Cryopreservation, the freezing and later warming of biological samples with minimal loss of viability, is important in many scientific disciplines. For some applications, particularly those where there is limited available material, it is critical to ensure the maximal survival rates of cryopreserved materials. Most of the challenges encountered with such techniques take place after the warming process where cryodamage affects cell viability and future development. Here we have used the nematode Caenorhabditis elegans to investigate the effects of cryodamage caused by slow-freezing. We find that freezing results in the death of some worms, with an approximately 40% reduction in the number of worms that develop in the frozen populations, but that the effects on worms that survive are limited. For example, there are no differences in the lifetime fecundity or in lifespan between frozen and control worms, although early fecundity and body size was reduced in frozen worms. Similarly, analyses of body wall muscle structure and of pharyngeal function indicates that muscle development and function are not significantly affected by freezing. We do however determine that freezing increases the rates of matricidal hatching, where progeny hatch within the mother. Overall, these results indicate that, for worms that survive, cryopreservation produces limited long-term effects, but do indicate that some phenotypes could be used in further analyses of the cellular damage induced by cryopreservation.

Spermatogenesis is central to successful sexual reproduction, producing large numbers of haploid motile male gametes. Throughout this process, a series of equational and reductional chromosome segregation precedes radical repackaging of the haploid genome. Faithful chromosome segregation is thus crucial, as is an ordered spatio-temporal ‘dance’ of packing a large amount of chromatin into a very small space. Ergo, when the process goes wrong, this is associated with an improper chromosome number, nuclear position and/or chromatin damage in the sperm head. Generally, screening for overall DNA damage is relatively commonplace in clinics, but aneuploidy assessment is less so and nuclear organisation studies form the basis of academic research. Several studies have focussed on the role of chromosome segregation, nuclear organisation and analysis of sperm morphometry in human subfertility observing significant alterations in some cases, especially of the sex chromosomes. Importantly, sperm DNA damage has been associated with infertility and both extrinsic (e.g. lifestyle) and intrinsic (e.g. reactive oxygen species levels) factors, and while some DNA-strand breaks are repaired, unexpected breaks can cause differential chromatin packaging and further breakage. A ‘healthy’ sperm nucleus (with the right number of chromosomes, nuclear organisation and minimal DNA damage) is thus an essential part of reproduction. The purpose of this review is to summarise state of the art in the fields of sperm aneuploidy assessment, nuclear organisation and DNA damage studies.

Human assisted reproductive technology procedures are routinely performed in clinics globally, and some of these approaches are now common in other mammals such as cattle. This is currently not the case in pigs. Given that the global population is expected to increase by over two billion people between now and 2050, the demand for meat will also undoubtedly increase. With this in mind, a more sustainable way to produce livestock; increasing productivity and implementing methods that will lead to faster genetic selection, is imperative. The establishment of routine and production scale pig embryo in vitro production could be a solution to this problem. Producers would be able to increase the overall number of offspring born, animal transportation would be more straightforward and in vitro produced embryos could be produced from the gametes of selected elite. Here we review the most recent developments in pig embryology, outline the current barriers and key challenges that exist, and outline research priorities to surmount these difficulties.

The ultimate aim of a genome assembly is to create a contiguous length of sequence from the p- to q- terminus of each chromosome. Most assemblies are however highly fragmented, limiting their use in studies of gene mapping, phylogenomics and genomic organisation. To overcome these limitations, we developed a novel scaffold-to-chromosome anchoring method combining reference-assisted chromosome assembly (RACA) and fluorescence in situ hybridisation (FISH) to position scaffolds from de novo genomes onto chromosomes. Using RACA, scaffolds were ordered and orientated into ‘predicted chromosome fragments’ (PCFs) against a reference and outgroup genome. PCFs were verified using PCR prior to FISH mapping. A universal set of FISH probes developed through the selection of conserved regions were then used to map PCFs of peregrine falcon (Falco peregrinus Tunstall, 1771), pigeon (Columba livia Gmelin, 1789), ostrich (Struthio camelus Linnaeus, 1758), saker falcon (Falco cherrug Gray, 1834) the budgerigar (Melopsittacus undulatus Shaw, 1805). Using this approach, we were able to improve the N50 of genomes seven-fold. Results revealed that Interchromosomal breakpoint regions are limited to regions with low sequence conservation, shedding light on why most avian species have very stable karyotypes. Our combined FISH and bioinformatics approach represents a step-change in the mapping of genome assemblies, allowing comparative genomic research at a higher resolution than was previously possible. The universal probe set facilitates research into avian karyotype evolution and the role of chromosome rearrangements in adaptation and phenotypic diversity in birds. Indeed, they have been used on over 20 avian species plus non-avian reptiles (including turtles), shedding light into the evolution of dinosaur species. Non-avian dinosaurs remain subjects of intense biological enquiry while pervading popular culture and the creative arts. While organismal studies focus primarily on their morphology, relationships, likely behaviour, and ecology there have been few academic studies that have made extensive extrapolations about the nature of non-avian dinosaur genome structure prior to the emergence of modern birds. We have used multiple avian whole genome sequences assembled at a chromosomal level, to reconstruct the most likely gross genome organization of the overall genome structure of the diapsid ancestor and reconstruct the sequence of inter and intrachromosomal events that most likely occurred along the Archosauromorpha-Archosauria-Avemetatarsalia-Dinosauria-Theropoda-Maniraptora-Avialae lineage from the lepidosauromorph-archosauromorph divergence ~275 million years ago through to extant neornithine birds.

Breed-specific ideal bodyweight range information is widely used by dog owners and breeders as a guideline to ensure animals are within a healthy weight range. Body Condition Scoring, a method used by veterinarians to assess an animal's overall shape with regard to weight is considered to be an excellent method to determine an animal's overall body condition; these values, however, do not always correspond to published weight ranges. Here, the weight, neuter status, age and a nine-point Body Condition Score of a population of 140 purebred dogs were recorded and subsequently analysed to determine whether bodyweight was an effective predictor for Body Condition Scores. This comparison indicated that published recommended, breed-specific body weight ranges are not a good predictor for an ideal BCS and as such, guidelines for owners and breeders need to be systematically reviewed.

Most recent initiatives to sequence and assemble new species' genomes de novo fail to achieve the ultimate endpoint to produce contigs, each representing one whole chromosome. Even the best-assembled genomes (using contemporary technologies) consist of subchromosomal-sized scaffolds. To circumvent this problem, we developed a novel approach that combines computational algorithms to merge scaffolds into chromosomal fragments, PCR-based scaffold verification, and physical mapping to chromosomes. Multigenome-alignment-guided probe selection led to the development of a set of universal avian BAC clones that permit rapid anchoring of multiple scaffolds to chromosomes on all avian genomes. As proof of principle, we assembled genomes of the pigeon (Columbia livia) and peregrine falcon (Falco peregrinus) to chromosome levels comparable, in continuity, to avian reference genomes. Both species are of interest for breeding, cultural, food, and/or environmental reasons. Pigeon has a typical avian karyotype (2n = 80), while falcon (2n = 50) is highly rearranged compared to the avian ancestor. By using chromosome breakpoint data, we established that avian interchromosomal breakpoints appear in the regions of low density of conserved noncoding elements (CNEs) and that the chromosomal fission sites are further limited to long CNE "deserts." This corresponds with fission being the rarest type of rearrangement in avian genome evolution. High-throughput multiple hybridization and rapid capture strategies using the current BAC set provide the basis for assembling numerous avian (and possibly other reptilian) species, while the overall strategy for scaffold assembly and mapping provides the basis for an approach that (provided metaphases can be generated) could be applied to any animal genome.

Artificial insemination is routinely used in commercial pig breeding, for which the use of high quality semen samples is imperative. Currently, semen quality is determined manually by morphological assessment. This method leads to high inter-operator variability due to its subjective nature. The development of a semi-automated software-based approach to assess sperm morphology would enable faster identification of morphological defects and permit identification of subtle differences that may affect fertilisation success. Here we have used a novel method to comprehensively analyse pig sperm nuclear morphology in greater detail than was previously possible. Semen samples from 50 fertile and 50 sub-fertile samples that had been previously manually categorised as fertile or sub-fertile were analysed using this new method, with at least 200 fixed and DAPI (4',6-diamidino-2-phenylindole) stained sperm heads imaged per sample. Differences in sperm nuclear morphology were observed between fertile and sub-fertile samples; specifically, fertile samples were associated with higher mean nuclear area, a consequence of a greater head width and a lower variability between sperm heads. This novel, unbiased and fast analysis method demonstrates a significant difference in sperm head morphology between fertile and sub-fertile animals, and has the potential to be further developed and used as a tool for sperm morphology assessment in the pig breeding industry.

Fluorescence in-situ hybridization (FISH) revolutionized cytogenetics using fluorescently labelled probes with high affinity with target (nuclear) DNA. By the early 1990s FISH was adopted as a means of preimplantation genetic diagnosis (PGD) sexing for couples at risk of transmitting X-linked disorders and later for detection of unbalanced translocations. Following a rise in popularity of PGD by FISH for sexing and the availability of multicolor probes (5-8 colors), the use of FISH was expanded to the detection of aneuploidy and selective implantation of embryos more likely to be euploid, the rationale being to increase pregnancy rates (referral categories were typically advanced maternal age, repeated IVF failure, repeated miscarriage or severe male factor infertility). Despite initial reports of an increase in implantation rates, reduction in trisomic offspring and spontaneous abortions criticism centered around experimental design (including lack of randomization), inadequate control groups and lack of report on live births. Eleven randomized control trials (RCTs) (2004-2010) showed that preimplantation genetic screening (PGS) with FISH did not increase delivery rates with some demonstrating adverse outcomes. These RCTs, parallel improvements in culturing and cryopreservation and a shift to blastocyst biopsy essentially outdated FISH as a tool for PGS and it has now been replaced by newer technologies (array CGH, SNP arrays, qRT-PCR and NGS). Cell-by-cell follow up analysis of individual blastomeres in non-transferred embryos is however usually prohibitively expensive by these new approaches and thus FISH remains an invaluable resource for the study of mosaicism and nuclear organization. We thus developed the approach described herein for the FISH detection of chromosome copy number of all 24 human chromosomes. This approach involves 4 sequential layers of hybridization, each with 6 spectrally distinct fluorochromes and a bespoke capturing system. Here we report previously published studies and hitherto unreported data indicating that 24 chromosome FISH is a useful tool for studying chromosome mosaicism, one of the most hotly debated topics currently in preimplantation genetics. Our results suggest that mosaic embryo aneuploidy is not highly significantly correlated to maternal age, probably due, in part, to the large preponderance of post-zygotic (mitotic) errors. Chromosome loss (anaphase lag) appears to be the most common mechanism, followed by chromosome gain (endoreduplication), however 3:1 mitotic non-disjunction of chromosomes appears to be rare. Nuclear organization (i.e. the spatial and temporal topology of chromosomes or sub-chromosomal compartments) studies indicate that human morula or blastocyst embryos (days 4-5) appear to adopt a "chromocentric" pattern (i.e. almost all centromeric signals reside in the innermost regions of the nuclear volume). By the blastocyst stage however, a more ordered organization with spatial and temporal cues important for embryo development appears. We have however found no association between aneuploidy and nuclear organization using this approach despite our earlier studies. In conclusion, while FISH is mostly "dead and buried" for mainstream PGS, it still has a place for basic biology studies; the development of a 24 chromosome protocol extends the power of this analysis.

Cryopreservation describes techniques that permit freezing and subsequent warming of biological samples without loss of viability. The application of cryopreservation in assisted reproductive technology encompasses the freezing of gametes, embryos, and primordial germ cells. Whilst some protocols still rely on slow-freezing techniques, most now use vitrification, or ultra-rapid freezing, for both oocytes and embryos due to an associated decreased risk of damage caused by the lack of ice crystal formation, unlike in slow-freezing techniques. Vitrification has demonstrated its use in many applications, not only following IVF procedures in human embryology clinics but also following in vitro production of embryos in agriculturally important, or endangered animal species, before embryo transfer. Here, we review the various cryopreservation and vitrification technologies that are used in both humans and other animals and discuss the most recent innovations in vitrification with a particular emphasis on their applicability to animal embryology.

The past 10–15 years have seen a revolution in the field of genomics, first with the human genome project, followed by those of key model and agricultural species (chicken, pig, cattle, sheep) and, most recently, ? 60 de novo avian genome assemblies. The ultimate aim of a genome assembly is to create a contiguous unbroken length of sequence from p- to q-terminus to facilitate studies of gene mapping, trait linkage, phylogenomics, and gross genomic organization/change. Chromosome rearrangements are biologically relevant both in the context of reduction in reproductive capability of individual animals and in the establishment in reproductive isolation as species evolve and diverge. Moreover, a karyotype effectively represents a low-resolution map of the genome of any species. In investigating all these aspects, FISH remains the tool of choice, and this study describes a step change in its use.

In vitro fertilisation is an effective method of assisted reproductive technology in both humans and certain non-human animal species. In most species, specifically, in humans and livestock, high in vitro fertilisation success rates are achieved via the transfer of embryos with the highest implantation and subsequent developmental potential. In order to reduce the risk of multiple gestation, which could be a result of the transfer of several embryos per cycle, restrictive transfer policies and methods to improve single embryo selection have been implemented. A non-invasive alternative to standard microscopic observation of post-fertilisation embryo morphology and development is time-lapse technology; this enables continuous, uninterrupted observation of embryo development from fertilisation to transfer. Today, there are several time-lapse devices that are commercially available for clinical use, and methods in which time-lapse could be used to improve embryology are continually being assessed. Here we review the use of time-lapse technology in the characterisation of embryogenesis and its role in embryo selection. Furthermore, the prospect of using this technology to identify aneuploidy in human embryos, as well as the use of time-lapse to improve embryological procedures in agriculturally important species such as the pig and cow are discussed.

There has been a recent explosion in avian genomics. In December 2014 the Beijing Genomics Institute in collaboration with a number of labs worldwide (including Kent) released 48 new de-novo avian genome sequences in a special edition of Science. This has led to a complete re-evaluation of the phylogenetic tree of birds and presents the opportunity to study avian comparative genomics in far more detail than before. Most of these genome sequences however exist only as “scaffolds” i.e. the depth of sequence and length of read produces contiguous fragments of sub-chromosomal size. This impedes insight into overall genome structure, which is particularly challenging, as one of the most interesting biological features of birds is the peculiarity of their karyotype. This project is an on-going effort to map scaffold assemblies to avian chromosomes using a combination of bioinformatics and Fluorescent in situ Hybridization (FISH). This has traditionally been a very time-consuming and costly procedure, however a combination of bioinformatic approaches coupled with novel hardware innovation has deconstructed the FISH protocol and re-invented it as a high throughput, cheaper procedure. Initial work has helped to reconstruct Pigeon and Peregrine Falcon genomes and will ultimately provide insight into various unanswered questions pertaining to avian gross genome rearrangement. These include why the unique overall genomic structure of birds is so evolutionarily conserved, why intra and inter-chromosomal rearrangements happen (e.g. in response to the development of traits such as vocal learning) and what the karyotypes of extinct species such as dinosaurs may have looked like.

Editorial on the Research Topic Food safety in low-and middle-income countriesThis is the first special edition on food safety in informal markets in low-and-middle income countries (LMICs).Despite their important public health and socio-economic impacts, foodborne diseases have only recently gained the attention of development institutes and initiatives (Grace, 2023).The is the result of growing appreciation of the enormous burden of foodborne disease in LMICs: the health burden is comparable to that of malaria, HIV/AIDs or tuberculosis and the economic cost is more than 100 billion USD a year (Havelaar et al., 2015;Jaffee et al., 2019).Most of the burden is caused by microbial and parasitic infections and most of these are acquired from fresh foods purchased in mass domestic markets in LMICs (Grace, 2015).These markets are easy to recognize but hard to define.They have been variously called informal, traditional, wet, embedded, and (more recently) territorial markets (Roesel and Grace, 2014;CFS., 2016).They typically include open public markets, kiosks/small shops, butchers, fruit stalls, street food and small-scale eateries.They often lack infrastructure, waste disposal is poor, and pests are common; much of the food sold is fresh, unpackaged, un-processed or traditionally processed and is cheaper than food sold in the modern sector; live animals (especially poultry and fish) may be sold and in some cases wildmeat and traditional foods such as insects; there is an absence of consistent or structured food safety and quality inspection.Informal markets are often supplied by small-scale farmers; workers are not salaried, lack qualifications and training, and often include women and youth; vendors of similar products tend to sell side-by-side with little differentiation of product or price; ready-to-eat food is often available; customers tend to shop frequently, buy in small quantities and be poorer and less educated than customers who patronize the modern food sector.Trust is a major factor in the customer-vendor relation, and vendors may provide added services such as credit or sale in small amounts.The size, variety, and impacts (both positive and negative) of these markets grow in importance as countries develop and urbanize.During this process food safety gets worse before it gets better (Jaffee et al., 2019).Despite their importance for health, nutrition, livelihoods, equity and the urban environment, these markets have been largely ignored in the research literature (Grace, 2015).Therefore, this Research Topic aimed to bring together studies on foodborne Frontiers in Sustainable Food Systems frontiersin.orgGrace et al. ./fsufs. .TABLE Summary of papers on food safety in informal markets.