Late Morula Research Articles

Meiotic and mitotic aneuploidies drive arrest of in vitro fertilized human preimplantation embryos

BackgroundThe high incidence of aneuploidy in early human development, arising either from errors in meiosis or postzygotic mitosis, is the primary cause of pregnancy loss, miscarriage, and stillbirth following natural conception as well as in vitro fertilization (IVF). Preimplantation genetic testing for aneuploidy (PGT-A) has confirmed the prevalence of meiotic and mitotic aneuploidies among blastocyst-stage IVF embryos that are candidates for transfer. However, only about half of normally fertilized embryos develop to the blastocyst stage in vitro, while the others arrest at cleavage to late morula or early blastocyst stages.MethodsTo achieve a more complete view of the impacts of aneuploidy, we applied low-coverage sequencing-based PGT-A to a large series (n = 909) of arrested embryos and trophectoderm biopsies. We then correlated observed aneuploidies with abnormalities of the first two cleavage divisions using time-lapse imaging (n = 843).ResultsThe combined incidence of meiotic and mitotic aneuploidies was strongly associated with blastocyst morphological grading, with the proportion ranging from 20 to 90% for the highest to lowest grades, respectively. In contrast, the incidence of aneuploidy among arrested embryos was exceptionally high (94%), dominated by mitotic aneuploidies affecting multiple chromosomes. In turn, these mitotic aneuploidies were strongly associated with abnormal cleavage divisions, such that 51% of abnormally dividing embryos possessed mitotic aneuploidies compared to only 23% of normally dividing embryos.ConclusionsWe conclude that the combination of meiotic and mitotic aneuploidies drives arrest of human embryos in vitro, as development increasingly relies on embryonic gene expression at the blastocyst stage.

A 3D-Printed Large Holding Capacity Device for Minimum Volume Cooling Vitrification of Embryos in Prolific Livestock Species

Although many devices have been developed to reduce sample volume, with an explosion of methods appearing in the literature over the last decade, commercially available devices with simultaneous vitrification of a larger number of embryos are scarce, with the apparent gap for their use in prolific livestock species. In this study, we investigated the effectiveness of a new three-dimensional (3D)-printed device that combines minimum volume cooling vitrification with simultaneous vitrification of a larger number of rabbit embryos. Late morulae/early blastocysts were vitrified with the open Cryoeyelet® device (n = 175; 25 embryos per device), the open Cryotop® device (n = 175; 10 embryos per device), and the traditional closed French mini-straw device (n = 125; 25 embryos per straw) and compared in terms of in vitro development and reproductive performance after transfer to adoptive mothers. Fresh embryos constituted the control group (n = 125). In experiment 1, there was no difference in the development rate to the blastocyst hatching stage between the CryoEyelet® and the other devices. In experiment 2, the CryoEyelet® device showed a higher implantation rate compared with the Cryotop® (6.3% unit of SD, p = 0.87) and French mini-straw® (16.8% unit of SD, p = 1.00) devices. In terms of offspring rate, the CryoEyelet® device was similar to the Cryotop® device but superior to the French straw device. Regarding embryonic and fetal losses, the CryoEyelet® showed lower embryonic losses compared to other vitrification devices. The analysis of bodyweight showed that all devices showed a similar outcomes-a higher birthweight but a lower body weight at puberty than those in the fresh transfer embryos group. In summary, the CryoEyelet® device can be used for the vitrification of many late morulae or early blastocyst stage rabbit embryos per device. Further studies should be performed to evaluate the CryoEyelet® device in other polytocous species for the simultaneous vitrification of a large number of embryos.

Effect of post-activation culture conditions on the development of parthenogenetic embryos in cattle

Purpose: to conduct a comparative analysis of the effect of commercial media BO-IVC and СR1aa at the stage of the activation and subsequent culture of artificially activated oocytes on the formation and quality of parthenogenetic bovine embryos.Materials and methods. 3 groups of disemeters of 50 goals in each were formed. In the first experimental group, the disemeted was in a meticulous manner with a ram-industrialist (artificial kriproporchid), in the second experimental-with a penEexctomed ram-industrialist. In the third (control) group, a producer ram was used. In the first experimental group of a ram-industrialist (artificial kriproporchid) with attached taps were released into a group of sheep twice a day for 1.5-2 hours. In the second experimental group of a penEctomed ram, it was placed in the corral to the disemetery in the morning for 3 hours. In the third group, the lamb producer was constantly with the disemets for two weeks, then he was changed on a new ram i.e. Used the methodology used in the farm. During the experiment, they observed the behavior of animals of all groups. In the experimental groups, after the detection of disemeters in the hunt, their natural insemination of the manufacturer was carried out. Based on the results of the subsequent oster, the effectiveness of the reproduction of sheep was evaluated.Results. The cleavage rate did not differ between the experimental groups, varying from 73,0 to 76,5%. Also, there was not found a significant effect of the conditions for post-activation culture of oocytes on their development before late morula and late blastocyst stage, which was for the CR1aa/CR1aa, CR1aa/BO-IVC and BOIVC/ BO-IVC groups 28,9±1,7, 40,4±7,5 and 36,0±6.4%, respectively. Meanwhile, we found out the effect of tested culture conditions on the ability of parthenogenetic embryos to overcome the 8-16 cell block and their quality on the late stages of embryo development. The rate of embryos with less than 16 nuclei was the highest in the CR1aa/CR1aa group (56,8±2,1 %). The replacement of CR1aa medium to BO-IVC medium (BO-IVC/BO-IVC group) significantly reduced this level (p<0,05). The positive effect was enhanced when CR1aa medium was used at the stage of culture in the presence of 6-DMAP and cycloheximide, and subsequent embryo development was in BO-IVC medium (CR1aa/BO-IVC group) (p<0.001). Furthermore, when we used the mixed variant of culture, the total cell number in parthenogenetic morula and blastocyst stage embryos increased (p<0.05).Conclusion. Thus, the BO-IVC medium at the stages of post-activation and subsequent development of artificially activated bovine oocytes is comparable to the CR1aa medium in terms of the efficiency of obtaining parthenogenetic embryos at the blastocyst stage. Nevertheless, its replacement at the post-activation stage with CR1aa medium makes it possible to improve the quality of parthenogenetic embryos.

Experimental Toxocara canis infection in chickens

The objective of this study was to determine the development phases of Toxocara canis eggs outside the host and the migration of larvae in the internal organs of chickens infected by ingestion of embryonated T. canis eggs. Under a microscope observation of T. canis eggs which were incubated in a petri dish containing 5 mL of distilled water at 30 – 33◦C with regularly aerated, this study showed the development of T. canis egg through various stages, including one cell, two cells, three cells, four cells, early morula, late morula, blastula, gastrula, tadpole, pre-larva, embryonated larva. In addition, it took approximately 9 days for T. canis eggs to hatch and for infective larvae to develop at 30 - 33◦C. A total of 50 chickens were randomly assigned to 3 groups including group I (10 chicks/group) was served as control group without T. canis eggs inoculation; two treatment groups II and III (20 chicks/group) were orally inoculated with 500 or 1000 T. canis eggs, respectively. On 1, 3, 6, 15 and 30 days post inoculation (dpi), two chickens/control group and 4 chickens/treatment group were necropsied. The results showed that the percentage of larvae recovered varied from 14.00 to 33.93% and 13.07 to 32.00% in treatment groups II and III, respectively. After 1, 3, 6, and 30 dpi, the significant differences about the number of larvae recovered in two treatment groups were found (P < 0.05). In both treatment groups, the percentage of larvae recovered from livers was higher than that in lung tissue. For 6 dpi, small white foci on the liver’s surface were seen. Interstitial pneumonia, petechial hemorrhages, dark or gray inflammatory nodules in the lung tissue and the atelectatic area were observed. Histopathology examination revealed infiltrations of leukocytes and eosinophil scattered in the liver and lung tissue.

MEIOTIC AND MITOTIC ANEUPLOIDIES DRIVE ARREST OF IN VITRO FERTILIZED HUMAN PREIMPLANTATION EMBRYOS

The high incidence of chromosome aneuploidy in early human development is the primary cause of pregnancy loss, miscarriage, and still birth following natural conception as well as in vitro fertilization (IVF). Preimplantation genetic testing for aneuploidy (PGT-A) of IVF embryos at the blastocyst stage has confirmed the prevalence of aneuploidies of putative meiotic and postzygotic mitotic origins. However, only about half of normally fertilized embryos develop to the blastocyst stage in vitro, while the others arrest at cleavage to late morula or early blastocyst stages.

Multiple ovulation and embryo transfer in sheep: Effects of embryo developmental stage and quality on viability in vivo under farm conditions.

Multiple ovulation and embryo transfer (MOET) technologies are integral to genetic improvement programs in the sheep industries. Despite the protocols being well established, previous findings regarding the effects of embryo properties on MOET success remain contradictory. The objective of this study was to determine the effects of embryo developmental stage and quality on embryo viability following transfer to recipient ewes. Data including details of 377 embryos collected from 45 Merino donor ewes were obtained from MOET trials conducted on three separate farms on day 6 after laparoscopic artificial insemination (AI). A total of 270 embryos were classified as being of transferrable grade (grade 1: n = 233; grade 2: n = 37). One or two transferrable grade embryos were transferred to each of 256 synchronised recipient ewes and pregnancy diagnosis was performed on day 36 after embryo transfer. Embryos at the hatched blastocyst stage tended to have greater viability in vivo compared to embryos at the late morula stage (59.0 ± 10.6% vs. 36.2 ± 9.7%; P = 0.083). The viability of grade 1 embryos was greater than that of grade 2 embryos (53.6 ± 7.8% vs. 35.9 ± 10.2%; P < 0.05). The results suggest that the success of the MOET trials was influenced by the transfer of embryos at the late morula stage, almost half of which were classified as grade 2 embryos. These findings highlight the importance of following strict embryo quality grading criteria to inform the most economical management of recipient ewes and maximize pregnancy outcomes.

O-099 TEAD4 regulates trophectoderm differentiation upstream of CDX2 in human preimplantation embryos

Abstract Study question What is the main pathway regulating trophectoderm (TE) differentiation during pre-implantation development in mouse versus human embryos? Summary answer TEAD4 is acting upstream of CDX2 and is involved in TE differentiation, as TEAD4-null human embryos exhibit compromised TE lineage differentiation. What is known already TEAD4 is the earliest transcription factor during early embryo development, required for the expression of TE-associated genes leading to successful TE differentiation and subsequent blastocoel formation in mouse. Functional knock-out studies in mouse, inactivating Tead4 by site-specific recombination have shown that Tead4-null embryos do not express TE specific genes, including Caudal-Type Homeobox Protein 2 (Cdx2) and GATA Binding Protein 3 (Gata3), but expression of inner cell mass (ICM)-specific genes, remains unaffected. Furthermore, ablation of Tead4 compromises embryonic development and subsequent blastocoel formation in mouse. The role of TEAD4, during human pre-implantation development has not been functionally characterized yet. Study design, size, duration CRISPR-Cas9 was introduced in mouse zygotes and editing efficiency was evaluated by next-generation sequencing (NGS) on 4.5dpc embryos (n = 55). Developmental kinetics were monitored in CRISPR-Cas9 targeted (n = 83), sham-injected (n = 26) and non-injected media-control (n = 51) mouse embryos. Immunofluorescence analysis was performed in Tead4 targeted (n = 57) and non-injected media-control embryos (n = 94). The same methodology was applied in human donated in vitro matured (IVM) metaphase-II (MII) oocytes, which were CRISPR-Cas9 targeted (n = 74) during ICSI or used as media-Control (n = 33). Participants/materials, setting, methods A gRNA-Cas9 mixture targeting exon 2 of Tead4/TEAD4 was microinjected in respectively mouse 2PN (pronuclear) stage zygotes, or human IVM MII oocytes along with the sperm. Generated embryos were cultured in vitro for 4 days in mouse or 6.5 days in human. Embryonic development and morphology were assessed daily, followed by a detailed scoring at the late blastocyst stage. Successful targeting following CRISPR-Cas9 introduction was assessed by immunostaining and NGS analysis of the targeted locus. Main results and the role of chance In mouse, we confirmed previous findings, as the developmental capacity of Tead4 targeted embryos was significantly reduced starting from the morula stage and blastocyst formation rates were 8.97% in the targeted group, compared to 87.23% in the control and 87.50% in the sham group, respectively. Immunofluorescence analysis of late morula and blastocyst stage embryos confirmed the absence of Tead4, Cdx2 and Gata3, resulting from the successful interruption of the Tead4 locus (n = 57). Exon 2 of TEAD4 was successfully targeted in human. In total, 21 embryos from various developmental stages were successfully NGS analyzed and 90,48% (19 out of 21) of the embryos carried genetic modifications as a result of CRISPR-Cas9 genome editing and seven blastocysts were identified carrying exclusively frameshift mutations. In contrast to mouse, the developmental capacity of human targeted embryos (25%) did not differ significantly from the control group (23%). However, the blastocyst morphology and quality were compromised in the targeted group showing mostly grade C TE scores, containing very few cells. Immunofluorescence analysis of targeted blastocysts (n = 6) confirmed successful editing by complete absence of TEAD4 and its downstream TE marker CDX2. Limitations, reasons for caution CRISPR-Cas9 germline genome editing results in multiple editing outcomes with variable phenotypic penetrance, the mosaic nature of which complicates the phenotypic analysis and developmental behaviour of the injected embryos. Wider implications of the findings Elucidation of the evolutionary conserved molecular mechanisms that regulate self-renewal of the trophoblast lineage can give us fundamental insights on early implantation failure. Trial registration number Not Applicable

COMPARATIVE ANALYSIS OF THE MORPHOLOGICAL CONDITION OF IN VIVO OBTAINS OF EMBRYOS FROM DONOR COWS OF DIFFERENT BREEDS

The aim of the study was a comparative analysis of the morphological status of embryos of cattle obtained in vivo from purebred and local donor cows of the I and II generations.&#x0D; The object of experimental studies were obtained embryos of cattle in vivo. They were obtained in the experimental farm "Ukrainka" of the Institute of Animal Science of the UAAS of Ukraine from 1985 to 1990. Information about animals and the results of research are stored in the archives of the Private Company "Bioservice". In order to study the stages of development of seven-day-old embryos of cattle, the embryo productivity of 11 purebred and 72 local donor cows was analyzed. Purebred cows were Ayshir (4 heads), Simmental (1 head) and Ukrainian Black-and-White dairy (6 heads) breeds. Crossbreeds of the first generation were donor cows from crossing red steppe and Holstein (2 goals), Simmental and Holstein (12 goals) and Ukrainian Black-and-White dairy and Holstein (9 goals) breeds. The crossbreeds of the second generation were donor cows from crossing red steppe and Holstein (14 goals), Simmental and Montbeliard (22 goals) and Ukrainian Black-and-White dairy and Holstein (13 goals) breeds.&#x0D; According to the results of the conducted research, a statistically significant difference (p &lt; 0.01) between purebred (7.8%) and local donor cows of the I and II generation (0.6 and 1.4%, respectively) was established by the number of embryos. obtained at the stage of expanded blastocyst. There was also a statistically significant difference (p &lt; 0.001) at the stage of late blastocyst between purebred (38.0%) and local donor cows of the I and II generation (7.1 and 9.2%, respectively). The analysis of the obtained research results shows that from purebred donor cows a significantly larger (p &lt; 0.001) total number of blastocysts was obtained by 60.4% compared to local donor cows of the I and II generation (19.6 and 24.2%, respectively).&#x0D; It was found that at the stage of late morula found significantly more embryos 44.7% and 32.9 (p &lt; 0.01), respectively, in donor cows of the second and fourth generation compared to purebred 19.4%. It should be noted that at the stage of early morula, a significantly larger number of embryos (p &lt; 0.001) was obtained from donor cows of the I and II generation (13.6 and 3.2%, respectively) compared to purebred 0.0%. According to the results of the comparative analysis of the total number of obtained morulae, a significantly higher number of embryos (p &lt; 0.001) obtained from donor cows of the I and II generation (48.6 and 50.0%, respectively) was found in comparison with purebred 21.7%.&#x0D; It was found that purebred donor cows received a greater 82.2% of the total number of suitable embryos compared to 68.2% (p &lt; 0.001) of local donor cows of the first and 73.5% (p &lt; 0.05) from local donor cows of the second generations.&#x0D; Further development of extracted morulae in vitro from purebred and local donor cows on the seventh day at a temperature of 37.5°C for 15 hours led to the formation of full-fledged blastocysts at 73.0% (400 of 548).&#x0D; It was found that purebred donor cows received more blastocysts (p &lt; 0.001) compared to local donor cows of the first generation by 40.8% and 36.2% compared to local donor cows of the second generation.&#x0D; It was found that from local donor cows of the second generation received 28.3%, and from local donor cows of the first generation by 26.9% more (p &lt; 0.001) morula compared to purebred donor cows.&#x0D; According to the results of the research, it was found that purebred donor cows received a higher total number of embryos (molula + blastocyst) compared to local donor cows of the first generation by 14.0% (p &lt; 0.001) and 8.7% &lt; 0.05) compared with local donor cows of the second generation.

Партеногенетическое развитие эмбрионов Bos taurus из ооцитов, созревших в различных системах культивирования

Abstract. Identification of the factors determining of donor’s oocyte competence to parthenogenetic development will allow developing an effective method for obtaining parthenotes to solve fundamental problems of regulating gene activity in ontogenesis, creating homozygous embryonic stem cell lines, improving the stages of cloning technology, and modeling of in vitro oocyte maturation media. The purpose of study is to evaluate the potencies of Bos taurus oocytes matured in different culture systems to cold shock-induced parthenogenesis. Methods. For oocyte maturation, culture systems of the following composition were used: 1 – TC-199 with 10 % fetal bovine serum (FBS), 50 μg/ml estradiol, 10 μg/ml luteinizing hormone, 10 μg/ml follicle-stimulating hormone; 2 – TC-199 with 10 % estrous serum of cows; 3 – TC-199 with 50 % fluid from follicles (Ø 3–8 mm); 4 – TC-199 with 50 % protein of follicular fluid (molecular weight of 65 kDa); 5 – TC-199 with 10 % FBS, 1×106 granulosa cells/ml medium; 6 – TC-199 with 10 % FBS and walls of follicles (Ø 6–8 mm); 7 – TC-199 with 10 % FBS, 1×106 granulosa cells/ml medium and walls of follicles (Ø 6–8 mm). After 24 hours of cultivation, the oocytes were activated by cold shock (exposure time 20 minutes, temperature 0…–4 °C. Results. The proportion of embryos at the stages of late morula and blastocysts from oocytes matured in system 7 was 45 % (58/129), which was significantly higher than in other systems: 1 – 28 % (39/141), P &lt; 0.05; 2 – 31 % (42/137), P &lt; 0.05; 3 – 25 % (33/133), P &lt; 0.01; 4 – 18 % (25/139), P &lt; 0.001; 5 – 31 % (41/132), P &lt; 0.05; 6 – 33 % (43/129). The introduction of estradiol or structural components of antral follicles into the oocytes maturation medium contributed to an increase in the proportion of parthenotes at the preimplantation stages of development, including blastocysts, and a decrease in the level of degenerated embryos. Scientific novelty. A comparative morphological analysis of the potentials for parthenogenesis of bovine oocytes matured in various culture systems and activated by cold shock was carried out for the first time. Optimal systems for in vitro maturation of female gametes were proposed. Based on the analysis of the results, we recommend before induction to parthenogenesis bovine oocytes culture in media supplemented with 50 ng/ml estradiol or structural components of antral follicles producing estradiol.

Experimental Evidence Reveals Both Cross-Infection and Cross-Contamination Risk of Embryo Storage in Liquid Nitrogen Biobanks.

Simple SummaryThis study was conducted to demonstrate the potential hazards of cross-infection and cross-contamination of embryos during storage in liquid nitrogen biobanks. For the harmless and successful cryopreservation of embryos, the vitrification method must be chosen meticulously to guarantee not only a high post-thaw survival of embryos, but also to reduce the risk of disease transmission when those embryos are in storage for long periods.In recent decades, gamete and embryo cryopreservation have become routine procedures in livestock and human assisted reproduction. However, the safe storage of germplasm and the prevention of disease transmission continue to be potential hazards of disease transmission through embryo transfer. This study aimed to demonstrate the potential risk of cross-infection of embryos from contaminated liquid nitrogen, and cross-contamination of sterile liquid nitrogen from infected embryos in naked and closed devices. Additionally, we examined the effects of antibiotic-free media on culture development of infected embryos. The study was a laboratory-based analysis using rabbit as a model. Two experiments were performed to evaluate both cross-infection (liquid nitrogen to embryos) and cross-contamination (embryos to liquid nitrogen) of artificially inoculated Salmonella Typhimurium, Staphylococcus aureus, Enterobacter aerogenes, and Aspergillus brasiliensis. Rapid cooling through vitrification was conducted on rabbit embryos, stored for a year, thawed, and cultured. In vivo produced late morulae–early blastocyst stages (72 h) embryos were used (n = 480). Embryos were cultured for 1 h in solutions with and without pathogens. Then, the embryos were vitrified and stored in naked and closed devices for one year in two liquid nitrogen biobanks (one pathogen-free and the other artificially contaminated). Embryos were warmed and cultured for a further 48 h, assessing the development and the presence of microorganism (chromogenic media, scanning electron microscopy). Embryos stored in naked devices in artificially contaminated liquid nitrogen became infected (12.5%), while none of the embryos stored in closed devices were infected. Meanwhile, storage of artificially infected embryos incurred liquid nitrogen biobank contamination (100%). Observations by scanning electron microscopy revealed that all the microorganisms were caught in the surface of embryos after the vitrification-thawed procedure. Nevertheless, embryos cultured in antibiotics and antimycotic medium developed to the hatched blastocyst stage, while artificially infected embryos cultured in antibiotic-free medium failed to develop. In conclusion, our findings support that both cross-contamination and cross-infection during embryo storage in liquid nitrogen biobanks are plausible. So, to ensure biosafety for the cryogenic storage, closed systems that avoid direct contact with liquid nitrogen must be used. Moreover, it seems essential to provide best practice guidelines for the cryogenic preservation and storage of gametes and embryos, to define appropriate quality and risk management procedures.

КОМПЕТЕНТНОСТЬ К ИНДУЦИРОВАННОМУ ПАРТЕНОГЕНЕЗУ ООЦИТОВ BOS TAURUS В ЗАВИСИМОСТИ ОТ ФУНКЦИОНАЛЬНОГО СТАТУСА ГАМЕТЫ И ЕЕ ПРОИСХОЖДЕНИЯ (ДИАМЕТРА ФОЛЛИКУЛА)

The development of effective methods for obtaining parthenogenetic embryos is currently being considered in a new perspective as a promising technology for creating homozygous lines of embryonic stem cells, genetically identical animals. Parthenogenetic embryos of animals of different species are used as a model for studying imprinting mechanisms. In the present study, Bos Taurus parthenogenetic embryos were obtained by cold shock induction (exposure for 20 minutes at a temperature of -0 - (-4 o ) C) of after oocytes culture in TC-119 medium with 10% fetal bovine serum, 50 ng / ml prolactin, 50 μg / ml of gentamicin together with 1 × 10 6 granulosa cells per ml of medium for 24 hours. Previously, the initial population of donor oocytes from the ovaries was evaluated with BCB test to assess functional status. For this, the oocyte-cumulus complexes were placed in a solution of 13 Mm brilliant crystalline blue - BCB (B-5388, Sigma) for 60 minutes at a temperature of 38.5 0 C in an atmosphere of 90% humidity. After the expiration of the BCB exposure time, the oocytes were ranked as follows: oocytes with a blue cytoplasm - BCB + oocytes (oocytes that completed their growth phase) and oocytes with unpainted ooplasm - BCB- oocytes (growing oocytes). It was found that the functional status (growing oocytes or oocytes that have finished growth phase) of oocytes and their origin (the diameter of the follicles from which the gametes were isolated) determine their competence for induced parthenogenesis. Oocytes that completed the growth phase have high potentials to cold shock-induced parthenogenesis, which is expressed in a significant increase in the proportion of embryos developed from them at all stages of preimplantation development (percentage of cleavage: 87% vs 11%, P 5 mm-8 mm) revealed low competence to cold shock-induced parthenogenesis of oocytes isolated from follicles less than 3 mm in diameter ( the percentage of cleavage: 20% vs 67% and 87%, P <0.001, respectively; level of late morula, blastocysts: 9% vs 22% and 37%, P <0.001, respectively).

Developmental stages and viability of Toxocara canis eggs outside the host

Toxocariasis is a soil-transmitted zoonotic disease caused mainly by ingestion of larvated eggs of Toxocara canis. To study the morphology of the intraovular developmental stages of Toxocara canis in culture, characterize non-viable eggs and the sequences of larval molting and compare the viability of eggs at the early stages of division and at reaching full maturation. Observation of developing embryos and characterization of non-viable eggs were done using light microscope. The proportions of viable eggs during embryonation were compared to the proportions of viable mature eggs. Cell division commenced after 24 hours of cultivation. Early stages were found to be present over a period of 3-5 days. The developmental stages identified were eggs with: One cell, two cells, three cells, four cells, early morula, late morula, blastula, gastrula, tadpole, pre-larva, first, second and third stage larva. Two larval molts occurred. Non-viable eggs had degenerated cytoplasm, thin or collapsed shell and the larvae did not move after exposure to light. No significant differences were found between the proportions of viable eggs from day five to day 21 as compared to viability of fully mature eggs (30 days). Developing embryos in the environment may be considered as a potential threat to the public health. The precise identification of developmental stages and the clear differentiation of viable and non-viable eggs can help in determining an accurate baseline rate of development that could be used in studies of ovicidal compounds.

The use of multiple ovulation, embryo cryopreservation and embryo transfer techniques to facilitate movement of genetic material between countries

The objective of the study was to evaluate the use multiple ovulation and embryo transfer techniques in an indigenous Greek dairy breed of sheep. We stimulated selected donor ewes of the Karagouniko breed to produce large numbers of embryos after the induction of multiple ovulations by gonadotropin treatment (superovulatory response). A total of 50 Greek Karagouniko ewes were synchronised into oestrus using progestagen pessaries and superovulated for embryo transfer using ovine FSH. Six days following laparoscopic insemination with fresh semen ewes were flushed surgically and embryos collected. Subsequently, the embryo recovery, along with embryo cryopreservation, embryo survival and quality were assessed. The Karagouniko donor ewes achieved a mean ovulation rate of 11.9 (SE. 0.89). The ova Recovery rate was 80,9% and 87,6% of the ova recovered being fertilised. A total of 327 (77,5%) of the viable embryos were assessed as being of sufficient quality for cryopreservation. The embryos ranged from late morulae to expanded blastocyst and were frozen via a 3-step process in 1.5 M ethylene glycol following repeated washing and trypsination. Cryopreserved embryos were frozen and then transported to Scotland, UK. There, embryos were thawed rapidly and re-hydrated via a 2 step sucrose/ethylene glycol gradient. A total of 92.4% of embryos frozen remained suitable for transfer semi-surgically into synchronized Scottish Blackface ewes. 183 embryos were transferred in total with a 66.1% survival rate. The survival rate of frozen thawed blastocysts(75%) was significantly greater than (P&lt;0.01) that for morulae (48%). It was concluded that MOET could be successfully applied in Greek dairy breeds of sheep as a means for genetic improvement. Frozenembryos could be a successful medium for the transportation of ovine genetic material from and to Greece, however, most likely the choice of embryonic stage for cryopreservation is crucial.

Our First Choice: Cellular and Genetic Underpinnings of Trophectoderm Identity and Differentiation in the Mammalian Embryo.

The trophectoderm (TE) is the first cell population to appear in the mammalian preimplantation embryo, as the result of the differentiation of totipotent blastomeres located on the outer surface of the late morula. Trophectodermal cells arrange in a monolayer covering the expanding blastocyst and acquire an epithelial phenotype with distinct apicobasal polarity and a basal lamina placed toward the blastocyst interior. During later development through the periimplantation and gastrulation stages, the TE gives rise to extraembryonic membranes and cell types that will eventually form most of the fetal placenta, the specialized organ through which the embryo obtains maternal nourishment necessary for subsequent exponential growth. The specification of the TE is controlled by the combination of morphological cues arising from cell polarity with differential activity of signaling pathways such as Hippo and Notch, and the restriction to outer cells of lineage specifiers such as CDX2. This is possibly the first symmetry-breaking decision undertaken by the uncommitted cells produced by a handful of mitosis divisions from the newly fertilized zygote. Understanding how this cell lineage is specified will therefore provide unique information about development, differentiation, and how the interplay between cellular morphology and signaling and regulatory factors results in a correctly 3D-patterned embryo.

Role of chemokine (C-C motif) ligand 24 in spatial arrangement of the inner cell mass of the bovine embryo.

The process of spatial rearrangement of cells of the inner cell mass (ICM) that are destined to become hypoblast is not well understood. The observation that the chemokine (C-C motif) ligand 24 (CCL24) and several other genes involved in chemokine signaling are expressed more in the ICM than in the trophectoderm of the bovine embryo resulted in the hypothesis that CCL24 participates in spatial organization of the ICM. Temporally, expression of CCL24 in the bovine embryo occurs coincidently with blastocyst formation: transcript abundance was low until the late morula stage, peaked in the blastocyst at Day 7 of development and declined by Day 9. Treatment of embryos with two separate antagonists of C-C motif chemokine receptor 3 (the prototypical receptor for CCL24) decreased the percent of GATA6+ cells (hypoblast precursors) that were located in the outside of the ICM. Similarly, injection of zygotes with a CCL24-specific morpholino decreased the percent of GATA6+ cells in the outside of the ICM. In conclusion, CCL24 assists in spatial arrangement of the ICM in the bovine embryo. This experiment points to new functions of chemokine signaling in the bovine embryo and is consistent with the idea that cell migration is involved in the spatial organization of hypoblast cells in the blastocyst.

206 GENOME-WIDE ASSOCIATIONS FOR REPRODUCTIVE TRAITS IN RUSSIAN HOLSTEIN POPULATION

Reproductive health is an important trait in selection of dairy cattle. A genome-wide association study (GWAS) is a powerful tool for annotating phenotypic effects on the genome and to get knowledge of genes and chromosomal regions associated with reproductive performance (Cole et al. 2011). Combining GWAS and genetic profiling of embryos before implantation enables to develop new strategies to select elite breeding genotypes before transfer (Humblot et al. 2010; Ponsart et al. 2013). The aim of the current study was to evaluate the association between single nucleotide polymorphisms (SNP) and estimated breeding values (EBV) for reproductive traits [interval to insemination (EBVII) and interval between calving (EBVIC)] for Russian Holstein cattle and to evaluate the effect of the biopsy procedure on the viability of bovine embryos produced in vitro as a basis for pre-implantation genetic diagnosis (PGD). Ninty-six progeny-tested sires of artificial insemination station Moscowskoe were selected based on the reliability for EBVII and EBVIC. Estimations of breeding values of sires were performed by best linear unbiased prediction (BLUP) mixed model equations. DNA was extracted from sire semen samples. SNP genotyping was performed using the Illumina BovineSNP50 BeadChip (Illumina Inc., San Diego, CA, USA) containing 54609 SNPs. Quality control information was carried out in PLINK (v. 1.07; Purcell et al. 2007 Am. J. Hum. Genet. 81). Based on the quality control information, 41442 SNPs were selected for subsequent GWAS. We have identified 3270 SNPs having significant effect (P &lt; 0.05) on studied traits. The most significant associations with EBVII were found for SNPs Hapmap38548-BTA-97184 and ARS-BFGL-BAC-11821 with coefficient of determination (R2) of 0.2189 and 0.1937, and P-values 2.27 × 10–6 and 1.01 × 10–6, respectively. The most significant effect on EBVIC was detected for SNPs ARS-BFGL-NGS-59769 and ARS-BFGL-NGS-38020 with coefficient of determination (R2) of 0.236 and 0.2421, and P-values 1.49 × 10–8 and 7.24 × 10–8, respectively. The highest number of significant associations was found on BTA5, BTA12, BTA19, and BTA14. Bovine embryos were produced in vitro using a standard procedure. Six to 8 cell biopsies were carried out at Day 6.5 after fertilization. The viability of biopsied embryos was evaluated comparing the hatching rates to non-manipulated embryos. The study of embryos viability after biopsy showed that the hatching rate of biopsied embryos (the number of hatched embryos from the number of embryos in stages of late morula and early blastocyte) was 48% comparing to 67% for non-manipulated embryos. Embryo biopsy is not dramatically decreasing embryo viability. Combining our results of association studies, performed on Russian Holstein population, and technique of embryo biopsy will provide us a powerful tool for selection progress.This research was supported by the Russian Ministry of Education and Science, project No. 2014-14-576-0057-175.

МОДЕЛИРОВАНИЕ СИСТЕМ СОЗРЕВАНИЯ ООЦИТОВ СВИНЕЙ in vitro

A system of extracorporal maturation of growing and fully grown oocytes have been developed on the basis of research of a role of structural elements of porcine follicles in formation of mature oocytes and a preventive estimation of the functional status of oocytes (BCB-test). The cultivation of oocytes in the developed system allows to receive till 45 % of embryos at the stages of late morulae and blastocysts.

The Impact of Ovarian Stimulation on the Expression of Candidate Reprogramming Genes in Mouse Preimplantation Embryos

Ovarian stimulation with gonadotrophins is an integral part of assisted reproductive technologies in human subfertility/infertility treatment. Recent findings have associated ovarian stimulation with the increased incidence of imprinting disorders in humans as well as defects in genome-wide methylation reprogramming and, in particular, imprinting in mice. Here, we present the first study that determined the impact of ovarian stimulation on the expression of developmentally important reprogramming genes (Apex1, Lig1, Lig3, Mbd2, Mbd3, Mbd4, and Polb) in single early mouse morula embryos (16-cell stage). Using absolute quantification of mRNA by quantitative real-time PCR, we observed an association of ovarian stimulation with a downregulation of mRNAs encoding the base excision repair proteins APEX1 and POLB as well as the 5-methyl-CpG-binding domain protein MBD3 in individual morula embryos. Whole mount immunofluorescence staining of early and late morula embryos with an antibody against APEX1 revealed individual embryos with lower protein expression levels after ovarian stimulation and a correlation of mRNA expression with protein abundance. Our data argue for a negative impact of ovarian stimulation during female gametogenesis and/or early embryo development affecting the expression of candidate reprogramming factors.

CCN5 Expression in mammals. III. Early embryonic mouse development

CCN proteins play crucial roles in development, angiogenesis, cell motility, matrix turnover, proliferation, and other fundamental cell processes. Early embryonic lethality in CCN5 knockout and over-expressing mice led us to characterize CCN5 distribution in early development. Previous papers in this series showed that CCN5 is expressed widely in mice from E9.5 to adult; however, its distribution before E9.5 has not been studied. To fill this gap in our knowledge of CCN5 expression in mammals, RT-PCR was performed on preimplantation murine embryos: 1 cell, 2 cell, 4 cell, early morula, late morula, and blastocyst. CCN5 mRNA was not detected in 1, 2, or 4 cell embryos. It was first detected at the early morula stage and persisted to the preimplantation blastocyst stage. Immunohistochemical staining showed widespread CCN5 expression in post-implantation blastocysts (E4.5), E5.5, E6.5, and E7.5 stage embryos. Consistent with our previous study on E9.5 embryos, this expression was not limited to a particular germ layer or cell type. The widespread distribution of CCN5 in early embryos suggests a crucial role in development.

Vitrification of in vitro produced bovine embryos: Effect of embryonic block and developmental kinetics

In order to investigate whether the kinetics and stage of embryo development affect cryosurvival of in vitro produced bovine embryos, cleaved embryos were categorized in six groups based on their developmental kinetics regarding the stage of embryonic block in bovine (8–16 cell stage): I and II – early (day 2) and late (day 3) 5–8 cell, III and IV – early (day 3) and late (day 4) 8–16 cell, and V and VI – early (day 4) and late (day 5) morula. The cryosurvival and developmental competence of these embryos were compared with each other and also with the corresponding control groups. The potential of 5–8 cell stage embryos to survive vitrification and further develop towards blastocyst stage was significantly lower than vitrified and un-vitrified 8–16 cell and morula stage embryos. These results suggest that, the survival rate and potential of embryos to develop towards blastocyst stage might be affected by the kinetic of the embryo development. Moreover, the results of this study indicated that the optimal stages of early embryo vitrification are post-embryonic block.