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Contents Mitochondria play fundamental roles during oocyte development. The accumulation and spatial redistribution of these energy-producing organelles have been linked to the developmental competence of mammalian oocytes. Here, we assessed the copy number, distribution and activity of mitochondria within cat oocytes during folliculogenesis. In Experiment 1, oocytes were recovered from primordial ( n = 152), primary (112), secondary (95), early (131), small (118), antral (86) and advanced antral (5) stages follicles, and mitochondria DNA extracted and quantified using qPCR. In Experiment 2, oocytes from pre-antral ( n = 44), early antral ( n = 66), small antral ( n = 59), antral ( n = 41) and advanced antral ( n = 21) follicles were isolated and stained with CMXRos MitoTracker dye to assess mitochondrial distribution pattern and activity levels. Oocyte's mitochondria DNA (mt DNA) copy numbers gradually increased as folliculogenesis progressed, with a significant shift at the small antral stage (0.5 to <1 mm in diameter). The location of mitochondria gradually shifted from a homogeneous distribution throughout the cytoplasm in pre-antral oocytes to a pericortical concentration in the advanced antral stage. Quantification of CMXRos fluorescent intensity revealed a progressive increase in mitochondrial activity in oocytes from the pre-antral to the large antral follicles. Taken together, these findings demonstrated that cat oocytes undergo dynamic changes in mitochondrial copy number, distribution and activity during folliculogenesis. These significant modifications to this crucial cytoplasmic organelle are likely associated with the acquisition of developmental competency by cat oocytes. [ABSTRACT FROM AUTHOR]

Contents The objective of the study was to assess the efficacy of coculture with conspecific cumulus-denuded oocytes ( CDOs) during in vitro maturation in a three-dimensional system of barium alginate microcapsules on the in vitro embryo development of domestic cat cumulus-oocyte complexes ( COCs). In Experiment I, COCs were cocultured with conspecific CDOs or cultured separately in a 3D system for 24 hr of in vitro maturation, before assessing the meiotic progression. In Experiment II, the in vitro fertilization of COCs and CDOs was carried out with chilled epididymal spermatozoa and the presumptive zygotes were cultured in vitro separately for 7 days in 3D microcapsules before assesment of embryonic development. The results showed that the viability was maintained and that meiosis was resumed in the 3D culture system. The presence of CDOs during in vitro maturation improved the meiotic competence of the COCs, since the proportions of telophase I/metaphase II were higher than that in the groups cultured separately. The enrichment of the maturation system by companion oocytes also enhanced the ability of COCs to develop into embryos, and increased the percentages of morula and blastoycst stages. The COCs cocultured with CDOs developed at higher rates than the COCs cultured separately and the CDOs themselves. The beneficial effects of coculture with conspecific CDOs were presumably due to the paracrine action of some secreted factors that enhanced many molecular patterns related to the complex of cumulus oophorous cells. Further investigations to understand how the 3D microenvironment can influence the features of oocytes and embryos are required. [ABSTRACT FROM AUTHOR]

Contents The Indochinese leopard ( Panthera pardus delacouri) population, included in CITES Appendix I, has been declining for decades. Proper gamete preservation condition is critical for breeding programme management using artificial insemination or in vitro fertilization ( IVF). The present study aimed at investigating the impact of post-thawing treatment of leopard semen with extracellular adenosine 5′-triphosphate ( ATPe) on sperm quality (including morphological traits and ability to fertilize an oocyte). Semen from six adult male leopards was collected by electroejaculation (one ejaculation per cat). After the evaluation of the fresh sample quality, the semen was cryopreserved (10 × 106 cells per straw; two straws per cat). After thawing, the sperm sample from the first straw of each cat was divided into three aliquots: control (no ATPe), supplemented with 1.0 or 2.5 mM ATPe that were evaluated for sperm quality at 10, 30 min and 3 hr post-thawing. The sperm sample from the second straw, supplemented with 0, 1.0 or 2.5 mM ATPe for 30 min, was assessed for IVF with domestic cat oocytes. Sperm quality (all metrics) was negatively affected by the cryopreservation process ( p ≤ .05). However, the percentage of sperm motility, level of progressive motility and percentage of plasma membrane integrity did not differ ( p > .05) among post-thawing groups. The sperm mitochondrial membrane potential was enhanced ( p ≤ .05) by ATPe treatment (1.0 and 2.5 mM; 10 min to 3 hr of incubation). Furthermore, incubation of ATPe (1.0 and 2.5 mM) for 30 min could promote sperm velocity patterns (curvilinear velocity; VCL and straight line velocity; VSL) ( p ≤ .05). The percentage of pronuclear formation and cleaved embryos was increased ( p ≤ .05) after 1.0 ATPe treatment (49.8 ± 2.8; 45.9 ± 1.5) compared to 0 mM (41.4 ± 3.3; 38.9 ± 0.5) whereas the number of sperm binding/oocyte did not significantly differ among groups. In summary, we suggest that ATPe activated the velocity of Indochinese leopard sperm motility that may lead to faster sperm/oocyte binding and sperm penetration (factors of successful embryo development). [ABSTRACT FROM AUTHOR]

Contents Ovarian tissue cryopreservation followed by tissue culture is a promising approach to preserving the fertility of biomedical models and endangered species. The objective of this study was to investigate the impact of exposure time to vitrification solution and presence of sucrose using different exposure temperatures and base media on intra-ovarian follicle integrity. Peripubertal ovarian cortical pieces were obtained by isolating the cortex and dissecting it into 1 × 1 × 0.2 mm3 pieces. The cortical pieces were then exposed to equilibration solution and then vitrification solutions ( VS) in one of the conditions mentioned above, plunged directly into liquid nitrogen and stored for ≥24 hr in liquid nitrogen. After thawing, the cortical pieces were cultured in vitro for 0, 1 or 7 days to determine the follicle integrity (through histological assessment) and the ability of the tissue to recover from cryoinjury. Fresh controls maintained a constant level of normal morphology (>60% of the total follicles) throughout the culture period. Cortical pieces exposed to VS with sucrose for 10 min had the highest percentage of normal follicles (approximately 20% after 7 days of culture) throughout the culture period. Other conditions using different base medium, lower exposure temperatures or different thawing methods did not improve the follicle integrity. This protocol provides a solid foundation on which to optimize ovarian tissue cryopreservation in the domestic cat and to investigate the molecular effects of vitrification. [ABSTRACT FROM AUTHOR]

Contents The sperm centrosome is an essential organelle with a key role in organizing the sperm aster for proper syngamy and formation of the first mitotic spindle. The sperm cell acquires the functional capability during epididymal transit by incorporation of key factors. The objective of the study was to identify these key maturation proteins, such as ninein and centriolin as well as cenexin-a scaffold protein that serves to bind ninein and centriolin. Epididymal samples were dissected from 17 adult cat testes (>1 year old) and spermatozoa were extracted from the different regions, including rete testis, caput, corpus, cauda and vas deferens. Tissue samples and sperm cells were fixed separately in 4% paraformaldehyde before immunostaining with anticenexin, ninein or centriolin antibodies. Results showed that the proportion of sperm cells with cenexin localized at the centrosome progressively increased along the tract with the lowest percentage of stained cells in the testis (mean = 45%) and highest in the cauda (mean = 81%). Although not significant, the intensity of cenexin immunofluorescence in positive cells increased twofold from the testis to vas deferens. There was no significant difference in the proportion of sperm labelled with centriolin or ninein (ranges of 21%-26% and 33%-48% between segments, respectively) or the intensity (±58% and ±63% change as compared to testis, respectively). Cenexin may serve as a scaffold protein for centriolin and ninein, as the vast majority of spermatozoa only displayed colocalization of these proteins when cenexin was also present (mean = 85% and 91% colocalization, respectively). In summary, these results could be applied to future efforts to create an in vitro culture system capable of rescuing the impaired centrosome of an infertile male, with particular potential for wild felid conservation. [ABSTRACT FROM AUTHOR]

Contents Innovations are emerging from the growing field of fertility preservation for humans and laboratory animals that are relevant to protecting and propagating valuable domestic and wild carnivores. These extend beyond the 'classical' approaches associated with sperm, oocyte and embryo freezing to include gonadal tissue preservation combined with in vitro culture or xenografting, all of which have potential for rescuing vast amounts of unused and wasted germplasm. Here, we review approaches under development and predicted to have applied value within the next decade, including the following: (i) direct use of early-stage gametes for in vitro fertilization; (ii) generation of more mature gametes from gonadal tissue or stem cells; (iii) simplification, enhanced safety and efficacy of cryopreservation methods; and (iv) biostabilization of living cells and tissues at ambient temperatures. We believe that all of these fertility preservation strategies will offer knowledge and tools to better manage carnivores that serve as human companions, valuable biomedical models or require assistance to reverse endangerment. [ABSTRACT FROM AUTHOR]

Contents The aim of this study is to produce live kittens from oocytes fertilized by intracytoplasmic sperm injection ( ICSI) with frozen/thawed testicular spermatozoa. Spermatozoa were collected from thawed testicular tissue and subsequently injected into in vitro matured cat oocytes. At 24 h post- ICSI, presumptive zygotes/cleaved embryos were treated with 10 μ m forskolin for 24 h to reduce intracellular lipid content of embryos (delipidation). At 48 h after oocyte injection, cleaved embryos (2- to 8-cell stage) were frozen in 10% (v/v) ethylene glycol-based medium by a slow controlled rate method and stored in liquid nitrogen. To evaluate in vitro and in vivo developmental competence, frozen embryos were thawed and then cultured for 6 days (n = 155) or cultured for 2 h before transferred (n = 209) to hormonal (equine chorionic gonadotropin/h CG)-treated cat recipients. Cleavage frequency at day 2 after ICSI with frozen/thawed testicular spermatozoa was ~30%. The percentages of frozen/thawed embryos that developed to morula and blastocyst stage (on day 3 and day 6 of in vitro culture, respectively) were significantly lower than that of fresh ICSI embryos (22.6 vs 45.2% and 21.3 vs 38.7%, respectively; p < 0.05). However, no difference was found in the number of blastomeres between frozen/thawed (242.5 ± 43.1) and fresh (320.2 ± 28.1) blastocysts. Three of seven cat recipients were pregnant and one pregnant cat delivered two healthy kittens. This is the first report of the birth of kittens after the transfer of frozen-thawed embryos produced by ICSI with frozen/thawed testicular sperm. [ABSTRACT FROM AUTHOR]

Contents Clouded leopards ( Neofelis nebulosa) produced high proportion of abnormal spermatozoa (mainly tail defects) that can limit sperm movement and conception. The study aimed to better identify the origin of those defects using a demembranation approach. Ejaculates (1-2 ejaculations/male; n = 9) were allocated to simple washing (control; resulting in 11.7% ± 1.9% coiled tails) and processed through colloid centrifugation to reduce the number of sperm with tail defects (treatment, resulting in 5.9% ± 0.9% coiled tails). Aliquots of semen were incubated in hypo-osmotic solution ( HOS, 60 mOsm fructose solution) containing 5 mM dithiothreitol ( DTT) (a reducing agent) to prevent oxidation of sperm membrane. Thereafter, 20% Triton X-100 ( TX) (a detergent) was added to the HOS/ DTT-treated samples. After HOS/ DTT incubation, the control samples and sperm-selected samples presented 73.4% ± 3.1% and 73.9% ± 2.5% swollen sperm (bent and coiled) indicating membrane intact, respectively. Most of the coiled tail in the raw ejaculates could not be opened by TX indicating that the cause of coiled sperm tails may be from testicular origin. The proportion of sperm with tightly coiled tail tended to be lower in the sperm-selected group than control group (18.8% ± 3.8% and 26.5% ± 3.4%; p = .1), whereas the sperm opened up by TX tended to be higher in the sperm-selected group (53.6% ± 10.4% and 21.1% ± 7.9%; p = .06). The results indicated TX was able to uncoil half of the tightly coiled sperm in the semen undergone preparation. In conclusion, the coiled sperm in the clouded leopard semen were likely not a defect of sperm volume regulation during post-ejaculate (osmotic swelling) but pre-ejaculate origin. Semen preparation demonstrated its ability to lessen the primary sperm defects and selected spermatozoa that were prone to be mitigated after demembranation. [ABSTRACT FROM AUTHOR]

Contents Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p < 0.05) in embryos derived in the SCBI culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality. [ABSTRACT FROM AUTHOR]

Contents The objective of this study was to assess and compare the quality of cat blastocysts produced in vitro using commercial blastocyst growth media supplemented with different sources of proteins (serum protein substitute from in vitro maturation through embryo development vs 4 mg/ml of bovine serum albumin for maturation and 5% foetal calf serum for fertilization and embryo development). Impact was specifically examined on the proportion of blastocyst formation, total number of blastomeres, proportion of inner cell mass and expression of pluripotency marker proteins NANOG and OCT-4. Blastocyst formation per total cleaved embryos was similar (p > 0.05) regardless of the protein supplementation. There were no differences (p > 0.05) between culture conditions regarding average number of blastomeres and proportion of inner cell mass in each embryo. Presence of OCT-4 protein was detected in nuclei of both trophectoderm and inner cell mass region, with a stronger signal in the latter regardless of the culture medium. NANOG protein also was present in the inner cell mass regardless of the in vitro culture condition. We therefore demonstrated that serum protein substitute was as good as semi-defined protein sources for the production of good-quality blastocysts and embryonic stem cells. In addition, a single defined medium could be successfully used for cat oocyte maturation, in vitro fertilization and embryo development. [ABSTRACT FROM AUTHOR]

Contents This study identified specific changes in histone lysine methylation patterns of the feline germinal vesicle ( GV) during pre-antral-to-antral follicle transition, the latter being a key interval for competence acquisition. Oocytes from adult cats were isolated from pre-antral, early (≤0.5 mm diameter), small (0.6-1 mm) or large (1-3.5 mm) antral follicles and immuno-stained with anti-histones H3 trimethylated at lysine 9 ( H3 K9me3), lysine 4 ( H3 K4me3), lysine 27 ( H3 K27me3) or H3 dimethylated at lysine 79 ( H3 K79me2). The vast majority of oocytes (range, 72.2-85.4%; p > 0.05) contained a GV with H3 K9me3 or H3 K27me3, regardless of follicular stage/size. However, the proportion of GVs with H3 K4me3 or H3 K79me2 was higher in early antral follicles (42.6%; p < 0.05) compared with other stages (range, 12.1-15.2%). Therefore, H3 K4me3 and H3 K79me2 (both known to be associated with selective gene activation) appear to be reliable markers of onset of GV competence during the pre-antral-to-antral transition phase. By contrast, H3 K9me3 and H3 K27me3 (both known to be related to selective gene repression) seem more linked to expression patterns during the GV stage and are less useful indicators during the entire folliculogenesis interval. [ABSTRACT FROM AUTHOR]

Contents The culture of ovarian follicles is an important tool for understanding the mechanisms controlling follicle development and differentiation of the oocyte. The benefit of recovering meiotically and developmentally competent oocytes from early stage follicles (primordial, primary, pre-antral and early antral) also would be significant, ranging from rescue of genomes from endangered species to preserving fertility in women facing cancer treatments. This research field is at an early stage of scientific discovery. To-date, live offspring from cultured primordial follicles that produced fertilizable oocytes has occurred only in the mouse. Progress in other more complex species has been limited because larger animals have longer durations of natural folliculogenesis, thereby requiring more culture time to generate fully grown follicles and oocytes. We believe the dog and cat are excellent models for understanding more about folliculogenesis in vitro. This review highlights what is known about this topic for these two species as well as future priorities. We have discovered that it is more challenging to maintain viability of primordial follicles within ovarian tissues in vitro in the dog than the cat. Nonetheless, it is possible to grow both isolated cat and dog pre-antral follicles in culture. Although the follicles of both species have the capacity to increase in size and produce steroids, only cat oocytes appear morphologically normal. The reason for this striking difference between these two species is an area of high research priority. While much more fundamental data are required, we envision advanced technology that will allow harvesting oocytes from the vast, unused follicle stores sequestered within carnivore ovaries. These gametes have utility for reproducing genetically valuable dogs and cats that are 'companions' or biomedical models for investigating human disorders as well as for salvaging the genomes of rare canid and felid species that die before contributing to genetic management programs. [ABSTRACT FROM AUTHOR]

Contents The immature cat oocyte contains a large-sized germinal vesicle (GV) with decondensed chromatin that is highly susceptible to cryo-damage. The aim of the study was to explore an alternative to conventional cryopreservation by examining the influence of GV chromatin compaction using resveratrol (Res) exposure (a histone deacetylase enhancer) on oocyte survival during vitrification. In Experiment 1, denuded oocytes were exposed to 0, 0.5, 1.0 or 1.5 mmol/l Res for 1.5 h and then evaluated for chromatin structure or cultured to assess oocyte meiotic and developmental competence in vitro. Exposure to 1.0 or 1.5 mmol/l Res induced complete GV chromatin deacetylation and the most significant compaction. Compared to other treatments, the 1.5 mmol/l Res concentration compromised the oocyte ability to achieve metaphase II (MII) or to form a blastocyst. In Experiment 2, denuded oocytes were exposed to Res as in Experiment 1 and cultured in vitro either directly (fresh) or after vitrification. Both oocyte types then were assessed for meiotic competence, fertilizability and ability to form embryos. Vitrification exerted an overall negative influence on oocyte meiotic and developmental competence. However, ability to reach MII, achieve early first cleavage, and develop to an advanced embryo stage (8–16 cells) was improved in vitrified oocytes previously exposed to 1.0 mmol/l Res compared to all counterpart treatments. In summary, results reveal that transient epigenetic modifications associated with GV chromatin compaction induced by Res is fully reversible and beneficial to oocyte survival during vitrification. This approach has allowed the production of the first cat embryos from vitrified immature oocytes. [ABSTRACT FROM AUTHOR]

Contents Knowledge about reproduction is critical for predicting the viability of wildlife populations in nature and for managing breeding programmes in captivity. Intensive species-based studies are the priority, because reproductive mechanisms are extraordinarily diverse, even within the same taxonomic family. Carnivores deserve more attention as such species are highly vulnerable to environmental change and human persecution. The present review provides contemporary illustrations of how reproductive science is contributing to understand unique reproductive mechanisms that are both of fundamental and applied interest. In the case of the endangered African wild dog ( Lycaon pictus) free-living in South Africa, non-invasive faecal corticosteroid assessments have yielded new insights about the impact of animal relocation and reintroduction on adaptive responses, reproductive fitness and survival. For the maned wolf ( Chrysocyon brachyurus), advances have been made in characterizing and comparing reproductive traits in free-ranging vs captive individuals. For the cheetah ( Acinonyx jubatus), recent studies have focused on the cryosensitivity of sperm and the ability to develop a field-friendly sperm cryo-method. The by-product has been a large-scale frozen repository of sperm from wild-caught cheetahs useful for infusing new genes into ex situ populations. Finally, rigorous, multi-disciplinary and cross-institutional reproductive studies of the black-footed ferret ( Mustela nigripes), including the use of artificial insemination, have contributed to the remarkable recovery and restoration of this species, once on the brink of extinction. In summary, advances in reproductive science are not necessarily related to ‘assisted breeding’. However, understanding the unique ways of carnivore reproduction greatly contributes to species management and conservation. [ABSTRACT FROM AUTHOR]

Contents Our objective was to examine the influences of differing media, protein supplementation and the microenvironment on cat vs dog primordial follicle viability in vitro. Ovarian cortical slices were cultured for 3, 9 or 15 days in α- minimum essential medium (α- MEM) or MEM supplemented with 10% fetal bovine serum ( FBS), 10% knock-out serum replacement ( KSR) or 0.1% polyvinyl alcohol (protein free). In a separate study, cat and dog ovarian tissues were cultured in protein-free α- MEM and MEM, respectively, in cell culture inserts, on 1.5% agarose gel or in 24-well cell culture plates (control). Follicle viability was assessed in both studies using calcein AM/ethidium homodimer and histological evaluation with haematoxylin/eosin staining. No cat follicle sustained viability beyond 9 days of in vitro culture in α- MEM compared to 37.5% of those incubated for 15 days in MEM in protein-free condition (p < 0.05). In contrast, α- MEM was superior (p < 0.05) to MEM in maintaining dog follicle viability (32.7% vs 8.1%) in protein-free condition at 15 days. Serum was detrimental (p < 0.05) to follicle survival in both species. Knock-out serum replacement supplementation and a protein-free condition supported cat follicle viability, whereas the latter was superior (p < 0.05) to the former for sustaining dog follicle survival. Likewise, dog follicle viability was enhanced (p < 0.05) by the agarose gel compared to the cell culture insert and control groups after 3 and 9 days of culture. For the cat, the agarose gel better (p < 0.05) supported follicle viability compared to the control, but was equivalent to the cell culture insert. Therefore, sustaining primordial follicle survival from intracortical ovarian slices requires a different in vitro microenvironment for the cat vs the dog. A key factor to enhancing survival of these early stage follicles in culture appears to be the use of agarose gel, which enhances follicle viability, perhaps by promoting gas exchange. [ABSTRACT FROM AUTHOR]

The gut microbiome has a well‐documented relationship with host fitness, physiology, and behavior. However, most of what is known comes from captive animals where diets and environments are more homogeneous or controlled. Studies in wild populations that experience dynamic environments and have natural life history variation are less common but are key to understanding the drivers of variation in the gut microbiome. Here we examine a wild population of yellow‐bellied marmots (Marmota flaviventer), an obligate winter hibernator, to quantify multivariate associations between host‐associated factors (e.g., age, sex, environmental harshness, and social behavior) and gut microbial composition. Across 5 years and 143 individuals, we found that males had a higher relative abundance of microbes associated with mass gain and cellulose digestion, which suggests a metabolic investment in mass gain (such as phylum Firmicutes and family Lachnospiraceae). By contrast, females had higher relative abundances of microbes associated with inflammation and metabolism (from microbial groups such as Tenericutes and Ruminococcus), possibly reflecting the importance of lactation and offspring investment. Post hoc analyses of lactating females showed a negative relationship with the abundance of microbes associated with mass gain but a positive relationship with microbes associated with metabolic energy, suggesting a trade‐off between investment in pups and maternal mass gain. Older animals also had reduced Proteobacteria relative abundance, a phylum associated with reduced inflammation. Results demonstrate that sex and age‐based traits, not sociality or environmental harshness, are associated with microbe‐mediated metabolism and inflammation in a wild, hibernating mammal. [ABSTRACT FROM AUTHOR]

Research on the development of innovative cryobanking techniques can aid coral restoration and conservation. This study applied a customized freezing device and "cryojig" along with vitrification and laser warming techniques to create the first cryorepository for pelagic‐phase larvae of the corals Seriatopora caliendrum, Pocillopora verrucosa, Stylophora pistillata, and Pocillopora acuta. Three vitrification solutions (VSs) were prepared, with each composed of two of four cryoprotective agents. These included VS1: 2 M ethylene glycol (EG) and 1 M propylene glycol; VS2: 2 M EG and 1 M glycerol; VS3: 2 M dimethyl sulfoxide and 1 M EG. Larvae were then mixed with the VSs, 40% (w/v) Ficoll, and 10% (v/v) gold nanoparticles (GNPs) at a final concentration of 1.2 × 1018 particles/m3 and using our customized device for vitrification and laser warming. Over a thousand coral larvae (576 S. caliendrum, 376 P. verrucosa, 238 S. pistillata, and 524 P. acuta) were successfully stored in the cryorepository and the proportions of coral larvae that were noted to be actively swimming or settled after vitrification and laser warming. This is the first project to establish a coral larvae cryorepository, and the methods applied could represent a critical tool for reef restoration and maintenance of marine biodiversity. [ABSTRACT FROM AUTHOR]

Efforts to slow biodiversity loss have ushered in an era of proactive conservation for rare species, or species that have any combination of low relative abundance, high habitat specificity, and limited geographic range. Approaches to wildlife management and conservation were often developed for more abundant, generalist species, and frequently do not translate well to rare species. Conservation for rare species therefore requires adapting knowledge, action and inquiry tools for common species to rare species. We therefore present 10 lessons for adapting conservation tools to rare species, each with advice to implement the lessons. Several themes emerge from these lessons. Information scarcity necessitates high standards of information for rare species. Missing rare species information can be complemented with multiple forms of knowledge, particularly when the need for conservation action precludes additional data collection. Investment in depth (over breadth) of information for individuals of rare species can provide alternatives to population trend, which has lower reliability for rare species. Natural history documentation can improve rare species conservation strategies. Finally, curiosity‐driven inquiry can inspire rare species conservation innovation. We hope providing guidance that addresses challenges for rare species will help others bridge the gap between common wildlife approaches and proactive rare species conservation. [ABSTRACT FROM AUTHOR]

The issue of poor sexual performance of some male giant pandas seriously impairs the growth and the genetic diversity of the captive population, yet there is still no clear understanding of the cause of the loss of this ability and its underlying mechanism. In this study, we analyzed the gut microbiota and its function in 72 fecal samples obtained from 20 captive male giant pandas, with an equal allocation between individuals capable and incapable of natural mating. Additionally, we investigated fecal hormone levels and behavioral differences between the two groups. A correlation analysis was then conducted among these factors to explore the influencing factors of their natural mating ability. The results showed significant differences in the composition of gut microbiota between the two groups of male pandas. The capable group had significantly higher abundance of Clostridium sensu stricto 1 (padjusted =.0021, GLMM), which was positively correlated with fatty acid degradation and two‐component system functions (Spearman, padjusted <.05). Additionally, the capable group showed higher gene abundance in gut microbiota function including purine and pyrimidine metabolism and galactose metabolism, as well as pathways related to biological processes such as ribosome and homologous recombination (DEseq2, padjusted <.05). We found no significant differences in fecal cortisol and testosterone levels between the two groups, and no difference was found in their behavior either. Our study provides a theoretical and practical basis for further studying the behavioral degradation mechanisms of giant pandas and other endangered mammal species. [ABSTRACT FROM AUTHOR]

Most species will not be able to migrate fast enough to cope with climate change, nor evolve quickly enough with current levels of genetic variation. Exacerbating the problem are anthropogenic influences on adaptive potential, including the prevention of gene flow through habitat fragmentation and the erosion of genetic diversity in small, bottlenecked populations. Facilitated adaptation, or assisted evolution, offers a way to augment adaptive genetic variation via artificial selection, induced hybridization, or genetic engineering. One key source of genetic variation, particularly for climatic adaptation, are the core metabolic genes encoded by the mitochondrial genome. These genes influence environmental tolerance to heat, drought, and hypoxia, but must interact intimately and co‐evolve with a suite of important nuclear genes. These coadapted mitonuclear genes form some of the important reproductive barriers between species. Mitochondrial genomes can and do introgress between species in an adaptive manner, and they may co‐introgress with nuclear genes important for maintaining mitonuclear compatibility. Managers should consider the relevance of mitonuclear genetic variability in conservation decision‐making, including as a tool for facilitating adaptation. I propose a novel technique dubbed Conservation Mitonuclear Replacement (CmNR), which entails replacing the core metabolic machinery of a threatened species—the mitochondrial genome and key nuclear loci—with those from a closely related species or a divergent population, which may be better‐adapted to climatic changes or carry a lower genetic load. The most feasible route to CmNR is to combine CRISPR‐based nuclear genetic editing with mitochondrial replacement and assisted reproductive technologies. This method preserves much of an organism's phenotype and could allow populations to persist in the wild when no other suitable conservation options exist. The technique could be particularly important on mountaintops, where rising temperatures threaten an alarming number of species with almost certain extinction in the next century. [ABSTRACT FROM AUTHOR]

The brocket deer (Genus Mazama) is a highly diverse cervid group distributed from Mexico to Argentina, with a downward population trend. However, literature on the basic reproductive biology of the genus is scarce. This work aimed to study biometric, histological and stereological aspects of the testes of Dwarf Red Brocket (Mazama rufina). Testes from free-ranging adult brockets (n = 3) were retrieved from necropsies. Testes were histologically processed. From histological images, several stereological parameters were estimated, and seminiferous epithelium cycle morphology was described. Testes volumes were between 8.2 and 18.4 ml and weights from 8.3 to 19.4 g. Gonadosomatic index (% paired-testes weight to body weight) went from 0.17 to 0.64. The tubular cross-sectional diameter was 179.8 ± 2.8 µm. Estimated volume densities for parenchyma and interstitium were 78.8% and 21.2% respectively. There were (in millions/ml) 96.0 ± 13.1 germ cells and 37.7 ± 6.0 somatic cells. Specific cell densities were (all expressed in millions/ml) as follows: spermatogonia 13.1 ± 4.2; primary spermatocytes 43.1 ± 5.0; round spermatids 36.8 ± 8.0 (lower density near the caudal pole, p < 0.01); sustentacular (Sertoli) cells 16.8 ± 4.1 and interstitial endocrine (Leydig) cells 17.4 ± 3.4. Sertoli cell index (germ cells per Sertoli cell) was 6.72. Eight stages of the cycle were described, and frequencies estimated, resembling those of goats. M. rufina adult testis anatomy is similar to that of other cervids and domestic ruminants, with an apparently lower spermatogenic efficiency. This work is a first approximation to the physiology of the testis of M. rufina. Basic knowledge of the reproductive physiology of vulnerable species may allow biotechnological approaches for the restitution of animal populations., (© 2021 Wiley-VCH GmbH.)

The Indonesian archipelago, where the tropical rainforest ecosystem is home to 15% of the world's vertebrate species, has been the hardest hit by deforestation in Southeast Asia for the last decade. This situation makes ex situ conservation strategies, particularly biobanks, more important than ever to supplement in situ efforts which are becoming increasingly difficult. This study examines the status and future of biobanking implementation in Indonesian wildlife. The results show that current biobanking efforts in Indonesian wildlife are still limited, with a non‐uniform distribution among taxa, sex and geographical origin. Current work is largely focused on mammals, while avifauna, reptiles and amphibians, particularly from the eastern region, are generally neglected. Cryopreservation is primarily used as a low‐cost preservation method, but success rates are strongly influenced by intra‐ and interspecific characteristics, making the development of species‐specific protocols essential. This report identifies the challenges and suggests ways to establish a national biobanking facility and collaborate with stakeholders to promote sustainable biobanking in Indonesia. [ABSTRACT FROM AUTHOR]

An animal's gut microbiota plays an important role in host health, reproduction and digestion. However, many studies focus on only a few individuals or a single species, limiting our ability to recognize emergent patterns across a wider taxonomic grouping. Here, we compiled and reanalysed published 16S rRNA gene sequence data for 745 gut microbiota samples from 91 reptile species using a uniform bioinformatics pipeline to draw broader conclusions about the taxonomy of the reptile gut microbiota and the forces shaping it. Our meta‐analysis revealed the significant differences in alpha‐ and beta‐diversity across host order, environment, diet, habitat and conservation status, with host diet and order contributing the most to these differences. We identified the principal bacterial phyla present in the reptile gut microbiota as Bacteroidota, Proteobacteria (mostly Gamma class), and Firmicutes, and detected the bacterial genus Bacteroides in most reptile individuals, thus representing a putative 'core' microbiota. Our study provides novel insights into key drivers of the reptile gut microbiota, highlights existing knowledge gaps and lays the groundwork for future research on these fascinating hosts and their associated microbes. [ABSTRACT FROM AUTHOR]

Thirteen black agouti (Dasyprocta fuliginosa) male fetuses, acquired in two areas of the Amazon Forest, were used for the purpose of morphologically describing the fetal male urogenital organs with a focus on addressing histological and macroscopic aspects. The organs of interest were dissected and photographed in situ and ex situ. Fragments were collected and subjected to routine histological processing for inclusion in paraffin, which was cut and stained by haematoxylin and eosin and Gomori's Trichrome methods and subsequently analysed in a light microscope. The results showed that the urinary tract is like that of domestic animals and is composed of smooth unilobed kidneys covered by a dense capsule of connective tissue and divided into two well‐defined regions, cortical and medullary. Ureters, urethra and urinary bladder also showed macroscopic and microscopic characteristics similar to those of domestic animals. The penis of these animals has fibroelastic characteristics, with numerous keratinized structures at its apex. In the middle third of the penis, a "U" shaped penile flexure was seen; the glans penis is covered by a keratinized epidermis containing horny spicules. The presence of a penile bone in an endochondral ossification process was observed, being more developed in gestational ages greater than 76 days. The annex glands were not observed, probably because they were fetuses; only the ampulla of the ductus deferens was identified. [ABSTRACT FROM AUTHOR]

The declining population of the Antillean manatee caused by ecosystem degradation and rising pollution has prompted interest in developing conservation strategies for this species. Given this scenario, somatic tissue banks are important tools for acquiring knowledge about the species, as well as for obtaining somatic cells for biotechnological and ecotoxicological applications. Therefore, we aimed to assess the effects of slow freezing (SF) and solid‐surface vitrification (SSV) of the dermis of captive Antillean manatees on the histology and ultrastructure of the tissue and cell viability in culture. While the SSV did not change the dermis thickness, the SF maintained the tissue proliferative potential, assessed by the nucleolar organizer region area, similar to noncryopreserved tissues. Moreover, both techniques reduced the number of fibroblasts and increased the percentage of collagen fibers. Nevertheless, only tissues cryopreserved with SF and noncryopreserved tissues were able to produce cells after in vitro culture. Although SF did not alter cell viability and proliferative activity, cells derived from cryopreserved tissues showed decreased metabolism, altered apoptosis, increased levels of reactive oxygen species, and mitochondrial membrane potential compared to cells from noncryopreserved tissues. In summary, we demonstrated for the first time that Antillean manatee somatic tissues can be cryopreserved by SF, and cells can be obtained after in vitro culture. Improvements in cryopreservation conditions, especially vitrification, of somatic samples are needed to increase the quality of somatic tissue banks in this species. Research Highlights: Antillean manatee somatic tissues can be cryopreserved by slow freezing, and viable cells can be obtained after in vitro culture.Although Antillean manatee viable somatic cells are obtained from these tissues, they undergo alterations in their metabolism and oxidative stress.Solid‐surface vitrification was not efficient in cell recovery, requiring improvements in its use in Antillean manatee skin conservation. [ABSTRACT FROM AUTHOR]

Oocyte in vitro maturation is necessary for the study and application of animal‐assisted reproduction technology in animal reproduction and breeding. The comprehensive transcriptional profile of equine oocyte maturated in vitro has not been fully mined yet, which makes many key transcriptional events still unidentified. Here, Smart‐seq2 was performed to analyse the gene expression pattern and the underlying regulatory mechanism of horse germinal vesicle (GV) and in vitro metaphase II (MII) oocytes. The results showed that 6402 genes (2640 up‐regulated and 3762 down‐regulated in MII samples compared to GV) and 4021 lncRNA transcripts (1210 up‐regulated and 2811 down‐regulated in MII samples compared to GV) were differentially expressed in GV and MII oocytes. Further, GO and KEGG analysis found that differentially expressed mRNAs and lncRNAs were mainly enriched in the pathways related to energy and lipid metabolism. In addition, LGALS3 was found a key gene in mediating the regulation of oocyte meiosis recovery and fertilization ability. This study provides novel knowledge about gene expression and energy metabolism during equine oocyte maturation and a reference for the further study and application of assisted reproductive technology in horse reproduction and breeding. [ABSTRACT FROM AUTHOR]

Climate change is expected to have far‐reaching effects at both the global and regional scale, but local effects are difficult to determine from coarse‐resolution climate studies. Dynamical downscaling can provide insight into future climate projections on local scales. Here, we present a new dynamically downscaled dataset for Indiana and the surrounding regions. Output from the Community Earth System Model (CESM) version 1 is downscaled using the Weather Research and Forecasting model (WRF). Simulations are run with a 24‐hr reinitialization strategy and a 12‐hr spin‐up window. WRF output is bias corrected to the National Centers for Environmental Protection/National Center for Atmospheric Research 40‐year Reanalysis project (NCEP) using a modified quantile mapping method. Bias‐corrected 2‐m air temperature and accumulated precipitation are the initial focus, with additional variables planned for future releases. Regional climate change signals agree well with larger global studies, and local fine‐scaled features are visible in the resulting dataset, such as urban heat islands, frontal passages, and orographic temperature gradients. This high‐resolution climate dataset could be used for down‐stream applications focused on impacts across the domain, such as urban planning, energy usage, water resources, agriculture and public health. [ABSTRACT FROM AUTHOR]

The intensity of sperm competition, in which sperm compete within the female reproductive tract to reach and fertilize her eggs, varies in species with different mating systems. Sperm competition is more intense in species where males cannot monopolize access to reproductive females and females mate with multiple males. In this scenario, a morphological change that increases the ability of sperm to reach and fertilize eggs should rapidly spread in the population, leading to sperm morphological differences between closely related species. Differences in sperm morphology have been reported among primate species with different mating systems. However, due to the inherent logistical and ethical difficulties to sample sperm from wild primates, the extent of variation in sperm morphology within species and among closely related species remains understudied. Here, we compared sperm morphological traits from two sister howler monkey species (Alouatta palliata and Alouatta pigra) that have different mating systems to investigate the effect of sperm competition on sperm morphological traits. We predicted that sperm from A. palliata, where females have more opportunities to mate with multiple males, would show differences in traits associated with increase sperm competitiveness compared to A. pigra where females mostly mate with the central male. We used linear mixed models to determine species differences in sperm morphology, controlling for individual variation. We found that midpieces and heads in A. palliata sperm were on average 26.2% and 11.0% longer, respectively, than those of A. pigra. Differences in these traits are important for sperm speed and hydrodynamic movement in other species and can affect fertilization success. This study provides empirical evidence of sperm morphological traits that evolved through sexual selection in sister primate species with different mating systems. Highlights: We collected sperm from wild Alouatta palliata and Alouatta pigra males to analyze sperm morphology. These sister species differ in mating system and gonadosomatic index, such that A. palliata has larger testis than A. pigra, suggesting more intense sperm competition.We analyzed 100 sperm cells per individual and computed 10 measurements from the head, midpiece, and tail for each sperm.We found significant differences in sperm head length and midpiece length between species. Sperm head and midpiece are longer in A. palliata. These traits have been associated with sperm swimming speed in other species.Our findings are consistent with the hypothesis that sperm competition is more intense in A. palliata than in A. pigra. [ABSTRACT FROM AUTHOR]

Mammalian carnivores (Carnivora) are crucial components of landscapes, because of both their top‐down effects on lower trophic level species and their sensitivity to bottom‐up processes, such as limited food resources (e.g. due to climate instability). To understand their functional role in Iberian ecosystems more clearly, and to define effective plans for their management and conservation, it is crucial to sum up the available regional knowledge that can inform decision‐making processes.We review bio‐ecological research on wild Iberian carnivores over 30 years (1990–2020) and identify key knowledge gaps and priority avenues for future research. Based on a systematic review of the scientific literature, we aimed to: 1) summarise current knowledge; 2) assess species and ecoregion representativeness; 3) identify key research topics addressed and those lacking investment and 4) suggest key future research priorities.We examined 920 peer‐reviewed articles involving wild Iberian mammalian carnivores, focusing on different bio‐ecological issues. We found considerable heterogeneity in the topics and species investigated, as well as in the study areas (ecoregions) explored, with a mismatch between the research priorities identified by researchers and the knowledge gaps.We suggest that future research should prioritise: 1) rear‐edge populations that are at the southwestern limits of the species' Eurasian range, thus being particularly sensitive to the increasing fragmentation and aridity of Iberian ecosystems, and that were less studied (e.g. brown bear Ursus arctos, stoat Mustela erminea, European mink Mustela lutreola and pine marten Martes martes); 2) less‐studied topics, such as morphometry and body condition, ecophysiology, and reproductive biology, all of which provide essential information for species' management and conservation and 3) specific ecoregions for which studies on species' adaptations to environmental and anthropic contexts are lacking (e.g. northern ecoregions of Iberia, Iberian conifer forests and Northwest Iberian montane forests). Our review provides the necessary background to support future research on carnivore populations in Iberia. [ABSTRACT FROM AUTHOR]

Age‐, region‐, and year‐specific estimates of reproduction are needed for monitoring wildlife populations during periods of ecosystem change. Population dynamics of Steller sea lions (Eumetopias jubatus) in Southeast Alaska varied regionally (with high population growth and survival in the north vs. the south) and annually (with reduced adult female survival observed following a severe marine heatwave event), but reproductive performance is currently unknown. We used mark‐resighting data from 1006 Steller sea lion females marked as pups at ~3 weeks of age from 1994 to 1995 and from 2001 to 2005 and resighted from 2002 to 2019 (to a maximum age of 25) to examine age‐, region‐, and year‐specific reproduction. In the north versus the south, age of first reproduction was earlier (beginning at age 4 vs. age 5, respectively) but annual birth probabilities of parous females were reduced by 0.05. In an average year pre‐heatwave, the proportion of females with pup at the end of the pupping season peaked at ages 12–13 with ~0.60/0.65 (north/south) with pup, ~0.30/0.25 with juvenile, and ~0.10 (both regions) without a dependent. In both regions, reproductive senescence was gradual after age 12: ~0.40, 0.40, and 0.20 of females were in these reproductive states, respectively, by age 20. Correcting for neonatal mortality, true birth probabilities at peak ages were 0.66/0.72 (north/south). No cost of reproduction on female survival was detected, but pup production remained lower (−0.06) after the heatwave event, which if sustained could result in population decline in the south. Reduced pup production and greater retention of juveniles during periods of poor prey conditions may be an important strategy for Steller sea lions in Southeast Alaska, where fine‐tuning reproduction based on nutritional status may improve the lifetime probability of producing pups under good conditions in a variable and less productive environment. [ABSTRACT FROM AUTHOR]

The andrological study of a species involves the macro‐ and microscopic analyses of the internal reproductive organs and the evaluation of seminal parameters and ultrastructural characteristics of the spermatozoa. As in other vertebrates, the male reproductive tract in chondrichthyans consists of testes and reproductive ducts (efferent duct, epididymis, Leydig's gland, ductus deferens and seminal vesicle). In this study the authors used three adult specimens of Zapteryx brevirostris from wild capture kept at the Ubatuba Aquarium, Brazil. Semen was collected by abdominal massage over the location of the seminal vesicle, preceded by ultrasonographic evaluation. The semen collected was diluted 1:200 and subject to quantitative and morphological analyses. Ultrastructural analysis was performed using transmission and scanning electron microscopy. Correlation was observed between successful collection and ultrasonographic image of an engorged seminal vesicle, as well as testicles with easily delimitable margins and higher echogenicity. It was possible to identify free spermatozoa with helical filiform appearance, as well as spermatozeugmata. The average sperm concentration resulted in 5 million packets per millilitre and 140 million spermatozoa per millilitre. The sperm nucleus is described as follows: cone shaped, parachromatin sheath less dense than the chromatin of the nucleus, smooth depression of the nuclear fossa, abaxial axoneme 9 + 2 and accessory axonemal columns in positions 3 and 8 and oval shaped, with flattened inner surface in cross‐section. These results broaden the knowledge of the andrology of this species, contributing to ex situ breeding programmes. [ABSTRACT FROM AUTHOR]

Aim: Understanding changes in the behaviour of threatened species responding to rapidly increasing human disturbances is critical for biodiversity conservation. Here, we synthesize a meta‐analysis of the cumulative effect of human disturbances on the behaviour of global threatened terrestrial mammals. Location: Global terrestrial ecosystem. Time Period: Data collected from 1993 to 2021. Major Taxa Studied: Terrestrial mammals. Results: There were significant differences in behavioural changes among categories of human disturbances (i.e. biological invasion, climate change, grazing, habitat degradation, protection management, road traffic and tourism). The effect size of road traffic on behavioural change was the largest and particularly led habitat selection to be more specialized. The effect size for habitat degradation on foraging behaviour was the largest, and the effect mainly led to a shorter time spent in foraging and a change in food selection. Changes to behaviour increased with human disturbance intensity and varied among species according to their functional traits including body mass, food habits, migration and group type. Climate change, grazing, road traffic and tourism had a greater effect on larger species. The effect size for habitat degradation on omnivorous species was the largest, while carnivorous and solitary species were more sensitive to tourism, and migratory species were especially vulnerable to climate changes. Main Conclusions: The diverse human disturbances interact with disturbance intensity, and some species' functional traits significantly affected the behavioural change in threatened terrestrial mammals. Such behavioural changes away from predisturbance patterns may have consequences for their fitness and community interactions. The management and conservation of threatened species should incorporate knowledge of their behavioural responses to human disturbance and take into account the potential ecological consequences for biodiversity conservation. [ABSTRACT FROM AUTHOR]

The term 'cloning' refers to the production of genetically identical individuals but has meant different things throughout the history of science: a natural means of reproduction in bacteria, a routine procedure in horticulture, and an ever‐evolving gamut of molecular technologies in vertebrates. Mammalian cloning can be achieved through embryo splitting, somatic cell nuclear transfer, and most recently, by the use of induced pluripotent stem cells. Several emerging biotechnologies also facilitate the propagation of genomes from one generation to the next whilst bypassing the conventional reproductive processes. In this review, we examine the state of the art of available cloning technologies and their progress in species other than humans and rodent models, in order to provide a critical overview of their readiness and relevance for application in endangered animal conservation. [ABSTRACT FROM AUTHOR]

The role of ex situ conservation facilities or captivity through captive breeding programs is essential in the conservation of the endangered Javan banteng. The development of semen cryopreservation may assist on one side of the conservation plan. However, the male Javan banteng reproductive capability must be considered as it influences the targeted outputs. Studying the potential biomarker for fertility such as osteopontin gene expression is also expected to help predict male fertility. Therefore, this study aimed to analyze the quality of spermatozoa after thawing to help predict the male reproductive capability of Javan banteng. Furthermore, this study investigated the potential role of osteopontin gene expression in male Javan banteng fertility. A positive reinforcement approach was used to accustom the male and female animals as we focused on establishing a collection procedure using neither sedation nor anaesthesia. Semen samples were collected at Taman Safari Indonesia, Bogor, in accordance with the female banteng receptivity. Semen samples were then evaluated and then cryopreserved under field conditions. Our study showed the different predicted reproductive capability of the Javan banteng based on the post-thaw spermatozoa quality, which showed significant differences. The OPN gene showed positive correlations with the progressive motility (r = 0.711, p = 0.048), viability (r = 0.822, p = 0.012), and acrosomal integrity (r = 0.665, p = 0.072) of Javan banteng spermatozoa after thawing. Our study demonstrated the predicted Javan banteng reproductive capability based on various post-thaw spermatozoa variables. This finding is also the first report on the OPN gene potential to be developed as the assessment tool of post-thaw spermatozoa quality of the male Javan banteng. The findings in our study may help give recommendations for future breeding programs, especially in the ex situ conservation sites. [ABSTRACT FROM AUTHOR]

Urbanization has dramatically altered Earth's landscapes and changed a multitude of environmental factors. This has resulted in intense land‐use change, and adverse consequences such as the urban heat island effect (UHI), noise pollution, and artificial light at night (ALAN). However, there is a lack of research on the combined effects of these environmental factors on life‐history traits and fitness, and on how these interactions shape food resources and drive patterns of species persistence. Here, we systematically reviewed the literature and created a comprehensive framework of the mechanistic pathways by which urbanization affects fitness and thus favors certain species. We found that urbanization‐induced changes in urban vegetation, habitat quality, spring temperature, resource availability, acoustic environment, nighttime light, and species behaviors (e.g., laying, foraging, and communicating) influence breeding choices, optimal time windows that reduce phenological mismatch, and breeding success. Insectivorous and omnivorous species that are especially sensitive to temperature often experience advanced laying behaviors and smaller clutch sizes in urban areas. By contrast, some granivorous and omnivorous species experience little difference in clutch size and number of fledglings because urban areas make it easier to access anthropogenic food resources and to avoid predation. Furthermore, the interactive effect of land‐use change and UHI on species could be synergistic in locations where habitat loss and fragmentation are greatest and when extreme‐hot weather events take place in urban areas. However, in some instances, UHI may mitigate the impact of land‐use changes at local scales and provide suitable breeding conditions by shifting the environment to be more favorable for species' thermal limits and by extending the time window in which food resources are available in urban areas. As a result, we determined five broad directions for further research to highlight that urbanization provides a great opportunity to study environmental filtering processes and population dynamics. [ABSTRACT FROM AUTHOR]

Few observations exist for the Kisatchie painted crayfish (Faxonius maletae), and little is known about its distribution, habitat use, or population structure. The lack of basic ecological data has led to a 'Data Deficient' designation, a category of conservation concern that is often overlooked for management actions. This work provides updated data and practical management suggestions for similarly Data Deficient species.Comprehensive (i.e. beyond known collection sites) crayfish surveys paired with habitat assessments across north‐east Texas were conducted. Among 74 survey sites in 10 counties within and adjacent to the known range for F. maletae, eight total crayfish species were encountered within 35 of these sites (47.3% of the total). Thirteen F. maletae individuals were collected across seven sites (9.5%). Notably, two individuals were found where the species had not been previously observed.Stepwise multiple logistic regression indicated that conductivity and availability of exposed root masses were positively related to F. maletae presence, although neither factor was statistically significant on its own. Neither nonmetric multidimensional scaling nor canonical correspondence analysis identified patterns between F. maletae presence and the presence of other crayfish species.Overall, F. maletae is geographically rare and occurs in exceptionally low abundance, challenging the ability to understand drivers of its distribution. However, given the depth of this survey, we can confidently say that F. maletae is no longer Data Deficient; rather, there is compelling evidence that it has a limited range and small population size.The Data Deficient versus rarity debate has wide‐reaching impacts on the conservation of aquatic taxa; species that are Data Deficient require heightened research focus in general, whereas rare organisms require targeted management to protect specific habitats. Furthermore, we suggest that future management efforts should focus on maintaining current populations through active monitoring to inform the potential habitat suitability of new areas for protection. [ABSTRACT FROM AUTHOR]

Contents Sperm cryopreservation offers a long-term preservation of male genetic materials for future assisted reproductive technologies. However, dramatic changes in temperature during freezing and thawing injure sperm cells. While motility is essential for AI and membrane integrity is crucial for in vitro fertilization ( IVF), sperm DNA integrity is a common index of fertilizing capability required for AI, IVF and intracytoplasmic sperm injection ( ICSI). In endangered felids died unexpectedly, attempts have been made to recover as many DNA intact spermatozoa as possible from epididymis and testis to increase the opportunity to produce offspring in future. Although sperm from caudal epididymis has shown retained fertilizing capability after freezing and thawing (27.3% conception rate after unilateral intrauterine insemination), sperm recovery from the corpus epididymis has been suggested as an alternative to increase the amount of preserved genetic materials. To improve epididymal sperm quality, pre-treatment with single-layer centrifugation resulted in selection of sperm cells with intact DNA while post-thaw treatment with extracellular ATP incubation promoted the blastocyst rate. Cold storage of domestic cat testis for 7 days at 4°C demonstrated <1% of sperm cells with fragmented DNA. Moreover, isolated testicular sperm cells, stored for 7 days at 4°C, produced after ICSI poorer percentages of cleavage, morula and blastocyst than the fresh control. In wild felids, a death-to-necropsy time of 2 hr after a jungle cat ( Felis chaus) aged 10 years died during anaesthesia plus another necropsy-to-sperm recovery time of 25 hr has been reported to yield the post-thawed testicular sperm with 22.2% intact DNA. In summary, the chromatin structure of feline ejaculated and epididymal sperm seems to be tolerated to cold storage and cryopreservation; thus, fertilizing capability is well protected. In contrast, the cat testicular sperm DNA is generally damaged through the cryopreservation. [ABSTRACT FROM AUTHOR]

The disruption of animals' symbiotic bacterial communities (their microbiota) has been associated with myriad factors including changes to the diet, hormone levels, and various stressors. The maintenance of healthy bacterial communities may be especially challenging for social species as their microbiotas are also affected by group membership, social relationships, microbial transfer between individuals, and social stressors such as increased competition and rank maintenance. We investigated the effects of increased social instability, as determined by the number of group changes made by females, on the microbiota in free‐living, feral horses (Equus caballus) on Shackleford Banks, a barrier island off the North Carolina coast. Females leaving their groups to join new ones had fecal microbial communities that were similarly diverse but compositionally different than those of females that did not change groups. Changing groups was also associated with the increased abundance of a several bacterial genera and families. These changes may be significant as horses are heavily dependent upon their microbial communities for nutrient absorption. Though we cannot identify the particular mechanism(s) driving these changes, to the best of our knowledge, ours is the first study to demonstrate an association between acute social perturbations and the microbiota in a free‐ranging mammal. [ABSTRACT FROM AUTHOR]

Culture of domestic cat preantral follicles can be a suitable technology to assist oocyte conservation strategies in the family Felidae. This research was aimed to comparatively analyse cat preantral follicular development of follicles directly seeded on growth surface or encapsulated in 0.5 or 1% of sodium alginate in a serum‐free medium containing FSH, EGF and IGF‐I. Preantral follicles were isolated from cat ovarian cortical tissue after ovariectomy. Alginate was dissolved at 0.5 or 1% in PBS. Follicles, 4 per well, with 0% (G‐0%), 0.5% (G‐0.5%) or 1% (G‐1%) of sodium alginate were cultured in M199 with FSH (100 ng/mL), EGF (100 ng/mL) and IGF‐I (100 ng/mL) for 7 days at 37°C, 5% CO2 and 99% humidity. Culture medium was replaced every 48 h and samples were stored at −20°C until ELISA of steroid hormones. Morphometric evaluation of follicles was performed every 24 h. G‐0% follicles showed granulosa cell migration away from the oocyte and disrupted morphology, whereby they reached apparently larger diameters (203.70 ± 5.82 μm; p <.05) than G‐0.5% and G‐1% follicles (157.89 ± 8.47 μm and 95.23 ± 1.67 μm, respectively) which maintained three‐dimensional organization, being larger in G‐0.5% than in G‐1% (p <.05). G‐0.5% follicles attained the multi‐layer preantral follicle stage on day 7 of culture, whereas G‐1% follicles underwent progressive atresia. On day 6, steroid concentrations were higher (p <.05) in G‐0% than in G‐1%: 60 ± 19 vs 0.88 ± 0.32 pg/mL oestradiol; 2.6 ± 0.84 vs 0.04 ± 0.02 ng/mL progesterone; 1.3 ± 0.22 vs 0.61 ± 0.04 ng/mL testosterone and 1.6 ± 0.54 vs 0.22 ± 0.07 ng/mL androstenedione respectively. Steroid concentrations in G‐0.5% were comprised between those of G‐0% and G‐1% (p >.05). In conclusion, two‐layer cat preantral follicles encapsulated in 0.5% alginate cultured in medium containing FSH, EGF and IGF‐I can develop up to the multi‐layer preantral stage in 7 days of culture, whereas follicles directly seeded on growth surface or encapsulated in 1% alginate lost their three‐dimensional organization, and experienced regression with compromised steroidogenesis, respectively. [ABSTRACT FROM AUTHOR]

Research on microbiota dynamics in humans (Gilbert et al., 2018), model organisms (Douglas, 2019), and free‐ranging, wild animals (Grond et al., 2018) has taken off in the past decades, and even in nonmodel organisms, research has already shifted from initial characterization studies to those examining associations with behaviour and fitness (Bodawatta et al., 2022; Corl et al., 2020; Risely et al., 2018; Turjeman et al., 2020). The microbiota is known to change through pregnancy and parturition (Koren et al., 2012), and there is also evidence in humans that infertility may be associated with microbiota composition (Silva & Giacobini, 2019), but how the microbiota is related to reproductive fitness in free‐ranging species is largely understudied or primarily focused on pathogen transmission (sexually transmitted infection) (Lombardo, 1998; Sheldon, 1993). In a From the Cover article in this issue of Molecular Ecology, Leclaire et al. (2022) begin to tease apart the relationship between the microbiota and reproductive fitness using the black‐legged kittiwake (Rissa tridactyla) as their study species. Following characterization of the microbiota in multiple body sites of breeders and nonbreeders, they discovered that breeding and nonbreeding females had distinct microbiota, that higher performing female breeders had lower abundances of potentially pathogenic taxa, and that feathers of these birds were characterized by reduced microbiota diversity compared to low‐performance breeders. Leclaire and her colleagues provide some of the first evidence of body‐wide differences in microbiota composition in relation to breeding status. Their research further supports the relationship between the microbiota and host fitness, and additional studies focusing on this topic can continue to unravel intricacies in host‐microbiota‐reproductive strategy evolution (Comizzoli et al., 2021; Rowe et al., 2020). Here, I review the results of Leclaire et al. (2022) and provide a wider context for their research by reviewing other studies in the field, focusing on avian species. [ABSTRACT FROM AUTHOR]

The microbiota is suggested to be a fundamental contributor to host reproduction and survival, but associations between microbiota and fitness are rare, especially for wild animals. Here, we tested the association between microbiota and two proxies of breeding performance in multiple body sites of the black‐legged kittiwake, a seabird species. First we found that, in females, nonbreeders (i.e., birds that did not lay eggs) hosted different microbiota composition to that of breeders in neck and flank feathers, in the choanae, in the outer‐bill and in the cloacae, but not in preen feathers and tracheae. These differences in microbiota might reflect variations in age or individual quality between breeders and nonbreeders. Second, we found that better female breeders (i.e., with higher body condition, earlier laying date, heavier eggs, larger clutch, and higher hatching success) had lower abundance of several Corynebacteriaceae in cloaca than poorer female breeders, suggesting that these bacteria might be pathogenic. Third, in females, better breeders had different microbiota composition and lower microbiota diversity in feathers, especially in preen feathers. They had also reduced dispersion in microbiota composition across body sites. These results might suggest that good breeding females are able to control their feather microbiota—potentially through preen secretions—more tightly than poor breeding females. We did not find strong evidence for an association between reproductive outcome and microbiota in males. Our results are consistent with the hypothesis that natural variation in the microbiota is associated with differences in host fitness in wild animals, but the causal relationships remain to be investigated. see also the Perspective by Sondra Turjeman [ABSTRACT FROM AUTHOR]

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To evaluate the effects of four extenders on the post‐thaw quality and fertility of goat semen, six Yunshang Black bucks' semen was collected, pooled, diluted with Andromed® (Andr®), Optidyl® (Opt®), P3644 Sigma l‐phosphatidylcholine (l‐α SL), and skim milk‐based (Milk) extenders, and then cryopreserved. The sperm motilities, abnormalities, membrane and acrosome integrity, mitochondrial activity, apoptosis, and reactive oxygen species (ROS) production were evaluated after thawing. After exocervical insemination with the thawed semen, the pregnancy, lambing, and twinning rates were recorded and compared. The results showed that sperm motilities, membrane integrity, acrosome integrity, mitochondrial activity, and viable spermatozoa were significantly higher in the Andr® and Opt® groups than those in the l‐α SL and Milk groups (p <.05). Furthermore, there was no difference between Andr® and Opt® (p >.05). The sperm abnormality was lower in semen frozen with the Andr® or Opt® extenders, as compared to the l‐α SL or Milk extender (p <.05). Regarding, the viable cells with low ROS production, the optimal results were obtained in the semen frozen with Andr® and Opt® extenders. Following exocervical insemination, the pregnancy and lambing rates in the Milk group were significantly lower than those in the other groups (p <.05). No difference was found in the pregnancy and lambing rates between Andr®, Opt®, and l‐α SL (p >.05). Furthermore, the twinning rates were similar between these four groups (p >.05). In conclusion, egg yolk or skim milk can be substituted by soybean lecithin during cryopreservation of goat semen. [ABSTRACT FROM AUTHOR]

Genome resource banks (GRBs) have the potential to preserve the genetic diversity of a species over time, yet they are rarely utilized as effective components of conservation breeding programs. Advances have been made in reproductive biology, collection and storage techniques, and use of stored gametes for achieving successful reproduction, but there are few guidelines for integrating GRBs into established breeding programs. Here we present basic guidelines, focusing on strategies for the collection, maintenance, and use of semen GRBs for protecting genetic diversity. These guidelines should be applied in the context of the specific purposes and roles of a breeding program's GRB, which will differ among species depending on vulnerability to loss and the status of rescue and conservation efforts. We recommend establishing up to three types of collections: (1) a National Reserve to preserve a species' genetic diversity, to be used only as a last resort; (2) a Savings Account to be used periodically to invigorate a genetically depauperate population; and (3) a Checking Account to be used as a regular part of the breeding program. We present methods for identifying donors to maximize genetic diversity in a GRB, as well as strategies for maintaining and optimally using GRBs. Highlights: The establishment and judicious use of three levels of gamete banks, each serving different roles, can enhance the protection of genetic diversity in conservation breeding programs.A "National Reserve" serves as a protected repository holding to the fullest extent the genetic diversity of a species.A "Savings Account" serves as a resource to periodically reinvigorate populations suffering from genetic erosion.A "Checking Account" contains semen from both deceased and living males to be considered as mate options each generation.We present quantitative methods for determining which males should be sampled to secure the desired genetic diversity in each type of bank. [ABSTRACT FROM AUTHOR]

In ecological and conservation studies, responsible researchers strive to obtain rich data while minimizing disturbance to wildlife and ecosystems. We assessed if samples collected noninvasively can be used for faecal microbiome research, comparing microbiota of noninvasively collected faecal samples to those collected from trapped common cranes at the same sites over the same periods. We found significant differences in faecal microbial composition (alpha and beta diversity), which likely did not result from noninvasive sample exposure to soil contaminants, as assessed by comparing bacterial oxygen use profiles. Differences might result from trapped birds' exposure to sedatives or stress. We conclude that if all samples are collected in the same manner, comparative analyses are valid, and noninvasive sampling may better represent host faecal microbiota because there are no trapping effects. Experiments with fresh and delayed sample collection can elucidate effects of environmental exposures on microbiota. Further, controlled tests of stressing or sedation may unravel how trapping affects wildlife microbiota. [ABSTRACT FROM AUTHOR]

Aim: Hyper‐arid sandy and rocky fields rank among the least biologically diverse habitats of the desert biome, yet knowledge of local biodiversity patterns is also extremely poor. In the Sahara Desert, palaeoclimate oscillations affected the extent of hyper‐arid habitats, but it is unclear how these dynamics determined the evolution and distribution of local specialists. Herein, we assessed cryptic diversity, diversification patterns and spatial connectivity within a Sahara‐widespread group of dryland‐adapted lizards. Location: Sahara‐Sahel ecoregions. Taxon: Acanthodactylus scutellatus species group. Methods: Inter‐ and intraspecific phylogenetic structure, divergence times, spatial genetic patterns and cryptic diversity were assessed using nuclear and mitochondrial loci. The effects of topography and land cover on phylogeographic structure and diversity were tested with generalized linear models. Interspecific hybridization was evaluated using 11 microsatellites across the group's major sympatry zone, predicted based on ecological niche models. Results: Species of Acanthodactylus scutellatus group exhibit Late Miocene origins, followed by extensive intraspecific divergence throughout the Pliocene. The northern Sahara worked as a major diversification hotspot, harbouring a patchwork of small‐ranged, divergent lineages. These lineages are parapatric or sympatric and present concordant nuclear and mitochondrial differentiation, suggesting species status. Genetic connectivity increases in southern latitudes, with wide‐ranging lineages spanning from the Red Sea to the Atlantic coast. Within these potential corridors, mountain outskirts and sand fields in the Sahara interior seemingly acted as origins for recent population expansions. Genetic diversity and connectivity are favoured by terrain roughness and soft‐sand cover respectively. Three species inhabit the Atlantic Sahara sympatry zone without evidence of gene flow. Main conclusions: Overlooked species‐level diversity within a major specialist group of Sahara drylands exposes the recurrent knowledge shortfalls present in hyper‐arid desert environments. Humidity and sandy habitat shifts triggered potential successions of population isolation and re‐connectivity, which favoured cladogenesis in northern desert regions and population expansions across southern east–west corridors. [ABSTRACT FROM AUTHOR]

The Chiricahua leopard frog (Lithobates chiricahuensis) is a threatened species endemic to the southwestern United States and Northern Mexico. Captive breeding programs were established to support reintroduction efforts, yet reproductive output has been lower than needed for recovery of the species. This study aimed to evaluate the effects of captivity on amphibian reproduction by (1) determining if captive, semi‐captive, and wild male L. chiricahuensis produce sperm at similar rates and concentration in response to hormone treatment; and (2) evaluating the quality of sperm obtained over time from these populations. Males from captive, semi‐captive, and wild locations were administered a combination of human chorionic gonadotropin and gonadotropin‐releasing hormone to stimulate sperm production and release. A high percentage of males in the captive (60%), semi‐captive (100%), and wild (95.3%) populations produced sperm following treatment. Sperm quality (forward progressive motility and total sperm motility) did not differ between groups. However, sperm quantity (sperm/ml) differed (p <.05) between populations, with semi‐captive and wild males producing higher concentrations of sperm than captive males. These results suggest that Chiricahua leopard frog sperm quantity, but not quality, may become negatively impacted by long‐term captivity in indoor, controlled settings. [ABSTRACT FROM AUTHOR]