Yu-Ten Ju

Objective: Protocols for somatic cell nuclear transfer (SCNT) remain technically challenging and inefficient. Demecolcine and nocodazole (Noc) have been used for chemically assisted enucleation to improve cloning efficiency; however, the procedure continues to be labor-intensive. This study aimed to use lysophosphatidic acid (LPA) to enhance Noc-mediated chemical enucleation (CE), thereby simplifying the SCNT procedure.Methods: Porcine MII oocytes were subject to CE by incubation with Noc and various concentrations of LPA for 1 h. Actin signals were evaluated to determine the optimal LPA. After treatment with Noc and 25 μM LPA for 1 h, spindle reformation was assayed following subsequent washout. Noc/LPA CE-treated oocytes were used for SCNT via direct microinjection of donor cells into the ooplasm. Reconstructed embryos were recovered for 2 h in PZM-5 prior to activation with ionomycin and N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine. Activated reconstructed embryos were cultured in 6-DMAP for 4 h, then in chaetocin for an additional 20–24 h to promote demethylation and acetylation. Cleavage and blastocyst rates were evaluated on Day 2 and Day 6, respectively.Results: The culture of the matured oocytes in Noc/LPA for 1 h, the percentage of oocytes exhibiting actin signals increased significantly from 12.5% to 87.6% with the addition of 25 μM LPA. Although spindles reformed in 59.0% of oocytes 1 h after Noc/LPA washout, 83.9% of oocytes retained spindles enclosed by actin rings; enabling the direct microinjection of donor cells into the ooplasm within 30 min. Blastocyst development rates currently vary from 9.5% to 16.6% after 6 days of culture in PZM-5.Conclusion: Although the blastocyst rate requires further improvement, we have shown that enucleation of MII pig oocytes can be successfully achieved via Noc/LPA treatment. This protocol simplifies the SCNT procedure while ensuring consistent blastocyst development in pig in vitro maturation oocytes.

This article presents a real-time RGB-D imaging system for robot-assisted remote microsurgery, integrating advanced optics, mechatronics, and dynamic control theory. The system combines a swept-source optical coherence tomography (SS-OCT) probe with a micro RGB camera, exploiting the SS-OCT’s high depth resolution and fast scan rate to generate accurate depth maps co-registered with the camera’s color data. A dual-axis galvanometer scanner, controlled by a fractional-order repetitive control algorithm with a data-driven inverse filter, enables real-time imaging at 30 fps with dynamically adjustable scan areas. Experimental evaluations highlight the system’s exceptional depth-sensing capabilities, including sub-millimeter resolution, precise depth mapping, and smooth dynamic scanning. In addition, the system enhances visual guidance during robotic microsurgery, as validated in teleoperated vessel ligation on a phantom and a cadaveric porcine model.

Taiwan harbors five endemic species of salamanders (Hynobius spp.) that inhabit distinct alpine regions, contributing to population fragmentation across isolated “sky islands”. With an evolutionary history spanning multiple glacial-interglacial cycles, these species represent an exceptional paradigm for exploring biogeography and speciation. However, a lack of suitable genetic markers applicable across species has limited research efforts. Thus, developing cross-amplifying markers is imperative. Expressed sequence-tag simple-sequence repeats (EST-SSRs) that amplify across divergent lineages are ideal for species identification in instances where phenotypic differentiation is challenging. Here, we report a suite of cross-amplifying EST-SSRs from the transcriptomes of the five Hynobius species that exhibit an interspecies transferability rate of 67.67%. To identify individual markers exhibiting cross-species polymorphism and to assess interspecies genetic diversity, we assayed 140 individuals from the five species across 84 sampling sites. A set of EST-SSRs with a high interspecies polymorphic information content (PIC = 0.63) effectively classified these individuals into five distinct clusters, as supported by discriminant analysis of principal components (DAPC), STRUCTURE assignment tests, and Neighbor-joining trees. Moreover, pair-wise F_ST values > 0.15 indicate notable between-cluster genetic divergence. Our set of 20 polymorphic EST-SSRs is suitable for assessing population structure within and among Hynobius species, as well as for long-term monitoring of their genetic composition.

Context: Elucidating how demography and contemporary landscape features regulate functional connectivity is crucial to implementing effective conservation strategies. We assessed the impacts of landscape features on the genetic variation of a locally endangered carnivore, the leopard cat (Prionailurus bengalensis) in Taiwan. Objectives: We aim to evaluate the association between genetic structure and landscape features. We further predicted the changes in genetic diversity and suitable habitats in the future. Methods: We genotyped 184 leopard cats in western Taiwan using 12 nuclear microsatellites and a mitochondrial marker. We applied a landscape optimization procedure with two genetic distances to identify major genetic barriers and employed ecological niche modeling to predict the future distribution of the leopard cat. Results: Bayesian demographic inferences revealed a dramatic population decline for all leopard cat populations in Taiwan. Genetic clustering and resistance surface modeling supported that the population connectivity was influenced by highways and high elevation. Niche modeling indicated low temperature was one of the primary factors limiting the occurrence of leopard cats that may inhibit their movement in high elevations. We predicted the suitable habitats of leopard cats would shrink northward and towards higher altitudes with rugged topography in response to global warming. Conclusions: Our study provided genetic evidence that leopard cats in Taiwan had undergone a dramatic population decline that may be associated with anthropogenic impacts. We also inferred the anthropogenic linear feature compromised the connectivity and persistence of leopard cats in human-mediated landscapes. Our finding serves as a model for landscape genetic studies of island carnivores in subtropical regions.

Elucidating how demography and contemporary landscape features regulate functional connectivity and diversity is crucial to implementing effective conservation strategies, especially in highly fragmented landscapes. We analyzed the impacts of landscape features and demographic events on the dispersal and genetic structure of a locally endangered carnivore, the leopard cat (Prionailurus bengalensis), across subtropical Taiwan. We genotyped 184 samples from both human-impacted and natural habitats using 12 nuclear microsatellites and a mitochondrial marker. Bayesian analyses revealed a recent population decline within the past 200 years, and a current genetic hotspot appears to have arisen from interbreeding between previously diverged populations. Genetic clustering and resistance surface modeling support the notion that the population structure is influenced by manufactured linear and natural topographical features. We employed a landscape optimization procedure using two genetic distances to reveal that highways and elevation are the most significant barriers affecting leopard cat connectivity. Niche modeling indicated that low temperature is a primary factor limiting leopard cat occurrence, potentially explaining why their resistance to movement is positively correlated with elevation. Utilizing an ensemble approach, we predict that suitable leopard cat habitat will shrink northward and towards higher more rugged altitudes. Our study provides genetic evidence that anthropogenic landscape features compromise the connectivity and persistence of a vagile carnivore that is under severe pressure from habitat loss and human activities. Our findings serve as a model for landscape genetic studies of island carnivores in subtropical regions, aimed at understanding how manufactured landscapes affect intra-species diversity and dispersal.

The island of Taiwan represents an ideal context for studying the effects of climatic oscillations and topographic variation on large herbivores due to its varied tropical to sub-tropical climate zones at different elevational ranges. We explored the phylogenetics of Formosan sambar deer (Rusa unicolor swinhoii) using the control region of the mitochondrial genome. We detected 18 haplotypes among 454 sequences across the island and grouped them into six regions based on SAMOVA, with 68.78% variance among regions. A Bayesian phylogenetic dendrogram revealed two spatially segregated genetic clades. Neutrality tests and Bayesian skyline plots uncovered different demographic expansion histories for the two clades. We further tested divergence times and chronology to propose potential phylogenetic scenarios, which were examined using approximate Bayesian computation. Finally, we present a credible hypothesis for a glacial refugium in the northern part of the Central Mountain Range. Subsequent secondary contact between the two clades during interglacial periods has led to the extant genetic structure of Formosan sambar deer.

Deer genera around the globe are threatened by anthropogenic interference. The translocation of alien species and their subsequent genetic introgression into indigenous deer populations is particularly harmful to the species of greatest conservation concern. Products derived from deer, including venison and antler velvet, are also at risk of fraudulent labeling. The current molecular markers used to genetically identify deer species were developed from genome sequences and have limited applicability for cross-species amplification. The absence of efficacious diagnostic techniques for identifying deer species has hampered conservation and wildlife crime investigation efforts. Expressed sequence tag-simple sequence repeat (EST-SSR) markers are reliable tools for individual and species identification, especially in terms of cross-species genotyping. We conducted transcriptome sequencing of sambar (Rusa unicolor) antler velvet and acquired 11,190 EST-SSRs from 65,074 newly assembled unigenes. We identified a total of 55 unambiguous amplicons in sambar (n = 45), which were selected as markers to evaluate cross-species genotyping in sika deer (Cervus nippon, n = 30) and red deer (Cervus elaphus, n = 46), resulting in cross-species amplification rates of 94.5% and 89.1%, respectively. Based on polymorphic information content (>0.25) and genotyping fidelity, we selected 16 of these EST-SSRs for species identification. This marker set revealed significant genetic differentiation based on the fixation index and genetic distance values. Principal coordinate analysis and STRUCTURE analysis revealed distinct clusters of species and clearly identified red-sika hybrids. These markers showed applicability across different genera and proved suitable for identification and phylogenetic analyses across deer species.

Taxonomic identification of two Taiwanese salamanders, Hynobius sonani (Maki, 1922), and H. formosanus Maki, 1922, was clarified. Based on the descriptions and measurement data in the original description, we confirmed that H. sonani has been treated as H. formosanus and vice versa, which might have been caused by confusion of sampling localities of the two species. Because we could not find the holotypes of the two species and concluded these were lost, we herein assign neotypes for these species for precluding future taxonomic confusion.

Intra-striatal transplantation of fetal ventral mesencephalic (VM) tissue has a therapeutic effect on patients with Parkinson’s disease (PD). Sertoli cells (SCs) possess immune-modulatory properties that benefit transplantation. We hypothesized that co-graft of SCs with VM tissue can attenuate rejection. Hemi-parkinsonian rats were generated by injecting 6-hydroxydopamine into the right medial forebrain bundle of Sprague Dawley (SD) rats. The rats were then intrastriatally transplanted with VM tissue from rats or pigs (rVM or pVM), with/without a co-graft of SCs (rVM+SCs or pVM+SCs). Recovery of dopaminergic function and survival of the grafts were evaluated using the apomorphine-induced rotation test and small animal-positron emission tomography (PET) coupled with [¹⁸F] DOPA or [¹⁸F] FE-PE2I, respectively. Immunohistochemistry (IHC) examination was used to determine the survival of the grafted dopaminergic neurons in the striatum and to investigate immune-modulatory effects of SCs. The results showed that the rVM+SCs and pVM+SCs groups had significantly improved drug-induced rotational behavior compared with the VM alone groups. PET revealed a significant increase in specific uptake ratios (SURs) of [¹⁸F] DOPA and [¹⁸F] FE-PE2I in the grafted striatum of the rVM+SCs and pVM+SCs groups as compared to that of the rVM and pVM groups. SC and VM tissue co-graft led to better dopaminergic (DA) cell survival. The co-grafted groups exhibited lower populations of T-cells and activated microglia compared to the groups without SCs. Our results suggest that co-graft of SCs benefit both xeno- and allo-transplantation of VM tissue in a PD rat model. Use of SCs enhanced the survival of the grafted dopaminergic neurons and improved functional recovery. The enhancement may in part be attributable to the immune-modulatory properties of SCs. In addition, [¹⁸F]DOPA and [¹⁸F]FE-PE2I coupled with PET may provide a feasible method for in vivo evaluation of the functional integrity of the grafted DA cell in parkinsonian rats.