Time-lapse embryo imaging and morphokinetic profiling: towards a general characterisation of embryogenesis

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

Upgrading short read animal genome assemblies to chromosome level using comparative genomics and a universal probe set

Most recent initiatives to sequence and assemble new species’ genomes de-novo fail to achieve the ultimate endpoint to produce a series of contigs, each representing one whole chromosome. Even the best-assembled genomes (using contemporary technologies) consist of sub-chromosomal sized scaffolds. To circumvent this problem, we developed a novel approach that combines computational algorithms to merge scaffolds into chromosomal fragments, scaffold verification by PCR and physical mapping to chromosomes. Multi genome-alignment-guided probe selection led to the development of a set of universal avian BAC clones that permit rapid anchoring of multiple scaffold loci 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 level 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. Using chromosome breakpoint data, we established that avian interchromosomal breakpoints appear in the regions of low density of conserved non-coding 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 could be applied to any animal genome

Upgrading short read animal genome assemblies to chromosome level using comparative genomics and a universal probe set

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

Cryopreservation of animal oocytes and embryos: current progress and future prospects

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

The production of pig preimplantation embryos in vitro: Current progress and future prospects

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

Cryopreservation produces limited long-term effects on the nematode Caenorhabditis elegans

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

Correlation between Pupal Color Dimorphism and Gender Ratios of Papilio polytes romulus (Lepidoptera: Papilionidae) in Sri Lanka

Papilio polytes romulus (Common Mormon) is the most common swallowtail species widely distributed throughout Sri Lanka with females being polymorphic. Their larval food plants include several Citrus and Murraya species and they also show pupal color dimorphism. To study the correlation between pupal color dimorphism and gender ratios their life cycle was studied in the month July of 2021. A free ranging female butterfly was observed laying eggs on Citrus aurantiifolia (lime) in a home garden in Battaramulla.16 eggs were found and 3 days later the eggs hatched and the larvae were collected with leaves and reared in the lab. The duration and measurements of each stages were recorded. After eclosion the gender and wingspan of all adult butterflies were recorded and released back to the environment. The complete life cycle of Common Mormon is known to be about 32-36 days with adult stage about 8-10 days. The immature stages (N=16) duration was recorded as follows: Egg 3.5±0.5 days, 1st instar 3.5±0.5 days, 2nd instar 2.5±0.5 days, 3rd instar 2.5±0.5 days, 4th instar 2.5±0.5 days, 5th instar 5.5±0.5 days, pre-pupa 1.0±0.0 day and pupa 11.5±0.5 days. The length of immature stages (N=16) were recorded as follows: Egg 1.0±0.2 mm, 1st instar 4.0±1.0 mm, 2nd instar 8.0±2.0 mm, 3rd instar 14.5±1.5 mm, 4th instar 23.0±3.0 mm, 5th instar 38.0±8.0 mm, pre-pupa 27.5±2.5 mm and pupa 31.5±0.5 mm. Adult butterfly wingspan ranged from 80- 115mm. Pupae were observed in 2 colors: Green and Brown. Out of 16 pupae, 4 were brown (25%) and 12 were green (75%). 5 pupae (4 brown and 1 green) were made on sticks (rough surface) and the rest (11 green) were made on the wall of the glass container (smooth surface). This showed a strong relationship between pupal color and substrate texture. From the 4 brown pupae emerged 2 males (50%) and 2 females (50%) butterflies. From the 12 green pupae emerged 10 males (83%) and 2 females (17%) butterflies. The gender ratios regardless of the pupal color showed male dominance with 12 males (75%) and 4 females (25%). All four females were of form romulus. Further investigation into environmental factors influencing pupal color plasticity such as season, type of larval food plant, captive vs. non-captive setting, temperature, relative humidity, pupation on plant vs. off plant, substrate texture and background color needs to be studied to fully understand the basis behind this relationship. However, in this study no relationship between pupal color, gender and gender ratios was seen. Keywords: Common mormon, Pupal color, Gender ratios, Sri Lank