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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.

In memory of Ingemar Gustavsson 23rd International Colloquium on Animal Cytogenetics and Genomics (23 ICACG) took place in June 9–12, 2018 in Saint-Petersburg, Russia. Organized biennially, the Colloquium runs from 1970. From its very start this meeting is associated with the name of Ingemar Gustavsson to whom we dedicated the Colloquium 2018. The long and productive career of Ingemar Gustavsson had focused on chromosomes and their fundamental role in animal physiology, fertility, health and production in the context of agriculture and veterinary medicine. His meticulous analysis of breeding data performed back in 1964–69 resulted in the unequivocal identification of an association between heterozygosity for the 1/29 translocation in Swedish cattle and reduction in the fertility of the breed. Eventually, the argument in favor of selective elimination of bulls carrying the translocation from the breeding programs prevailed and the field of modern veterinary cytogenetics was established. Participants from fourteen different countries attended the 23 ICACG in Russia, the country having long lasting traditions in cytogenetics and the Scientific schools of N.K. Koltzov, S.S. Chetverikov and A.S. Serebrovsky, geneticists who made important conceptual contributions to studies of chromosomes and genes, population genetics and evolutionary theory as early as in the beginning of the XX-th century. All the abstracts received were subdivided between plenary and seven scientific sessions covering the issues in evolutionary and comparative cytogenetics, cytogenetics and genomes of domestic animals, meiosis studies, particular chromosome analyses, clinical cytogenetics, karyotypes and genomes of vertebrate and invertebrate animals, chromatin studies. In the abstract text below each presentation is marked with a capital letter: „L” stands for lectures, „O” for oral presentations and „P” for poster presentations. We gratefully acknowledge the support from the Saint-Petersburg Association of Scientists and Scholars (SPbSU), Veterinary Genetics Center ZOOGEN, Russian Foundation for Basic Research (RFBR), VEUK, Helicon, Axioma BIO, BioVitrum, Sartorius, DIA-M companies. The current collected abstracts comprise written contributions of the presentations during the 23 ICACG and were edited by Svetlana Galkina and Maria Vishnevskaya. The next Colloquium – 24 ICACG – will be held at the University of Kent in Canterbury (UK) in 2020. Please, cite abstracts as follows: Gall JG (2018) Giant chromosomes and deep sequences: what the amphibian egg tells us about transcription. In: Galkina SA, Vishnevskaya MS, Mikhailova EI (Eds) 23rd Inernational Colloquium on Animal Cytogenetics and Genomics (23rdICACG), June 9–12, 2018, St Petersburg, Russia. Comparative Cytogenetics 12(3): p–p. https://doi.org/10.3897/CompCytogen.v12i3.27748

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.

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.

It is now over 10 years since the first avian genome [International Chicken Genome Sequencing Consortium, 2004] and the first complete avian karyotype [Masabanda et al., 2004] were both published; however, until 2014, avian cytogenetics has focused heavily on descriptive studies [e.g. Griffin et al., 2007, 2008; Skinner et al., 2009; Volker et al., 2010] with less attention to its functional relevance. Last year, however, saw 2 landmark efforts in the chromosomal studies of birds: a special issue of Chromosome Research in April and the announcement of recently completed sequences of multiple new avian genomes in Science and the BMC journals (taking the total number sequenced to over 50) in December. Studying the chromosomes of birds is, perhaps for the first time, telling us more about avian biology, function and evolution than it ever has... Conclusions. The most recent advances in avian cytogenetics have culminated in great promise not only for the study of bird karyotypes, but also for providing insight into the mechanisms of chromosome evolution in general. New avenues for investigation include gene regulation; for instance, it will become necessary to map accurately the physical location of polyadenylation and transcription start sites, important reference points that define promoters and post-transcriptional regulation. It will also become possible to sequence full-length transcripts, to allow accurate identification of alternate splicing events and their controlling elements. The ENCODE (Encyclopedia of DNA Elements) project has helped to define functional elements of the human genome, including those aforementioned as well as other chromatin signals, e.g. active chromatin, enhancers, insulators, methylation domains, etc. An effort of agENCODE is underway to include agriculturally important birds such as chicken, turkey, duck, quail, and perhaps ostrich. The study of cytogenetics will be essential here in helping to define higher-order structures in nuclear organization that show regulatory interactions within and between chromosomes. Finally, reconstruction of evolutionary events allows us to study genome organization and function not only in extant but, by extrapolation, in extinct species also. Reconstruction of avian-reptilian ancestral karyotypes will allow us to define chromosomal rearrangements in long-dead species that have captured the public imagination. Here be dragons!

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.

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.

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 in vitro fertilisation (IVF) procedures in human embryology clinics, but also following in vitro production (IVP) of embryos in agriculturally important, or endangered animal species, prior to 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.

For 10,000 years pigs and humans have shared a close and complex relationship. From domestication to modern breeding practices, humans have shaped the genomes of domestic pigs. Here we present the assembly and analysis of the genome sequence of a female domestic Duroc pig (Sus scrofa) and a comparison with the genomes of wild and domestic pigs from Europe and Asia. Wild pigs emerged in South East Asia and subsequently spread across Eurasia. Our results reveal a deep phylogenetic split between European and Asian wild boars ∼1 million years ago, and a selective sweep analysis indicates selection on genes involved in RNA processing and regulation. Genes associated with immune response and olfaction exhibit fast evolution. Pigs have the largest repertoire of functional olfactory receptor genes, reflecting the importance of smell in this scavenging animal. The pig genome sequence provides an important resource for further improvements of this important livestock species, and our identification of many putative disease-causing variants extends the potential of the pig as a biomedical model., The research leading to these results has received funding from the European Research Council under the European Community’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement no. 249894 (SelSweep)

Background: The availability of multiple avian genome sequence assemblies greatly improves our ability to define overall genome organization and reconstruct evolutionary changes. In birds, this has previously been impeded by a near intractable karyotype and relied almost exclusively on comparative molecular cytogenetics of only the largest chromosomes. Here, novel whole genome sequence information from 21 avian genome sequences (most newly assembled) made available on an interactive browser (Evolution Highway) was analyzed. Results: Focusing on the six best-assembled genomes allowed us to assemble a putative karyotype of the dinosaur ancestor for each chromosome. Reconstructing evolutionary events that led to each species’ genome organization, we determined that the fastest rate of change occurred in the zebra finch and budgerigar, consistent with rapid speciation events in the Passeriformes and Psittaciformes. Intra- and interchromosomal changes were explained most parsimoniously by a series of inversions and translocations respectively, with breakpoint reuse being commonplace. Analyzing chicken and zebra finch, we found little evidence to support the hypothesis of an association of evolutionary breakpoint regions with recombination hotspots but some evidence to support the hypothesis that microchromosomes largely represent conserved blocks of synteny in the majority of the 21 species analyzed. All but one species showed the expected number of microchromosomal rearrangements predicted by the haploid chromosome count. Ostrich, however, appeared to retain an overall karyotype structure of 2n = 80 despite undergoing a large number (26) of hitherto un-described interchromosomal changes. Conclusions Results suggest that mechanisms exist to preserve a static overall avian karyotype/genomic structure, including the microchromosomes, with widespread interchromosomal change occurring rarely (e.g., in ostrich and budgerigar lineages). Of the species analyzed, the chicken lineage appeared to have undergone the fewest changes compared to the dinosaur ancestor. The authors would like to thank Alain Vignal and Thomas Faraut of INRA Toulouse (France) for access to the duck chromosome assembly data. This research was funded in part by PL-Grid Infrastructure (DML), Biotechnology and Biological Sciences Research Council BB/K008161 (DML, DKG), BB/K008226/1 (DML), BB/J010170/1 (DML, MF) and a knowledge transfer partnership award (DKG and Cytocell Ltd). The authors are grateful to Malcolm Ferguson-Smith’s lab (Cambridge, UK) for producing the flow-sorted chicken microchromosome paints. We also thank Cytocell Ltd (Cambridge, UK) for technical support in FISH technologies. This article was originally published in BMC Genomics 2014, 15:1060 doi:10.1186/1471-2164-15-1060

Background The ability to transport and store DNA at room temperature in low volumes has the advantage of optimising cost, time and storage space. Blood spots on adapted filter papers are popular for this, with FTA (Flinders Technology Associates) Whatman™TM technology being one of the most recent. Plant material, plasmids, viral particles, bacteria and animal blood have been stored and transported successfully using this technology, however the method of porcine DNA extraction from FTA Whatman™TM cards is a relatively new approach, allowing nucleic acids to be ready for downstream applications such as PCR, whole genome amplification, sequencing and subsequent application to single nucleotide polymorphism microarrays has hitherto been under-explored. Findings DNA was extracted from FTA Whatman™TM cards (following adaptations of the manufacturer’s instructions), whole genome amplified and subsequently analysed to validate the integrity of the DNA for downstream SNP analysis. DNA was successfully extracted from 288/288 samples and amplified by WGA. Allele dropout post WGA, was observed in less than 2% of samples and there was no clear evidence of amplification bias nor contamination. Acceptable call rates on porcine SNP chips were also achieved using DNA extracted and amplified in this way. Conclusions DNA extracted from FTA Whatman cards is of a high enough quality and quantity following whole genomic amplification to perform meaningful SNP chip studies.

Background Obesity, excess fat tissue in the body, can underlie a variety of medical complaints including heart disease, stroke and cancer. The pig is an excellent model organism for the study of various human disorders, including obesity, as well as being the foremost agricultural species. In order to identify genetic variants associated with fatness, we used a selective genomic approach sampling DNA from animals at the extreme ends of the fat and lean spectrum using estimated breeding values derived from a total population size of over 70,000 animals. DNA from 3 breeds (Sire Line Large White, Duroc and a white Pietrain composite line (Titan)) was used to interrogate the Illumina Porcine SNP60 Genotyping Beadchip in order to identify significant associations in terms of single nucleotide polymorphisms (SNPs) and copy number variants (CNVs). Results By sampling animals at each end of the fat/lean EBV (estimate breeding value) spectrum the whole population could be assessed using less than 300 animals, without losing statistical power. Indeed, several significant SNPs (at the 5% genome wide significance level) were discovered, 4 of these linked to genes with ontologies that had previously been correlated with fatness (NTS, FABP6, SST and NR3C2). Quantitative analysis of the data identified putative CNV regions containing genes whose ontology suggested fatness related functions (MCHR1, PPARα, SLC5A1 and SLC5A4). Conclusions Selective genotyping of EBVs at either end of the phenotypic spectrum proved to be a cost effective means of identifying SNPs and CNVs associated with fatness and with estimated major effects in a large population of animals.

Background The availability of the complete chicken (Gallus gallus) genome sequence as well as a large number of chicken probes for fluorescent in-situ hybridization (FISH) and microarray resources facilitate comparative genomic studies between chicken and other bird species. In a previous study, we provided a comprehensive cytogenetic map for the turkey (Meleagris gallopavo) and the first analysis of copy number variants (CNVs) in birds. Here, we extend this approach to the Pekin duck (Anas platyrhynchos), an obvious target for comparative genomic studies due to its agricultural importance and resistance to avian flu. Results We provide a detailed molecular cytogenetic map of the duck genome through FISH assignment of 155 chicken clones. We identified one inter- and six intrachromosomal rearrangements between chicken and duck macrochromosomes and demonstrated conserved synteny among all microchromosomes analysed. Array comparative genomic hybridisation revealed 32 CNVs, of which 5 overlap previously designated "hotspot" regions between chicken and turkey. Conclusion Our results suggest extensive conservation of avian genomes across 90 million years of evolution in both macro- and microchromosomes. The data on CNVs between chicken and duck extends previous analyses in chicken and turkey and supports the hypotheses that avian genomes contain fewer CNVs than mammalian genomes and that genomes of evolutionarily distant species share regions of copy number variation ("CNV hotspots"). Our results will expedite duck genomics, assist marker development and highlight areas of interest for future evolutionary and functional studies.