Differences In Embryos Research Articles

P-267 The morula stage. Is it time to repair?

Abstract Study question Are there statistically significant differences in embryo’s fate when exclusion or extrusion of blastomeres is detected along development? Summary answer According to our results, exclusion or extrusion of blastomeres affects the embryo’s subsequent viability, suggesting that different biological processes are involved in its appearance. What is known already The morula stage is a clear example of embryo plasticity, being a critical step towards blastocyst development. One morphokinetic parameter observed at this stage is the exclusion (before compaction) and extrusion (during or after compaction) of blastomeres. Exclusion and extrusion seem to be related to embryo self-correction phenomena and they are considered negative criteria due to potential associations with tight junction failures, gene expression, aneuploidies, or a high degree of mosaicism. However, the moment in which those phenomena are detected, can have different origins and though different consequences in embryo development. Study design, size, duration A total of 668 fertilized embryos and cultured to the blastocyst stage were included in the study. Embryo cohorts correspond to 158 consecutive patients that underwent an IVF treatment with fresh single blastocyst transfer and all their cohorts, between June 2017 and September 2022. Data recovered during culture in time-lapse incubator regarding the extrusion or exclusion of cells and the final fate of the transferred and the cohort embryos were retrospectively analysed. Participants/materials, setting, methods Embryos were cultured in single culture medium (CSCC®, Irvine Scientific) in a time-lapse incubator (MIRI-TL®, ESCO Medical). Embryos were evaluated for the time of exclusion/extrusion, number and volume of excluded/extruded cells and their final fate (transferred and the result, vitrified, discarded). For those transferred, Beta-hCG was performed 14 days after transfer and if positive clinical pregnancies were confirmed 3 weeks later. Data were analysed using IBM SPSS Statistics v25.0, with p < 0.05 indicating statistical significance. Main results and the role of chance Of the 668 analysed embryos, 340 were deemed “useful”: 225 were transferred (158 fresh, 67 cryotransferred after fresh negative pregnancy test), and 115 remain still vitrified. The rest, 328, were considered “non-useful”: 84 discarded and 244 blocked. 37% of the embryos presented total compaction and 63% partial, being 70.1% exclusion, 9.7% extrusion and 20.2% both phenomena. Exclusion/extrusion was significantly higher (p < 0.05) in non-useful embryos compared to the useful ones. In the distribution with regards to the final fate, significant differences were found (p < 0.05) being 78% exclusion, 8.8% extrusion and 13.3% both for useful, and 53.8% exclusion, 5.8% extrusion and 40.4% both for non-useful. There were statistically significant differences (p < 0.05) in the volume and number of cells excluded/extruded and the fate, being higher in those that resulted non-useful. ROC analysis showed that the volume occupied by excluded cells influences the embryo’s fate (AUC=0.738). Regarding transferred embryos, those that presented the phenomenon and resulted in a positive beta-hCGs, 95.8% was exclusion, none of them extruded cells and 4.2% both. For the negatives, 71% of exclusion, 11.4% of extrusion and 17.1% both, resulting in non-significant differences (p = 0.055). No significative differences were found either for the ones that resulted in a clinical pregnancy. Limitations, reasons for caution Although no statistical differences were found according to the moment of exclusion of blastomeres, before or after compaction, and the pregnancies, there is a clear trend in the sooner it happens the more embryo viability. And increase in the sample size might be helpful to confirm it. Wider implications of the findings The establishment of new parameters can improve embryo selection to help embryologists discriminating between embryos that appear to be of similar quality. Trial registration number not applicable

Sire modulates developmental kinetics and transcriptome of the bovine embryo.

The first week of gestation is a period of major pregnancy loss in cattle, this study reveals that the male plays a key role in regulating embryonic development during this time. The impact of sire on preimplantation embryonic development in cattle remains poorly understood. This study evaluated differences in embryos produced in vitro from sires with varying capacities to produce blastocysts. Sires classified as high (HP) and low performing (LP) based on their ability to produce embryos were used to better understand how sire regulates embryonic development. By monitoring development, it was determined that the most common arrest stage was the five- to six-cell stage. Embryos (four to six cells) from HP and LP sires were then analyzed for autophagic activity, where embryos for LP sires exhibited increased autophagy than HP-derived embryos. Transcriptome analysis of four-cell embryos found that embryos from LP sires might have issues in sperm mitochondrial clearance, histone retention, and DNA damage, while HP sires had increased expression of genes involved in transcription, chromosome segregation, and cell division. In conclusion, LP sires had an increased proportion of embryos arresting at the five- to six-cell stage, and these embryos had higher rates of cellular stress due to paternal contributions from the spermatozoon.

Endometriosis and infertility

In this article, the causes and mechanisms of infertility in endometriosis are fully explained, the four main factors of infertility related to endometriosis, the importance of the embryo factor in infertility in endometriosis, the fundamental differences in embryos.

Lifestyle intervention prior to IVF does not improve embryo utilization rate and cumulative live birth rate in women with obesity: a nested cohort study.

Lifestyle intervention prior to IVF does not improve embryo utilization rate and cumulative live birth rate in women with obesity: a nested cohort study.

Mitochondrial DNA analysis of HVRI region in human blastocysts

Introduction Mitochondrial DNA plays an important role in energy production, which affects chromosome segregation. Embryos with high levels of mitochondrial DNA (mtDNA) have difficulty implanting and increased aneuploidy, causing a decreased chance of pregnancy. Some haplogroups, which can be determined by sequencing the hypervariable region I (HVRI) of mtDNA, have a higher susceptibility to aneuploidy than others. The objective of this study was to determine if there are any genetic mutations within the HVRI region of mtDNA that causes a genetic predisposition to having elevated levels of mtDNA in euploid embryos. Materials and Methods The DNA used in this experiment was obtained from embryo biopsies and was whole genome amplified via SurePlex. We used polymerase chain reaction (PCR) to amplify the HVRI region of embryos from patients with two normal mtDNA euploid embryos and at least one elevated mtDNA euploid embryo. Elevated versus normal levels of mtDNA were previously determined via Mitograde testing. Three repeats of PCR were performed per embryo. Amplicons were purified by ExoSAP-IT and sequenced via Sanger sequencing. Results Gel electrophoresis of amplicons revealed the HVRI region was successfully amplified in 96.7% of embryos (264/273). Embryos from 1 out of 35 patients, labeled patient X, failed to amplify. 21 embryos from 7 patients were Sanger sequenced with an efficiency of 61.3% per amplified embryo. 11 embryos were successfully sequenced with over 675 out of 750 nucleotides readable, 8 were partially sequenced with over 350 out of 750 nucleotides readable, and 2 failed sequencing with less than 350 nucleotides readable. The two failed sequences belonged to embryos of patient X. Haplogroup analysis via HaploGrep 2.0 was successful in all 21 sequenced embryos. As expected, haplogroups were the same for all embryos from the same patient with an exception of patient X who had differing haplogroups for her elevated and normal mtDNA embryos. The failed amplification and sequencing of patient X indicate a possible mutation where the forward or reverse primer binds. Conclusions Sequencing revealed no nucleotide differences in embryos with elevated versus normal levels of mtDNA from the same patient, indicating there is no genetic predisposition caused by insertions/deletions or single nucleotide polymorphisms to having elevated levels of mtDNA encoded within the mtDNA HVRI region. 6 out of 7 patients sequenced belonged to haplogroup H, which was previously found to have lower incidences of aneuploidy. This suggests that embryos from patients within haplogroup H will have a higher percentage of euploidy but may still have fertility issues caused by elevated levels of mtDNA.

Morphological differences in embryos of goldfish (Carassius auratus auratus) under different incubation temperatures.

The goldfish (Carassius auratus auratus) is a useful species for embryonic micromanipulations because of its large egg size and wide temperature tolerance. Here, we describe in detail the rate of development and morphological characteristics of goldfish embryos incubated at temperatures between 10 °C and 30 °C. The cleavage speed increased rapidly as temperature increased. Synchronized cell divisions occurred at 131 min intervals at 10 °C, at 33 min intervals at 20 °C, and at 19 min intervals at 30 °C during the cleavage period. The rate of hatched abnormal embryos significantly increased at temperatures of 26 °C and above, while there was no change in the number of abnormal embryos at temperatures less than 24 °C. Moreover, the blastomeres around the center of the blastodisc rose in the direction of the animal pole at temperatures less than 14 °C. At the lower temperatures, clusters of maternally-supplied germplasm were visualized both at the ends of the first three cleavage furrows and at the border between the lower and upper tiers at the 16- to 32-cell stage, with injection of artificial mRNA and vasa in situ hybridization. This study showed that temperature affects not only developmental speed but also the shape of the blastodisc and the distribution of maternally-supplied materials in the blastodisc. By controlling the temperature, it is possible for researchers to prepare many stages of embryos and shapes of the blastodisc from a single batch of eggs.

Retinoic acid and meiosis induction in adult versus embryonic gonads of medaka.

In vertebrates, one of the first recognizable sex differences in embryos is the onset of meiosis, known to be regulated by retinoic acid (RA) in mammals. We investigated in medaka a possible meiotic function of RA during the embryonic sex determination (SD) period and in mature gonads. We found RA mediated transcriptional activation in germ cells of both sexes much earlier than the SD stage, however, no such activity during the critical stages of SD. In adults, expression of the RA metabolizing enzymes indicates sexually dimorphic RA levels. In testis, RA acts directly in Sertoli, Leydig and pre-meiotic germ cells. In ovaries, RA transcriptional activity is highest in meiotic oocytes. Our results show that RA plays an important role in meiosis induction and gametogenesis in adult medaka but contrary to common expectations, not for initiating the first meiosis in female germ cells at the SD stage.

210 DNA METHYLATION AND HYDROXYMETHYLATION ANALYSIS IN A MODEL OF OOCYTE DIFFERENTIAL DEVELOPMENTAL COMPETENCE IN SHEEP

DNA methylation is an important epigenetic mark that plays a role in gene regulation by the addition of a methyl group to CpG islands in the DNA. Despite being relatively stable in somatic cells, DNA methylation is subject to reprogramming during embryo development and gametogenesis. The aim of this work was to evaluate different aspects of DNA methylation in relation to oocyte quality in the ovine species. A model of differential developmental competence consisting in ovine oocytes and in vitro produced (IVP) blastocysts derived from adult (AD) and prepubertal (PR) donors, was used. The methylation was analysed in terms of: expression of a panel of genes involved in DNA methylation [DNA methyltransferases (DNMTs)] and demethylation [ten-eleven translocation dioxygenases (TET)] in oocytes and blastocysts; global methylation and hydroxymethylation by direct immunofluorescence; locus-specific methylation of 2 imprinted genes by pyrosequencing. Gene relative quantification was performed by RNA reverse transcription followed by real-time PCR. Pools of 10 immature (GV) and in vitro-matured (MII) oocytes and (IVP) blastocysts derived from AD and PR donors (4 replicates per class) were analysed. Lower expression of TET1, TET2, and TET3 was observed in PR GV oocytes (ANOVA; P < 0.05), while no significant differences were found for the enzymes involved in methylation (DNMT1, DNMT3A, DNMT3B; ANOVA; P > 0.05). The levels of all the genes studied showed no significant differences in embryos at blastocyst stage (ANOVA; P > 0.05). Methylation and hydroxymethylation immunostaining were performed in GV and MII oocytes using anti-5-methylcytosine mouse mAb and 5-hydroxymethylcytosine rabbit pAB. High levels of DNA methylation were observed in both AD and PR GV and MII oocytes, while hydroxymethylation immunopositivity was scattered evident throughout the gamete chromatin. Pyrosequencing of bisulfite converted DNA was used to determine the methylation status within differentially methylated regions (DMR) of maternally imprinted H19 (CTCF binding site IV; 11 CpG sites) and paternally imprinted IGF2R (17CpG sites within intron 2). No differences were observed between classes of oocytes for each gene (pools of 40 oocytes per replicate, 3 replicates per class; ANOVA; P > 0.05). Our work shows no differences in the expression of the enzymes involved in methylation, in accordance with the results of global and locus specific methylation analysis. Conversely, we observed lower expression of the TET genes in PR GV oocytes (ANOVA; P > 0.05). TET1, TET2, and TET3, whose expression has never been studied in ovine, generate 5-hydroxymethlcytosine (5hmC) by oxidation of 5-methylcytosine (5mC), and are involved in active DNA demethylation during early embryo development. Our observation of lower expression of the TET genes in lower competence PR GV oocytes suggests that epigenetic mechanisms may affect oocyte quality and paves the way to better understand methylation dynamics during sheep pre-implantation development.

Gene expression profile differences in embryos derived from prepubertal and adult Japanese Black cattle during in vitro development

The present study was carried out to compare the gene expression profiles of in vitro-generated embryos derived from adult and prepubertal Japanese Black cattle oocytes using GeneChip Bovine Genome Array (containing 24072 probe sets representing over 23000 transcripts). Microarray experiments were performed on populations of 8- to 16-cell stage embryos and blastocysts derived from adult (24-35 months old) versus prepubertal (9-10 months old) Japanese Black cattle oocytes matured and fertilised in vitro. In total, 591 (2.4%) and 490 (2.0%) genes were differentially expressed in prepubertal and adult bovine in 8- to 16-cell and blastocyst stage embryos, respectively. Out of these, 218 and 248 genes were upregulated, while 373 and 242 were downregulated in prepubertal and adult 8- to 16-cell and blastocysts stage embryos, respectively. Gene ontology classification regarding biological process, molecular functions and cellular component revealed diversity in transcript abundances between prepubertal and adult groups in both the distinct developmental stages. Quantitative reverse transcription-PCR validated the expression differences of some selected transcripts as identified by microarray analysis. To our knowledge, this is the first report indicating the significant number of genes differentially expression (>2-fold, P<0.01) in preimplantition embryos between adult and prepubertal Japanese Black cattle during in vitro development.

125 EFFECT OF GLYCERALDEHYDE-3-PHOSPHATE DURING BOVINE IN VITRO EMBRYO CULTURE

Most systems for producing mammalian embryos in vitro use glucose as an energy source in the media despite putative toxic effects (Schini and Bavister 1988 Biol. Reprod. 39, 1183–1192; Takahashi and First 1992 Theriogenology 37, 963–978). Currently there is a tendency to identify other suitable energy sources in an attempt to replace glucose from culture media. Glyceraldehyde-3-phosphate (G3P), a glucose-derived high-energy compound, is the end product of the energy-consuming phase of glycolysis that enters the pay-off phase of the pathway characterised by a net gain of energy. The aim of this study was to determine whether G3P is a valid energy source for supporting in vitro embryo development in cattle. Abattoir-derived oocytes (n = 832, over 4 replicates) were matured in vitro in TCM-199 with 15% bovine serum (BS), 0.5 μg mL–1 FSH, 5 μg mL–1 LH, 0.8 mM L-glutamine, and 50 mg mL–1 gentamicin. Mature COC were fertilized in Tyrode’s modified medium, with 30 mg mL–1 heparin, 30 mM penicillamine, 15 mM hypotaurine, 0.15 mM epinephrine, and 1% BS. Both IVM and IVF were carried out at 39°C and 5% CO2 in air. After 20 to 22 h of gamete co-incubation, presumptive zygotes were denuded and cultured in SOF containing either 1.5 mM glucose (control group) or G3P at 3 different concentrations (0.125, 0.5, and 1.5 mM). It is worth specifying that in the 3 G3P-supplemented groups small amounts of glucose were left (0.15 mM) because it is known that a complete removal would affect embryo development by interfering with ribose synthesis through the pentose–phosphate pathway. In vitro culture was carried out at 39°C under humidified air with 5% CO2, 7% O2, and 88% N2 in air for 7 days, when the percentages of tight morulae-blastocysts (TMBL) and superior quality blastocysts (BL) were recorded. Differences in embryo yields among groups were analysed by chi-square test. Supplementation of IVC medium with 1.5 mM G3P reduced (P &lt; 0.01) TMBL (5.0%) and BL (5.0%) rates compared with all other groups, indicating a toxic effect. However, when G3P was added at lower concentrations, no differences in TMBL (37.3 and 26.1, respectively, with 0.125 and 0.5 mM G3P) and in BL rates (35.3 and 25.5%, respectively, with 0.125 and 0.5 mM G3P) were observed compared with the control (32.7% TMBL and 31.4% BL, respectively). Within G3P-treated groups, the higher embryo yields were recorded with 0.125 mM compared with 0.5 mM (P &lt; 0.05) and 1.5 mM (P &lt; 0.01). Interestingly, embryos produced with G3P at the lower concentrations (0.125 and 0.5 mM) seemed to show a faster development compared with the control. In conclusion, these results demonstrated that G3P is a valid energy source for bovine preimplantation embryos and, hence, that G3P supplementation of IVC medium may be a suitable option for reducing glucose concentration in the media. However, further studies are needed to investigate lower concentrations of G3P and to better evaluate embryo viability.

The utility of embryo banking in order to increase the number of embryos available for preimplantation genetic screening in advanced maternal age patients

to determine if embryo banking with PGS is more optimal than proceeding with PGS regardless of embryo number. patients were divided into 2 groups, group 1 were those that banked embryos and proceeded through another round of IVF prior to PGS, and group 2 underwent PGS regardless of embryo number. Group 2 was divided into group 2A (patients with >10 embryos) and group 2B (patients who had <10 embryos). there was no difference in embryos biopsied, normal embryos, number transferred, and pregnancy rate between group 1 and 2. A significant number of patients did not have a transfer in group 2B (6/11) compared to group 1 (3/19) (P = 0.0419). There was no significance between pregnancy rates per transfer between group 1 (6/16) and group 2B (2/5). our data suggests that banking will increase the odds of going to transfer but there was no increase in pregnancy rates.

Establishment of a bovine blastocyst-derived cell line collection for the comparative analysis of embryos created in vivo and by in vitro fertilization, somatic cell nuclear transfer, or parthenogenetic activation

Tools and methods for analyzing differences in embryos resulting from somatic cell nuclear transfer (NT) in comparison to those derived from normal fertilization are needed to define better the nature of the nuclear reprogramming that occurs after NT. To this end, a collection of bovine blastocyst-derived cell lines was created. In vitro expanded or hatched blastocysts, used as primary culture tissue, were from NT; in vitro maturation, fertilization, and culture (IVF); or parthenogenetic (P) activation. Also, five in vivo-fertilized and developed blastocysts were collected by uterine flushing on the eighth d postfertilization. Whole blastocysts were physically attached to STO feeder layers to initiate all of the cell lines generated. The majority of the cell lines in the collection are trophectoderm, 38 NT-derived, 6 in vivo-derived, 20 IVF-derived, and 13 P-derived. Trophectoderm identity was ascertained by morphology and, in many cases, interferon-tau production. Several visceral endoderm cell lines and putative parietal endoderm cell lines were also established. At approximately 5% efficiency, epiblast masses from NT and IVF blastocysts survived and were isolated in culture. Two epiblast masses were also isolated from P blastocysts. Spontaneous differentiation from the epiblast outgrowths resulted in the establishment of fibroblast cell lines. The use of the trophectoderm cell lines as a comparative in vitro model of bovine trophectoderm and placental function is discussed in relation to NT reprogramming.

Effects of breed on kinetics of ovine FSH and ovarian response in superovulated sheep

Embryo production is a useful tool for ex situ conservation of endangered species and breeds, despite a high variability in the ovarian response to superovulatory treatments. The current study evaluated the incidence and mechanisms of genetic factors in such variability, by determining the pharmacokinetics and pharmacodynamics of a standard treatment with ovine FSH (oFSH) in two endangered Spanish sheep breeds (Rubia del Molar, R, and Negra de Colmenar, N) in comparison to Manchega ewes (M, control group). In the first experiment, pharmacokinetics of an i.m. single dose of 1.32mg of oFSH was evaluated in seven animals of each breed. Plasma FSH concentrations reached their maximum at 4h post-administration in all the ewes, but several of the kinetic parameters (plasma FSH concentration at 4h post-administration, maximum plasma FSH concentration, Cmax, and both the area under the plasma concentration-time curve extrapolated to the infinite, AUCinf, and to the last moment of sampling, AUClast) were higher in the N group. In the second trial, 10 animals of each breed were superovulated using eight decreasing doses of oFSH (3× 1.32mg, 2× 1.10mg, and 3× 0.88mg). The R group, when compared to N and M, showed both a higher number of corpora lutea (13.7±0.6 versus 10.0±0.4 in N and 9.8±0.6 in M, P<0.05 for both) and embryos (7.9±0.8 versus 4.3±0.4 in N, P<0.05, and 6.7±0.5 in M, n.s.). Evaluation of pharmacokinetic and dynamic parameters showed that, although there was a trend for a higher hormone availability in R sheep, mean FSH plasma concentrations were similar between breeds (0.54±0.08ng/ml for R, 0.45±0.05ng/ml for N and 0.35±0.05ng/ml for M). However, differences were found in the number of preovulatory follicles growing in response to the FSH treatment between R (24.4±2.2), M (18.9±1.5, n.s.) and N sheep (14.1±1.4; P<0.01). Thus, differences in embryo yields between breeds would be related to differences in the pattern of follicular growth in response to FSH treatment.

Lack of physiological plasticity in the early chicken embryo exposed to acute hypoxia

By exposing chicken embryos to hypoxia (10%) acutely (2, 4, and 6 hr) during early development (2, 3, and 4 days) we tested the hypothesis that hypoxia has an impact on embryonic growth and impairs cardiac development at the time cardiac morphogenesis is taking place. After the hypoxic perturbation, the embryos were allowed to develop until day 9, when embryo mass, heart mass, and rate of oxygen consumption were recorded. Four-day-old embryos exposed to 6 hr of hypoxia showed an increased mortality (38.9% versus 18% for controls), indicating the immediate effect of hypoxia on survivability. While only 8% of the controls displayed morphological abnormalities, 3- and 4-day-old embryos exposed for 6 hr showed more frequent developmental abnormalities (25% and 30% respectively). No significant differences in embryo or heart mass were found except in 4-day-old embryos exposed for 2 hr. Mass-specific oxygen consumption was not different between controls and embryos exposed to hypoxia at 2 or 3 days of development, but it was increased in 4-day-old embryos exposed for 4 hr (P < 0.05). These results suggest that an acute hypoxic episode does not have an impact when occurring very early in development (days 2 or 3). However, when the hypoxic episode occurs on day 4, survivability is largely decreased. Considering the lack of permanent effects on the surviving embryos, we suggest that the early embryo resorts to a simple strategy of death or survival, and the individual capacity for survival must be based on interindividual differences rather than the existence of compensatory mechanisms. J. Exp. Zool. 286:450-456, 2000.

Regeneration of somatic embryos and roots from quince leaves cultured on media with different macroelement composition.

The effects of different macroelement combinations on somatic embryogenesis of quince (Cydonia oblonga Mill.) were tested. Leaves were excised from shoot cultures of quince clones and cultured on macroelement combinations of 8 different growth media. Callus production varied depending on the medium and the clone combinations. The influence of genotype and macronutrient combination on somatic embryo and root regeneration was also observed. Clone BA 29 showed the highest embryogenic properties and Murashige and Skoog-based medium appeared to be the most favourable for somatic embryo formation. Root regeneration was higher on Woody Plant Medium and Schenck and Hildebrandt-based media. Interactive effects between genotypes and macroelement combinations were also detected both for embryo and root formation. In all treatments, somatic embryos underwent early developmental arrest and failed to convert into plants. Differences in embryo and root regeneration observed among macroelement combinations may be ascribable to different levels of medium nitrogen and probably to the ratio between nitrate and ammonium.

Effects of superovulated heifer diet type and quantity on relative mRNA abundances and pyruvate metabolism in recovered embryos

This study investigated the effects of quantity and type of diet fed to superovulated donor heifers on molecular and metabolic indices of embryonic development. These effects included the relative abundances of mRNAs for the alpha 1 subunit of Na/K-ATPase and the antioxidant enzyme Cu/Zn-SOD, as well as pyruvate utilization in bovine morulae and blastocysts developed in vivo. Heifers were fed a daily ration of either grass silage and a citrus-beet pulp-based concentrate or grass silage and a barley-based concentrate for 116 days, both at 3 kg per day or ad libitum. In embryos derived from heifers fed the pulp-based diets, the relative abundances of the transcripts were not affected by either day of collection or quantity of diet. In embryos derived from heifers fed the barley-based diets, the relative abundances of the Na/K-ATPase transcripts were also not changed by these main effects, while the relative abundances of the Cu/Zn-SOD transcripts were affected by day of collection and by the quantity of diet. Pyruvate metabolism was affected by day of collection, and was significantly increased in day 8 embryos compared with day 7 and day 6 embryos. Diet quantity did not affect pyruvate utilization, whereas diet type did increase pyruvate metabolism in the barley group when compared with the pulp group. The results of this study show for the first time that molecular and metabolic variations may exist in embryos derived in vivo and developed in donor heifers on nutritional regimens differing in type and quantity. Differences in embryos collected on different developmental days may be attributed to varying cell numbers. Alterations in the relative abundances of the Cu/Zn-SOD transcripts and pyruvate metabolism caused by the quantity of diet fed to the donor animal were likely to have been due to alterations in metabolic end products that accumulate in reproductive tract fluids, whereas differences in embryonic metabolism caused by type of diet are related to the composition of the diet. These findings characterize embryos produced in vivo at the molecular level, indicating that the molecular markers used in the present study can differentiate between populations of embryos produced under different nutritional regimens and determine conditions conductive to the production of good quality embryos.

What is a juvenile sea urchin? A comparative and phylogenetic survey of post-metamorphic juveniles

Echinoid development progresses through embryonic and larval stages to metamorphosis and the adult form. Despite vast differences in embryos and larval forms, including bilaterally symmetric echinopluteus larvae, ovoid non-feeding larvae and brooded embryos, all metamorphose into juvenile sea urchins with pentaradial symmetry. The adult sea urchin body plan is initiated as the juvenile rudiment. The rudiment has been called the phylotypic stage for the class Echinoidea, a designation that implies little variation at this midpoint in development (e.g. Raff et al., 1991; Richardson, 1995; Raff, 1996). However, right at metamorphosis (upon eversion of the juvenile rudiment), variations in test symmetry, shape and number of spines, and number of skeletal plates, podia and pedicellariae are present in juveniles. This variation suggests either that there is no phylotypic stage or that such a stage occurs earlier in rudiment formation. To distinguish between these possibilities, I explored the patterns by which the juvenile rudiment is formed as well as the variation among juveniles approximately 1 day after metamorphosis in 19 echinoid taxa covering a broad taxonomic range including cidaroids, diadematids, irregular echinoids (spatangoids and clypeasteroids), arbaciids, temnopleurids, echinometrids and strongylocentrotids. Most of the material for analysis of juveniles was obtained by the author. Additional information was gathered from classical studies of metamorphosis. Data were collected on the number and shape of dorsal pedicellariae, juvenile and adult spines, primary and secondary podia, and juvenile test shape. When possible multiple individuals within a species were examined, revealing no or only minor trait variation. These data were mapped on a well-resolved phylogeny established from adult characters.

Variations in gynogenic response among long-day onion ( Allium cepa L.) accessions

Long-day onion genotypes originating from Europe, North America and Japan were tested for gynogenic induction ability. Haploid induction using an efficient single-step flower culture induction procedure was at least as productive as the previously studied double-step flower/ovary culture, while undesired callusing of flower bases did not substantially lower the induction frequency. Differences in embryo yields among different accessions and among different donor plants within accessions were very pronounced. Three extremely responsive accessions were found, with average embryo yields of 18.6–22.6%, while individual donor plants produced up to 51.7% embryos. In terms of geographic origin, genetic material bred in America was on average almost five and nine times more responsive than European and Japanese material, respectively. The stability of high induction frequency was confirmed by culturing flowers from the same individual donor plants in two subsequent growth years. Of the regenerants, 90.5% were haploid and 88.2% of analysed diploid regenerants were homozygous. The results indicate that the genotype of donor plants has a crucial influence on haploid induction ability and that the less labour intensive single-step flower induction procedure is an efficient method of obtaining a high-frequency homozygous-embryo induction rate.

Using radiography to attempt to screen for haploid embryos in apple seeds

Obtaining apple haploid plants should offer new possibilities for genetic studies and breeding in this perennial species. Apple seeds were examined in search of an early screening for haploid embryos. Two varieties, Erovan and Golden Delicious, were studied by using X-ray radiography of mature seeds. Measures of length and width of integument and embryo and also length of radicle were performed. No relationship between the level of ploidy and the size of embryo was found. In addition, there was no morphological difference in embryos between seeds that germinated or did not germinate. Observations were also made on plantlets showing the pale green lethal factor (pglf). The morphology of seed cannot be used to predict the lethal phenotype.

Effect of insemination volume on embryo recovery in mares

Summary The objective of this study was to determine the effect of insemination volumes on embryo recovery in mares. Sixty-two light-horse mares were randomly assigned to 1 of 3 insemination treatments as they exhibited estrus. Mares were artificially inseminated with 100 × 10 6 progressively motile spermatozoa (pms) extended in 10, 100 or 200 ml of dried skim milk extender (E-Z Mixin). Embryo-recovery attempts were performed 6 or 7 days post-ovulation. 6 Due to recovery of a low number of embryos, a second experiment was conducted with the same mares using a greater number of progressively motile spermatozoa (250 × 10 6 pms) suspended in either 10 or 100 ml of E-Z Mixin extender. In Experiment 1, there was no difference (P>0.05) in numbers of embryos recovered between mares inseminated with 10 (40%) and 100 ml (10.0%) of extender, nor was there a difference (P>0.05) between mares inseminated with 100 (10.0%) and 200 ml (0.0%). However, there was a significant difference in embryos recovered between mares inseminated with 10 (40.0%) and 200 ml (0.0%) (P