Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer

BACKGROUND: Somatic cell nuclear transfer (SCNT) provides an appealing alternative for the preservation of genetic material in non-domestic and endangered species. An important prerequisite for successful SCNT is the availability of good quality donor cells, as normal embryo development is dependent upon proper reprogramming of the donor genome so that embryonic genes can be appropriately expressed. The characteristics of donor cell lines and their ability to produce embryos by SCNT were evaluated by testing the effects of tissue sample collection (DART biopsy, PUNCH biopsy, post-mortem EAR sample) and culture initiation (explant, collagenase digestion) techniques. RESULTS: Differences in initial sample size based on sample collection technique had an effect on the amount of time necessary for achieving primary confluence and the number of population doublings (PDL) produced. Thus, DART and PUNCH biopsies resulted in cultures with decreased lifespans (<30 PDL) accompanied by senescence-like morphology and decreased normal chromosome content (<40% normal cells at 20 PDL) compared to the long-lived (>50 PDL) and chromosomally stable (>70% normal cells at 20 PDL) cultures produced by post-mortem EAR samples. Chromosome stability was influenced by sample collection technique and was dependent upon the culture's initial telomere length and its rate of shortening over cell passages. Following SCNT, short-lived cultures resulted in significantly lower blastocyst development (≤ 0.9%) compared to highly proliferative cultures (11.8%). Chromosome stability and sample collection technique were significant factors in determining blastocyst development outcome. CONCLUSION: These data demonstrate the influence of culture establishment techniques on cell culture characteristics, including the viability, longevity and normality of cells. The identification of a quantifiable marker associated with SCNT embryo developmental potential, chromosome stability, provides a means by which cell culture conditions can be monitored and improved

Exploiting Microfluidics for Extracellular Vesicle Isolation and Characterization: Potential Use for Standardized Embryo Quality Assessment

Recent decades have seen a growing interest in the study of extracellular vesicles (EVs), driven by their role in cellular communication, and potential as biomarkers of health and disease. Although it is known that embryos secrete EVs, studies on the importance of embryonic EVs are still very limited. This limitation is due mainly to small sample volumes, with low EV concentrations available for analysis, and to laborious, costly and time-consuming procedures for isolating and evaluating EVs. In this respect, microfluidics technologies represent a promising avenue for optimizing the isolation and characterization of embryonic EVs. Despite significant improvements in microfluidics for EV isolation and characterization, the use of EVs as markers of embryo quality has been held back by two key challenges: (1) the lack of specific biomarkers of embryo quality, and (2) the limited number of studies evaluating the content of embryonic EVs across embryos with varying developmental competence. Our core aim in this review is to identify the critical challenges of EV isolation and to provide seeds for future studies to implement the profiling of embryonic EVs as a diagnostic test for embryo selection. We first summarize the conventional methods for isolating EVs and contrast these with the most promising microfluidics methods. We then discuss current knowledge of embryonic EVs and their potential role as biomarkers of embryo quality. Finally, we identify key ways in which microfluidics technologies could allow researchers to overcome the challenges of embryonic EV isolation and be used as a fast, user-friendly tool for non-invasive embryo selection

The oxidative stress adaptor p66Shc is required for permanent embryo arrest in vitro

<p>Abstract</p> <p>Background</p> <p>Excessive developmental failure occurs during the first week of <it>in vitro </it>embryo development due to elevated levels of cell death and arrest. We hypothesize that permanently arrested embryos enter a stress-induced "senescence-like" state that is dependent on the oxidative stress-adaptor and lifespan determinant protein p66Shc. The aim of this study was to selectively diminish p66Shc gene expression in bovine oocytes and embryos using post-transcriptional gene silencing by RNA-mediated interference to study the effects of p66Shc knockdown on <it>in vitro </it>fertilized bovine embryos.</p> <p>Results</p> <p>Approximately 12,000–24,000 short hairpin (sh)RNAi molecules specific for p66Shc were microinjected into bovine germinal vesicle stage oocytes or zygotes. Experiments were comprised of a control group undergoing IVF alone and two groups microinjected with and without p66Shc shRNAi molecules prior to IVF. The amount of p66Shc mRNA quantified by Real Time PCR was significantly (P < 0.001) lowered upon p66Shc shRNAi microinjection. This reduction was selective for p66Shc mRNA, as both histone H2a and p53 mRNA levels were not altered. The relative signal strength of p66Shc immuno-fluorescence revealed a significant reduction in the number of pixels for p66Shc shRNAi microinjected groups compared to controls (P < 0.05). A significant decrease (P < 0.001) in the incidence of arrested embryos upon p66Shc shRNAi microinjection was detected compared to IVF and microinjected controls along with significant reductions (P < 0.001) in both cleavage divisions and blastocyst development. No significant differences in p66Shc mRNA levels (P = 0.314) were observed among the three groups at the blastocyst stage.</p> <p>Conclusion</p> <p>These results show that p66Shc is involved in the regulation of embryo development specifically in mediating early cleavage arrest and facilitating development to the blastocyst stage for in vitro produced bovine embryos.</p

Reproduction in female wild cattle: Influence of seasonality on ARTs

Wild cattle species, often considered less alluring than certain conservation-dependent species, have not attracted the same level of interest as the charismatic megafauna from the general public, private or corporate donors, and other funding agencies. Currently, most wild cattle populations are vulnerable or threatened with extinction. The implementation of reproductive technologies to maintain genetically healthy cattle populations in situ and ex situ has been considered for more than 30 years. Protocols developed for domestic cattle breeds have been used with some success in various wild cattle species. However, inherent differences in the natural life history of these species makes extrapolation of domestic cattle protocols difficult, and in some cases, minimally effective. Reproductive seasonality, driven by either photoperiod or nutritional resource availability, has significant influence on the success of assisted reproductive technologies (ARTs). This review focuses on the physiological processes that differ in breeding (ovulatory) and non-breeding (anovulatory) seasons in female cattle, and the potential methods used to overcome these challenges. Techniques to be discussed within the context of seasonality include: estrus synchronization and ovulation induction, ovarian superstimulation, artificial insemination (AI), multiple ovulation embryo transfer (MOET), and ovum pick-up (OPU) with in vitro fertilization (IVF) and embryo transfer (ET)

Fecal adrenal hormone patterns during ovulatory and non-ovulatory reproductive cycles in female veiled chameleons (Chamaeleo calyptratus)

The relationship between the reproductive (hypothalamic-pituitary-gonadal; HPG) and adrenal (hypothalamicpituitary- adrenal; HPA) hormone axes is complex and can vary depending on the species and environmental factors affecting an individual. In an effort to understand this relationship in female veiled chameleons (Chamaeleo calyptratus), the patterns of fecal metabolites of corticosterone (C), estradiol (E), testosterone (T), and progesterone (P) were analyzed by enzyme immunoassay (EIA) during ovulatory (OC; eggs laid) and nonovulatory cycles (NOC; no eggs laid). Glucocorticoid (GC) metabolites in the fecal extracts were characterized by HPLC and corticosterone EIA performance was assessed by parallelism, accuracy and precision tests. The results indicated that the assay chosen reliably measured the hormone metabolites present in the fecal extracts. Regular, cyclical hormone metabolite patterns consisting of an E peak followed by peaks of T, P and C in close succession were observed during both ovulatory and non-ovulatory cycles; relative levels of P and C, however, were higher during ovulatory cycles. Corticosterone metabolite levels, in particular, increased throughout vitellogenesis and peaked in late vitellogenesis (in non-ovulatory cycles) or around the time of ovulation, and remained elevated throughout the gravid period, falling just prior to oviposition. The results provide evidence of variation in glucocorticoid production throughout different stages of the reproductive cycle, including a role in the ovulatory process; the physiology, however, remains unclear

Effects of captivity, diet, and relocation on the gut bacterial communities of white-footed mice.

Microbes can have important impacts on their host's survival. Captive breeding programs for endangered species include periods of captivity that can ultimately have an impact on reintroduction success. No study to date has investigated the impacts of captive diet on the gut microbiota during the relocation process of generalist species. This study simulated a captive breeding program with white-footed mice (Peromyscus leucopus) to describe the variability in gut microbial community structure and composition during captivity and relocation in their natural habitat, and compared it to wild individuals. Mice born in captivity were fed two different diets, a control with dry standardized pellets and a treatment with nonprocessed components that reflect a version of their wild diet that could be provided in captivity. The mice from the two groups were then relocated to their natural habitat. Relocated mice that had the treatment diet had more phylotypes in common with the wild-host microbiota than mice under the control diet or mice kept in captivity. These results have broad implications for our understanding of microbial community dynamics and the effects of captivity on reintroduced animals, including the potential impact on the survival of endangered species. This study demonstrates that ex situ conservation actions should consider a more holistic perspective of an animal's biology including its microbes

Museum specimen corticosterone data

File contains corticosterone levels extracted from the hair of museum specimens of deer mouse (Peromyscus maniculatus), that had been collected over an approximately 75-year period

A perspective on the role of emerging technologies for the propagation of companion animals, non-domestic and endangered species

Assisted reproductive technologies (ART) have been used successfully in humans, domestic and laboratory species for many years. In contrast, our limited knowledge of basic reproductive physiology has restricted the application of ART in companion animal, non-domestic and endangered species (CANDES). Although there are numerous benefits, and in some cases a necessity, for applyingART for the reproductive and genetic management of CANDES, the challenges encountered with even the most basic procedures have limited the rate of progress. In this foreword we discuss the status of conventional ART, such as artificial insemination and in vitro fertilisation, as well as their benefits and inherent difficulties when applied to CANDES. It is upon these techniques, and ultimately our knowledge of basic reproductive physiology, that the success of emerging technologies, such as those described in this special issue, are dependent for success

A path forward in the investigation of seabird strandings attributed to light attraction

Abstract A variety of anthropogenic threats cause mortality and population declines of procellariiform seabirds. Globally, fledglings of many colonial procellariiforms become stranded in towns and cities during their first flights from the nest, which occur at night. Since the 1960s, when the phenomenon became widely known, these strandings have been largely attributed to attraction toward artificial lights at night (ALAN). Artificial light attraction has been blamed due to the predictable, annual nature of strandings; the large numbers of birds found in lighted areas during stranding events; and the inexperience of fledglings in interpreting sensory stimuli. However, up‐to‐date, few alternative hypotheses to that of light attraction have been suggested, and few if any have been explored experimentally. In this paper, we do not seek to refute the light attraction hypothesis. Instead, our objectives are threefold. We wish to (1) highlight the current evidence for light attraction in procellariiforms; (2) identify where evidence may be lacking or subject to confirmation bias; and (3) suggest alternative hypotheses and possible experimental approaches to study them. Given the imperiled nature of many of the affected species and the need to explore and address this source of mortality, our goal in this review is to accelerate and diversify research efforts on this topic