar, Boulder Colorado Oak Avenue, West Th Avenue Butterfly PavilionAbstract:Activity patterns provide insight into the overall relationship between a species and its environment. Despite declines in populations of Argali and some attention on ecological questions, limited information exists on Argali behavior. We measured diurnal activity budgets of Argali for 12 months by recording behavior (i.e., foraging, bedding, standing, traveling, other) with instantaneous scan sampling at 5-minute intervals. Argali exhibited seasonal differences in activity budgets. The greatest proportion of daytime in winter was spent foraging. The greatest proportion of daytime in summer was spent bedding. Argali reduced all other behaviors in winter in favor of foraging. In summer Argali reduced their foraging to a seasonal low and increased bedding. Behavior was constrained by forage in winter and by temperatures in summer. Females fed more than males in all seasons. Other behaviors varied according to season and reproductive periods. Females demonstrated highest vigilance in spring and summer, while males exhibited highest vigilance during the autumn rut. These data are the first regarding detailed behavior of Argali and are valuable to their management and conservation by providing information on constraints faced by the species.
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Desert pastoralists' negative and positive effects on rare wildlife in the Gobi.
Conservation biology : the journal of the Society for Conservation Biology, 2017Co-Authors: L. Stefan Ekernas, Richard P. Reading, Sukh Amgalanbaatar, Ganchimeg Wingard, Wesley M. Sarmento, Hannah S. Davie, James D. Murdoch, Joel BergerAbstract:In arid regions of the developing world, pastoralists and livestock commonly inhabit protected areas, resulting in human–wildlife conflict. Conflict is inextricably linked to the ecological processes shaping relationships between pastoralists and native herbivores and carnivores. To elucidate relationships underpinning human–wildlife conflict, we synthesized 15 years of ecological and ethnographic data from Ikh Nart Nature Reserve in Mongolia's Gobi steppe. The density of Argali (Ovis ammon), the world's largest wild sheep, at Ikh Nart was among the highest in Mongolia, yet livestock were >90% of ungulate biomass and dogs >90% of large-carnivore biomass. For Argali, pastoral activities decreased food availability, increased mortality from dog predation, and potentially increased disease risk. Isotope analyses indicated that livestock accounted for >50% of the diet of the majority of gray wolves (Canis lupus) and up to 90% of diet in 25% of sampled wolves (n = 8). Livestock composed at least 96% of ungulate prey in the single wolf pack for which we collected species-specific prey data. Interviews with pastoralists indicated that wolves annually killed 1–4% of Ikh Nart's livestock, and pastoralists killed wolves in retribution. Pastoralists reduced wolf survival by killing them, but their livestock were an abundant food source for wolves. Consequently, wolf density appeared to be largely decoupled from Argali density, and pastoralists had indirect effects on Argali that could be negative if pastoralists increased wolf density (apparent competition) or positive if pastoralists decreased wolf predation (apparent facilitation). Ikh Nart's Argali population was stable despite these threats, but livestock are increasingly dominant numerically and functionally relative to Argali. To support both native wildlife and pastoral livelihoods, we suggest training dogs to not kill Argali, community insurance against livestock losses to wolves, reintroducing key native prey species to hotspots of human–wolf conflict, and developing incentives for pastoralists to reduce livestock density.
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Ecological interactions shape the distribution of a cultural ecosystem service: Argali sheep (Ovis ammon) in the Gobi-Steppe of Mongolia
Biological Conservation, 2017Co-Authors: James D. Murdoch, Richard P. Reading, Sukh Amgalanbaatar, Ganchimeg Wingard, Badamjav LkhagvasurenAbstract:Abstract Cultural ecosystem services, such as the enjoyment and satisfaction of viewing a species in the wild, are often underrepresented in conservation planning. Understanding the spatial distribution of wildlife-related services can inform management, which is especially important for declining species. We examined how natural and human features of a landscape influence the distribution of Argali sheep in Ikh Nart Nature Reserve, Mongolia, a popular destination for tourists seeking the opportunity to see and experience this declining species. We collected 1999 Argali locations from 2009 to 2011 and developed an occupancy model for each season and across seasons. Occupancy probability equates to the chance of seeing the species, and provides a measure that not only describes the distribution of Argali, but also the cultural service they provide. We developed a set of 67 candidate models and the top-ranking model each season and across seasons was the interaction of proximity to water and proportion of rocky outcrop at a site. Argali occupancy increased as distance to water decreased and amount of rocky outcrop increased. The simulated removal of water sources resulted in a 98% loss in landscape quality. Our results provide among the first occupancy models for Argali, which can inform decision-making at multiple spatial scales. They also reveal the importance of water sources, which are intensively used by people and livestock, and indicate that careful management of these resources will be important to sustaining Argali in the landscape.
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Creating a model of habitat suitability using vegetation and ruggedness for Ovis ammon and Capra sibirica (Artiodactyla: Bovidae) in Mongolia
Journal of Asia-Pacific Biodiversity, 2017Co-Authors: Nanette Bragin, Ganchimeg Wingard, Sukh Amgalanbaatar, Richard P. ReadingAbstract:Abstract Spatially-explicit wildlife habitat models, such as a Habitat Suitability Index Model (HSIM), are increasingly used to understand optimal environmental conditions needed for a species survival and viability. HSIM compares different environmental variables, including vegetation, slope, and aspect to determine optimal habitat for a species. HSIM can compare a species’ use of resources with availability and determine risks for a species viability. Furthermore, decision makers can use HSIM to make decisions regarding resources needed for species of concern and development of protected areas. We used a geographic information system to create a HSIM for Argali sheep ( Ovis ammon ) and Siberian ibex ( Capra siberica ), species of conservation concern in Mongolia. We used vegetation and ruggedness layers and compared Argali and ibex use with habitat availability. Argali and ibex presence correlated with three habitat classes: dense rock, low-density shrub, and short grass/forb. We found no significance in correlation for ruggedness.
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conservation presence not socioeconomics leads to differences in pastoralist perceived threats to Argali
Journal of Asia-Pacific Biodiversity, 2016Co-Authors: Wesley Sarmento, Richard P. ReadingAbstract:Abstract Community-based conservation approaches that keep people on landscapes increasingly complement preservationist models of reserves without people. Evaluations of community conservation have shown that economic incentives and socioeconomics primarily drive people’s attitudes and perceptions. Work at Mongolia’s Ikh Nart Nature Reserve demonstrates how to achieve successful conservation by integrating local people into the overall program. Using a short questionnaire, we interviewed pastoralist families across two soums (similar to a U.S. county) in Ikh Nart. We examined (1) pastoralists’ perceived threats to Argali sheep (Ovis ammon), (2) socioeconomic differences among pastoralists, and (3) differences between pastoralists from different soums. We found that 15 years of conservation activities—education, research, and modest ecotourism—that occurred in the northern soum led to influences on people’s perceptions toward Argali conservation. Compared with pastoralists from southern Ikh Nart, pastoralists from the northern part of the reserve more likely knew that Argali are protected and understood primary threats to the species. Socioeconomic factors, such as age, sex, and wealth, did not significantly influence responses. The negligible economic incentives in Ikh Nart did not lead to response differences. Our results demonstrate that conservation can influence people across socioeconomic classes without providing large economic incentives.
Kenneth G. Boykin - One of the best experts on this subject based on the ideXlab platform.
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Habitat assessment of Marco Polo sheep (Ovis ammon polii) in Eastern Tajikistan: Modeling the effects of climate change.
Ecology and evolution, 2018Co-Authors: Eric Ariel L. Salas, Raul Valdez, Stefan Michel, Kenneth G. BoykinAbstract:Identifying the factors predicting the high-elevation suitable habitats of Central Asian Argali wild sheep and how these suitable habitats are affected by the changing climate regimes could help address conservation and management efforts and identify future critical habitat for the species in eastern Tajikistan. This study used environmental niche models (ENMs) to map and compare potential present and future distributions of suitable environmental conditions for Marco Polo Argali. Argali occurrence points were collected during field surveys conducted from 2009 to 2016. Our models showed that terrain ruggedness and annual mean temperature had strong correlations on Argali distribution. We then used two greenhouse gas concentration trajectories (RCP 4.5 and RCP 8.5) for two future time periods (2050 and 2070) to model the impacts of climate change on Marco Polo Argali habitat. Results indicated a decline of suitable habitat with majority of losses observed at lower elevations (3,300-4,300 m). Models that considered all variables (climatic and nonclimatic) predicted losses of present suitable areas of 60.6% (6,928 km2) and 63.2% (7,219 km2) by 2050 and 2070, respectively. Results also showed averaged habitat gains of 46.2% (6,106 km2) at much higher elevations (4,500-6,900 m) and that elevational shifts of habitat use could occur in the future. Our results could provide information for conservation planning for this near threatened species in the region.
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Open-Access Geographic Data for the Argali Habitat in the Southeastern Tajik Pamirs
Data, 2016Co-Authors: Eric Ariel L. Salas, Raul Valdez, Kenneth G. BoykinAbstract:Seven Geographic Information System (GIS) layers comprise this dataset intended for understanding the Marco Polo Argali habitat in the southeastern Tajikistan Pamirs (37°33′ N, 74°09′ E). Extensive remote sensing habitat data processing and field data analysis of the Marco Polo sheep study area have yielded these layers that are now available online to download and for use by other researchers interested in studying the Argali patterns and habitat suitability in the southeastern Tajik Pamirs. It is important to note that the layers were generated using a 30-m Landsat ETM image and field data from 2012.
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Geographic Layers as Landscape Drivers for the Marco Polo Argali Habitat in the Southeastern Pamir Mountains of Tajikistan
ISPRS International Journal of Geo-Information, 2015Co-Authors: Eric Ariel L. Salas, Raul Valdez, Kenneth G. BoykinAbstract:We described in this report the essential geographic layers used as landscape drivers for the Marco Polo Argali habitat in the eastern Tajik Pamirs. Using remote sensing techniques and geographic information systems (GIS), individual layers were generated in order to acquire more information on Argali patterns and habitat suitability and to make the dataset available online. We introduced an improved object-based image analysis in our mapping of the vegetation cover by utilizing spectral, topographic, and texture variables. We exhausted every Landsat image band and texture feature combination to select the best pairing of band-texture components. For vegetation class alone, the producer’s accuracy was 90.8% and the user’s accuracy was 91.6%.
Zhongde Wang - One of the best experts on this subject based on the ideXlab platform.
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genetic inactivation of the sry gene in Argali wild and romney domestic sheep with crispr cas and talen systems for producing sex reversed female animals
Reproduction Fertility and Development, 2014Co-Authors: Zhongde WangAbstract:The Tibetan Argali (Ovis ammon hodgsoni), a wild sheep of the subfamily Caprinae (Bovidae), primarily found in the Tibetan Plateau, is categorized as near threatened on the International Union for Conservation of Nature (IUCN) Red List. For the conservation of this species, we have achieved the cloning of a full-term live Argali lamb (died shortly after birth) from a male Argali cell line that has been cryopreserved for more than 20 years. While working towards the goal of cloning a live Argali, we also recognised the need to establish a reproductive herd of animals by producing both fertile males and females. However, as in the case of Argali, it is difficult, and in some cases impossible, to obtain male and female cell lines from an endangered mammalian species. Therefore, for demonstrating a proof of concept of using an assisted reproduction technology (ART) for the conservation of endangered species or reviving extinct species, we used the Argali as an experimental model to produce both fertile males and females through ART. The Sry gene plays a central role in mammalian sex determination: mutations in the Sry gene result in the development of XY females; in mice, Sry knockout (KO) results in fertile XY females. Therefore, we created sex-reversed Argali cell lines through the KO of the Sry gene. In parallel, for potentially comparing the fertility of sex-reversed XY females between the Argali and domestic sheep [Romney (Ovis aries)], we also knocked out the Sry gene in the domestic sheep. Specifically, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system to introduce mutations to the high-mobility group (HMG) box of the Sry gene. Skin fibroblasts from either adult Argali or Romney sheep were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (FBS). Cells were harvested at 100% confluence with 0.25% trypsin-EDTA and transfected with the CRISPR/Cas DNA constructs by using the Amaxa Nucleofector system. For each experiment, 106 cells were transfected with 5 μg of CRISPR/Cas DNA constructs. After 72 hours post-transfection, cells were harvested, with half being used for genomic DNA isolation (for examining KO efficiency) and the other half for limited dilution for obtaining single-cell-derived colonies. With the Surveyor assay and a restriction enzyme digestion assay (successfully knocked-out cells lose the Dde I site in the HMG box of the Sry gene, rendering resistance to Dde I digestion), we demonstrated that the Sry KO efficiency was about 10% both in Argali and Romney sheep. By screening single-cell-derived colonies from the transfected Romney sheep cells, an Sry-KO cell line harboring a 2-nucleotide deletion in the HMG box was successfully established. Multiple vials of cells from this Sry-KO cell line have been cryopreserved and will be used for animal cloning via somatic cell nuclear transfer (SCNT) in the sheep-breeding season this fall.
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Genetic Inactivation of the Sry Gene in Argali Wild and Romney Domestic Sheep with CRISPR/Cas and TALEN Systems for Producing Sex-Reversed Female Animals
Reproduction Fertility and Development, 2014Co-Authors: Zhongde WangAbstract:The Tibetan Argali (Ovis ammon hodgsoni), a wild sheep of the subfamily Caprinae (Bovidae), primarily found in the Tibetan Plateau, is categorized as near threatened on the International Union for Conservation of Nature (IUCN) Red List. For the conservation of this species, we have achieved the cloning of a full-term live Argali lamb (died shortly after birth) from a male Argali cell line that has been cryopreserved for more than 20 years. While working towards the goal of cloning a live Argali, we also recognised the need to establish a reproductive herd of animals by producing both fertile males and females. However, as in the case of Argali, it is difficult, and in some cases impossible, to obtain male and female cell lines from an endangered mammalian species. Therefore, for demonstrating a proof of concept of using an assisted reproduction technology (ART) for the conservation of endangered species or reviving extinct species, we used the Argali as an experimental model to produce both fertile males and females through ART. The Sry gene plays a central role in mammalian sex determination: mutations in the Sry gene result in the development of XY females; in mice, Sry knockout (KO) results in fertile XY females. Therefore, we created sex-reversed Argali cell lines through the KO of the Sry gene. In parallel, for potentially comparing the fertility of sex-reversed XY females between the Argali and domestic sheep [Romney (Ovis aries)], we also knocked out the Sry gene in the domestic sheep. Specifically, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system to introduce mutations to the high-mobility group (HMG) box of the Sry gene. Skin fibroblasts from either adult Argali or Romney sheep were cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (FBS). Cells were harvested at 100% confluence with 0.25% trypsin-EDTA and transfected with the CRISPR/Cas DNA constructs by using the Amaxa Nucleofector system. For each experiment, 106 cells were transfected with 5 μg of CRISPR/Cas DNA constructs. After 72 hours post-transfection, cells were harvested, with half being used for genomic DNA isolation (for examining KO efficiency) and the other half for limited dilution for obtaining single-cell-derived colonies. With the Surveyor assay and a restriction enzyme digestion assay (successfully knocked-out cells lose the Dde I site in the HMG box of the Sry gene, rendering resistance to Dde I digestion), we demonstrated that the Sry KO efficiency was about 10% both in Argali and Romney sheep. By screening single-cell-derived colonies from the transfected Romney sheep cells, an Sry-KO cell line harboring a 2-nucleotide deletion in the HMG box was successfully established. Multiple vials of cells from this Sry-KO cell line have been cryopreserved and will be used for animal cloning via somatic cell nuclear transfer (SCNT) in the sheep-breeding season this fall.