Embryonic Death Research Articles

Biotesting of decabromodiphenyl oxide using a multi-component test system

The results of biotesting of decabromodiphenyl oxide using a multicomponent test sys- tem are presented. It has been demonstrated that its aqueous solutions exhibit a reverse dose- dependent effect in stimulating the growth of colonies of the green algae Chlorella vulgaris and show no signs of toxic effects on Daphnia magna. During the thermal decomposition of decabro- modiphenyl oxide at a temperature of 400°C, a mixture of products is formed, one of which is bromine. The decomposition product is released into the air and possesses toxic properties, as evi- denced by stimulation of the growth of colonies of the green algae Chlorella vulgaris, as well as the death and growth abnormalities of Planorbis mollusk embryos. Therefore, water samples con- taining decabromodiphenyl oxide can be assessed for toxicity by biotesting methods, using cul- tures of green algae Chlorella vulgaris and 24-hour-old Planorbis embryos as test subjects. It is noteworthy that in the context of the prevailing perception of the safety of decabromodiphenyl ox- ide, based on its poor water solubility and low toxicity to biological organisms, there arises a need to study embryotoxic effects of the thermal decomposition products on both animals and humans.

P-530 Precise quantification of DNA in spent culture media sheds light on associations between DNA quantity, embryonic ploidy status and morphological quality

Abstract Study question Is it possible to accurately measure cell-free DNA (cfDNA) quantity in embryo spent culture media, and are amounts correlated with embryo morphological and/or chromosomal status? Summary answer A sensitive DNA quantification method was created, demonstrating that cfDNA quantity is not associated with embryo ploidy, but increases when embryos are of poor morphology. What is known already Preimplantation genetic testing involves embryo biopsy, necessitating specialist equipment and highly trained staff, increasing costs. Additionally, there have been concerns over potential embryo damage to the embryo resulting from biopsy. The discovery of cfDNA in medium in which embryos were cultured, led to suggestions that non-invasive PGT (niPGT) might be possible. However, the amount of cfDNA in media samples, and potential correlations with other aspects of embryo biology, require clarification. We aimed to provide the first accurate measurement of the amount of DNA in spent culture medium, providing insights into whether niPGT is likely to provide acceptable accuracy rates. Study design, size, duration This prospective analysis involved media samples from 151 fresh embryos, which were produced at a single IVF clinic over the course of one year. Embryos were cultured individually, with a medium change on day-3 of development. Trophectoderm biopsy was undertaken on day-5/-6, followed by PGT-A. Embryo morphology was assessed according to ASEBIR criteria (ranked A-C). Participants/materials, setting, methods The culture and biopsy protocols used were standard for the clinic. Media was collected prior to biopsy and underwent a novel quantitative whole genome amplification method, allowing precise measurement of DNA with respect to a standard curve generated using samples containing known amounts of DNA. PGT-A involved the use of a highly validated method, based upon next generation sequencing. Correlation between DNA quantity and embryo morphological quality was also studied. Data were analyzed using ANOVA. Main results and the role of chance There was no difference in the quantity of DNA in media samples associated with euploid and aneuploid embryos (mean DNA quantity and standard deviations were 1.72 ± 3.23 pg and 1.54 ± 3.66 pg for aneuploid and euploid embryos, respectively; p = 0.75). Overall, 91% of media samples contained less than the equivalent of one cell (<5pg of DNA) and almost two-thirds of samples (65%) contained less than 1pg of DNA. Given such low quantities of embryonic DNA, achieving accurate genetic testing is likely to be challenging. Drops that held embryos until day-6 had significantly higher levels of DNA than those associated with culture to day-5 (P < 0.0001), suggesting cfDNA accumulates with time. Thus, longer culture periods are likely to yield more DNA for analysis. Comparing the amount of cfDNA with morphological status revealed a significant association. Embryos of fair quality (C) tended to have higher quantities of DNA in spent culture medium, whereas top-quality embryos (classified as A or B) typically had less (mean and standard deviation of DNA quantity: 1.23 ± 3.14 pg for class A and B versus 2.60 ± 3.85 pg for class C; p < 0.05). This may reflect increased cell death and DNA release in poorer quality embryos. Limitations, reasons for caution This project is limited by its single-centre nature and relatively small sample size. A large-scale, multicentre study would be necessary to confirm these results. Wider implications of the findings It has been hypothesised that cfDNA quantity in culture medium may be predictive of embryo chromosomal status. We found no evidence to support this. Although DNA quantities were higher after culture to day-6, levels remained low, calling into question the ability of niPGT to deliver high accuracy diagnosis of embryos. Trial registration number not applicable

The effect of hydroxy-selenomethionine on the productive and reproductive performance of old broiler breeders.

Selenium (Se) is a rare essential element that plays a vital role in the health and performance of animals. By interfering in the production of antioxidant enzymes such as glutathione peroxidase, thioredoxin reductase and methionine sulfoxide, Se plays a role in reducing the effects of oxidative stress and animal performance. This study aimed to investigate the effect of hydroxy-selenomethionine (OH-SeMet) in the diet of broiler breeder and old broiler breeder roosters on productive performance, reproduction and sperm quality parameters. For this purpose, 260 broiler breeders of the Ross 308 strain were used in a completely randomized design with four treatments and five replications (13 hens and one rooster in each replication). Experimental treatments included: (1) a basal diet without OH-SeMet (T1:control), (2) a broiler breeder diet without OH-SeMet and a rooster diet containing 0.1mg/kg OH-SeMet (T2), (3) broiler breeder diet containing 0.1mg/kg OH-SeMet and rooster diet without OH-SeMet (T3) and (4) broiler breeder and rooster diet contained 0.1mg/kg OH-SeMet (T4). The results showed that T3 and T4 treatments improved egg production, egg weight, egg mass and feed conversion ratio (FCR) compared to the control treatment (p<0.05). The fertility and hatchability percentages of T4 and T2 treatments increased compared to T1 and T3 treatments (p<0.05). The rate of embryonic losses in T1 was higher than in other treatments. However, grade one chickens were higher in T4 than in other treatments (p<0.05). Total motility and viability of sperms were significantly higher in T2 and T4 treatments than in T1 and T3 treatments. The sperm abnormality percentage and sperm MDA concentration decreased in T2 and T4 treatments. Therefore, using OH-SeMet may be a practical approach to help old broiler breeders' production and reproduction performance.

Describing endosymbiont-host interactions within the parasitism-mutualism continuum.

Endosymbionts are widespread in arthropods, living in host cells with effects that extend from parasitic to mutualistic. Newly acquired endosymbionts tend to be parasitic, but vertical transmission favors coevolution toward mutualism, with hosts sometimes developing dependency. Endosymbionts negatively affecting host fitness may still spread by impacting host reproductive traits, referred to as reproductive "manipulation," although costs for hosts are often assumed rather than demonstrated. For cytoplasmic incompatibility (CI) that involves endosymbiont-mediated embryo death, theory predicts directional shifts away from "manipulation" toward reduced CI strength; moreover, CI-causing endosymbionts need to increase host fitness to initially spread. In nature, endosymbiont-host interactions and dynamics are complex, often depending on environmental conditions and evolutionary history. We advocate for capturing this complexity through appropriate datasets, rather than relying on terms like "manipulation." Such imprecision can lead to the misclassification of endosymbionts along the parasitism-mutualism continuum.

Anti-Angiogenic Activity of Sabungai (Gynura procumbens) using a Cancer-induced Chicken Embryo

Cancer has been increasing over recent years and is considered the primary cause of mortality following cardiovascular disease. Angiogenesis, the biological process by which new blood vessels are generated, plays a crucial role in the growth and spread of tumors. The objective of this study was to assess the anti-angiogenic activity of different Sabungai (Gynura procumbens) leaf extract concentrations in the Chorioallantoic Membrane (CAM) of cancer-induced chicken embryos. The results of a one-way analysis of variance (25.20 ± 2.70, 27.20 ± 1.08, and 22.80 ± 1.66) indicated a statistically significant difference in CAM vascularization between the various treatments. Post hoc analysis utilizing Tukey's Honest Significant Difference (HSD) test revealed that Treatment 2 (300ppt) significantly affected CAM vascularization, as evidenced by an average vascularization count of 27.20 ± 1.08, followed by Treatment 1 (100ppt) and Treatment 3 (500ppt). The results indicated that the extract from Sabungai leaves effectively hinders the formation of new blood vessels in a dose-dependent manner. The highest concentration of leaf extract (500 ppt) resulted in an average vascularization of 22.80 ± 1.66. However, excessively high doses can cause bleeding and embryo death. The results demonstrated that sabungai leaf extract affected blood vessel development in chicken embryos. The results of this research have various implications that contribute to scientific knowledge and potential applications in cancer treatment.

Embryonic death as a probable reason for the collapse of population densities in Lymantria dispar (Linnaeus, 1758) (Lepidoptera: Erebidae, Lymantriinae)

For the first time the causes of mass death of embryos in a population of Lymantria dispar (Linnaeus, 1758) with biochemical and molecular methods are investigated. In this study egg masses were collected in the territory of Russia from two populations, with low (the West Siberian population) and high (Trans-Ural) density and analyzed. Spring hatching of larvae from the eggs stored under constant laboratory conditions at 2º C was two times lower for insects of the Trans-Ural (TU) population compared to the West Siberian (WS) population (43 ± 6 % and 86 ± 7 % accordingly). The level of virus defined by PCR for TU and WS populations was 76±9 % and 36±6%, respectively. The major components of eggs were the same between populations. However, we found decreased concentrations of proteins important for embryogenesis in TU population, such as an arilforin-like molecule and no vitellin 2-like component. Field data were comparable to those of the laboratory, and in TU populations there was a decrease of density. The obtained results suggest that alteration in hormonal balance of the infected with nucleopolyhedrovirus (NPV) insects may lead to a decrease or absence of some key proteins of embryogenesis in eggs. It can influence hatching of larvae from eggs and, accordingly, population dynamics of the lepidopteran.

ZFHX3 variants cause childhood partial epilepsy and infantile spasms with favourable outcomes

BackgroundThe ZFHX3 gene plays vital roles in embryonic development, cell proliferation, neuronal differentiation and neuronal death. This study aims to explore the relationship between ZFHX3 variants and epilepsy.MethodsWhole-exome sequencing was...

Integrated OMICs approach reveals energy metabolism pathway is vital for Salmonella Pullorum survival within the egg white.

Pullorum disease, causing serious embryo death and chick mortality, results in substantial economic losses worldwide due to transovarial transmission. Egg-borne outbreaks are frequently reported in many countries. The present study has filled the knowledge gap regarding how the specific chicken-adapted pathogen Salmonella Pullorum behaves within the challenging environment of egg white. The deletion of the fim fimbrial system can increase survival in the albumen, possibly by reprogramming metabolism-related gene products, which reveals a new adaptive strategy of pathogens. Moreover, the comparison, including previous research on Salmonella Enteritidis, capable of vertical transmission, aims to provide diversified data sets in the field and further help to implement reasonable and effective measures to improve both food safety and animal health.

Identifying Regions of the Genome Associated with Conception Rate to the First Service in Holstein Heifers Bred by Artificial Insemination and as Embryo Transfer Recipients.

Heifer conception rate to the first service (HCR1) is defined as the number of heifers that become pregnant to the first breeding service compared to the heifers bred. This study aimed to identify loci associated and gene sets enriched for HCR1 for heifers that were bred by artificial insemination (AI, n = 2829) or were embryo transfer (ET, n = 2086) recipients, by completing a genome-wide association analysis and gene set enrichment analysis using SNP data (GSEA-SNP). Three unique loci, containing four positional candidate genes, were associated (p < 1 × 10-5) with HCR1 for ET recipients, while the GSEA-SNP identified four gene sets (NES ≥ 3) and sixty-two leading edge genes (LEGs) enriched for HCR1. While no loci were associated with HCR1 bred by AI, one gene set and twelve LEGs were enriched (NES ≥ 3) for HCR1 with the GSEA-SNP. This included one gene (PKD2) shared between HCR1 AI and ET services. Identifying loci associated or enriched for HCR1 provides an opportunity to use them as genomic selection tools to facilitate the selection of cattle with higher reproductive efficiency, and to better understand embryonic loss.

BaP/BPDE exposure causes human trophoblast cell dysfunctions and induces miscarriage by up-regulating lnc-HZ06-regulated IL1B

Exposure to environmental BaP or its metabolite BPDE causes trophoblast cell dysfunctions to induce miscarriage (abnormal early embryo loss), which might be generally regulated by lncRNAs. IL1B, a critical inflammatory cytokine, is closely associated with adverse pregnancy outcomes. However, whether IL1B might cause dysfunctions of BaP/BPDE-exposed trophoblast cells to induce miscarriage, as well as its specific epigenetic regulatory mechanisms, is completely unexplored. In this study, we find that BPDE-DNA adducts, trophoblast cell dysfunctions, and miscarriage are closely associated. Moreover, we also identify a novel lnc-HZ06 and IL1B, both of which are highly expressed in BPDE-exposed trophoblast cells, in villous tissues of recurrent miscarriage patients, and in placental tissues of BaP-exposed mice with miscarriage. Both lnc-HZ06 and IL1B suppress trophoblast cell migration/invasion and increase apoptosis. In mechanism, lnc-HZ06 promotes STAT4-mediated IL1B mRNA transcription, enhances IL1B mRNA stability by promoting the formation of METTL3/HuR/IL1B mRNA ternary complex, and finally up-regulates IL1B expression levels. BPDE exposure promotes TBP-mediated lnc-HZ06 transcription, and thus up-regulates IL1B levels. Knockdown of either murine lnc-hz06 (which down-regulates Il1b levels) or murine Il1b could alleviate miscarriage in BaP-exposed mice. Collectively, this study not only discovers novel biological mechanisms and pathogenesis of unexplained miscarriage but also provides novel potential targets for treatment against BaP/BPDE-induced miscarriage.

Early Pregnancy Loss in an Era of “Wrongful Embryonic Death”

This Viewpoint examines the possible legal consequences of pregnancy loss following the Alabama Supreme Court’s decision to extend personhood to laboratory-conceived embryos.

The novel lnc-HZ12 suppresses autophagy degradation of BBC3 by preventing its interactions with HSPA8 to induce trophoblast cell apoptosis

ABSTRACT Abnormal expression of long non-coding RNAs (lncRNAs) is associated with the dysfunctions of human trophoblast cells and the occurrence of miscarriage (abnormal early embryo loss). BBC3/PUMA (BCL2 binding component 3) plays significant roles in regulation of cell apoptosis. However, whether specific lncRNAs might regulate BBC3 in trophoblast cells and further induce apoptosis and miscarriage remains completely unclear. Through screening, we identified a novel lnc-HZ12, which was significantly highly expressed in villous tissues of recurrent miscarriage (RM) patients relative to their healthy control (HC) group. Lnc-HZ12 suppressed chaperone-mediated autophagy (CMA) degradation of BBC3, promoted trophoblast cell apoptosis, and was associated with miscarriage. In mechanism, lnc-HZ12 downregulated the expression levels of chaperone molecules HSPA8 and LAMP2A in trophoblast cells. Meanwhile, lnc-HZ12 (mainly lnc-HZ12-SO2 region in F2 fragment) and HSPA8 competitively bound with the 169RVLYNL174 patch on BBC3, which prevented BBC3 from interactions with HSPA8 and impaired the formation of BBC3-HSPA8-LAMP2A complex for CMA degradation of BBC3. Thus, lnc-HZ12 upregulated the BBC3-CASP9-CASP3 pathway and induced trophoblast cell apoptosis. In villous tissues, lnc-HZ12 was highly expressed, CMA degradation of BBC3 was suppressed, and the apoptosis levels were higher in RM vs HC villous tissues, all of which were associated with miscarriage. Interestingly, knockdown of murine Bbc3 could efficiently suppress placental apoptosis and alleviate miscarriage in a mouse miscarriage model. Taken together, our results indicated that lnc-HZ12 and BBC3 played important roles in trophoblast cell apoptosis and miscarriage and might act as attractive targets for miscarriage treatment. Abbreviation: 7-AAD: 7-aminoactinomycin D; BaP: benzopyrene; BBC3/PUMA: BCL2 binding component 3; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HC: healthy control; HSPA8: heat shock protein family A (Hsp70) member 8; IP: immunoprecipitation; LAMP2A: lysosomal associated membrane protein 2; LncRNA: long non-coding RNA; mRNA: messenger RNA; MT: mutant-type; NC: negative control; NSO: nonspecific oligonucleotide; PARP1: poly(ADP-ribose) polymerase 1; RIP: RNA immunoprecipitation; RM: recurrent miscarriage; TBP: TATA-box binding protein; WT: wild-type.

Evidence for metabolism of creatine by the conceptus, placenta, and uterus for production of adenosine triphosphate during conceptus development in pigs†.

In pigs, the majority of embryonic mortality occurs when free-floating conceptuses (embryos/fetuses and associated placental membranes) elongate, and the uterine-placental interface undergoes folding and develops areolae. Both periods involve proliferation, migration, and changes in morphology of cells that require adenosine triphosphate (ATP). We hypothesize that insufficient ATP in conceptus and uterine tissues contributes to conceptus loss in pigs. Creatine is stored in cells as phosphocreatine for ATP regeneration through the creatine-creatine kinase- phosphocreatine pathway. However, the expression of components of this pathway in pigs has not been examined throughout gestation. Results of qPCR analyses indicated increases in AGAT, GAMT, CKM, CKB, and SLC6A8 mRNAs in elongating porcine conceptuses, and immunofluorescence microscopy localized guanidinoacetate N-methyltransferase, creatine kinase M, and creatine kinase B proteins to the trophectoderm of elongating conceptuses, to the columnar chorionic epithelial cells at the bottom of chorioallantoic troughs, and to endometrial luminal epithelium at the tops of the endometrial ridges of uterine-placental folds on Days 40, 60, and 90 of gestation. Guanidinoacetate N-methyltransferase protein is expressed in endometrial luminal epithelium at the uterine-placental interface, but immunostaining is more intense in luminal epithelium at the bottoms of the endometrial ridges. Results of this study indicate that key elements of the pathway for creatine metabolism are expressed in cells of the conceptus, placenta, and uterus for potential production of ATP during two timepoints in pregnancy with a high demand for energy; elongation of the conceptus for implantation and development of uterine-placental folding during placentation.

PPARG is dispensable for bovine embryo development up to tubular stages†.

Following blastocyst hatching, ungulate embryos undergo a prolonged preimplantation period termed conceptus elongation. Conceptus elongation constitutes a highly susceptible period for embryonic loss, and the embryonic requirements during this process are largely unknown, but multiple lipid compounds have been identified in the fluid nourishing the elongating conceptuses. Peroxisome proliferator-activated receptors mediate the signaling actions of prostaglandins and other lipids, and, between them, PPARG has been pointed out to play a relevant role in conceptus elongation by a functional study that depleted PPARG in both uterus and conceptus. The objective of this study has been to determine if embryonic PPARG is required for bovine embryo development. To that aim, we have generated bovine PPARG knock-out embryos in vitro using two independent gene ablation strategies and assessed their developmental ability. In vitro development to Day 8 blastocyst was unaffected by PPARG ablation, as total, inner cell mass, and trophectoderm cell numbers were similar between wild-type and knock-out D8 embryos. In vitro post-hatching development to D12 was also comparable between different genotypes, as embryo diameter, epiblast cell number, embryonic disk formation, and hypoblast migration rates were unaffected by the ablation. The development of tubular stages equivalent to E14 was assessed in vivo, following a heterologous embryo transfer experiment, observing that the development of extra-embryonic membranes and of the embryonic disk was not altered by PPARG ablation. In conclusion, PPARG ablation did not impaired bovine embryo development up to tubular stages.

Developmental Disruption of Mef2c in Medial Ganglionic Eminence–Derived Cortical Inhibitory Interneurons Impairs Cellular and Circuit Function

BackgroundMEF2C is strongly linked to various neurodevelopmental disorders including autism, intellectual disability, schizophrenia, and attention-deficit/hyperactivity disorder. Mice that constitutively lack 1 copy of Mef2c or selectively lack both copies of Mef2c in cortical excitatory neurons display a variety of behavioral phenotypes associated with neurodevelopmental disorders. The MEF2C protein is a transcription factor necessary for cellular development and synaptic modulation of excitatory neurons. MEF2C is also expressed in a subset of cortical GABAergic (gamma-aminobutyric acidergic) inhibitory neurons, but its function in those cell types remains largely unknown. MethodsUsing conditional deletions of the Mef2c gene in mice, we investigated the role of MEF2C in parvalbumin-expressing interneurons (PV-INs), the largest subpopulation of cortical GABAergic cells, at 2 developmental time points. We performed slice electrophysiology, in vivo recordings, and behavior assays to test how embryonic and late postnatal loss of MEF2C from GABAergic INs impacts their survival and maturation and alters brain function and behavior. ResultsLoss of MEF2C from PV-INs during embryonic, but not late postnatal, development resulted in reduced PV-IN number and failure of PV-INs to molecularly and synaptically mature. In association with these deficits, early loss of MEF2C in GABAergic INs led to abnormal cortical network activity, hyperactive and stereotypic behavior, and impaired cognitive and social behavior. ConclusionsMEF2C expression is critical for the development of cortical GABAergic INs, particularly PV-INs. Embryonic loss of function of MEF2C mediates dysfunction of GABAergic INs, leading to altered in vivo patterns of cortical activity and behavioral phenotypes associated with neurodevelopmental disorders.

Analysis of survival and morphometric parameters of loach embryos and prelarvae under the action of the polyethyleneglycol derivative polymeric nanocarrier

Background. Our previous studies have shown a minor embryotoxic effect of polymeric poly(VEP-co-GMA)-graft-mPEG carrier on Missgurnus fossilis L. embryos and prelarvae. At the same time, it was found that a 3-day exposure to 10-15 M polymer carrier slowed developing, which is characterized by a delayed hatching of larvae. However, in the 10-day experiment on loach larvae exposed to polymeric carrier, anomalies and defects of development have not been identified. The aim of this work was to analyze the embryos and prelarvae survival, as well as determine the morphometric indicators of the loach embryos under the action of a PEG-modified polymer carrier. Materials and Methods. Ovulation in loach females (Misgurnus fossilis L.) was stimulated by intramuscular injection of female chorionic gonadotropin (500 units), eggs were obtained 36 h after stimulation, fertilized in Petri dishes with a suspension of sperm according to A. A. Neifach. The stages of development were observed visually under a binocular microscope MBS-9 with a photo camera in a real-time mode. The experimental embryos were incubated in Goltfreter’s solution with the addition of PEG-containing carrier to a final concentration of 10 and 100 μmol/L. The morphological development of the embryo groups was evaluated according to the T. Fujimoto deve­lopment tables (2004), and the morphological parameters (diameter and area of roes, blastomers, embryos, embryo sac and blastodisk) were measured using ImageJ and Photoshop (CC 2014v15) programs. Results. Addition of the 10 μmol/L PEG-containing carrier to the medium initiated significant positive changes in the survival of loach embryos and prelarvae, in contrast to exposure to 100 μmol/L carrier. A significant increase in the area and diameter of blastomers and the embryos themselves under the influence of 100 μmol/L PEG carrier was established, which may indicate swelling of the embryos and a disruption of water-salt exchange that causes the early death of embryos. Conclusions. (1) Low embryotoxicity of the PEG-polymer was confirmed on the studied model of the loach embryos. Based on the obtained data and the original data of J. Maes et al. (2) PEG is an attractive polymeric carrier for the delivery of a variety of compounds to both embryos and prelarvae as well as other model subjects. Despite the fact that we investigated only two concentrations of the PEG-containing carrier, it is quite likely that these concentrations of the carrier are (3) biologically active in themselves and, therefore, a thorough selection of the PEG-carrier concentration for each objective of the model is needed.

Different narcotic gases and concentrations for immobilization of ostrich embryos for in-ovo imaging

In-ovo imaging using avian eggs has been described as a potential alternative to animal testing using rodents. However, imaging studies are hampered by embryonal motion producing artifacts. This study aims at systematically comparing isoflurane, desflurane and sevoflurane in three different concentrations in ostrich embryos. Biomagnetic signals of ostrich embryos were recorded analyzing cardiac action and motion. Ten groups comprising eight ostrich embryos each were investigated: Control, isoflurane (2%, 4%, and 6%), desflurane (6%, 12%, and 18%) and sevoflurane (3%, 5%, and 8%). Each ostrich egg was exposed to the same narcotic gas and concentration on development day (DD) 31 and 34. Narcotic gas exposure was upheld for 90 min and embryos were monitored for additional 75 min. Toxicity was evaluated by verifying embryo viability 24 h after the experiments. Initial heart rate of mean 148 beats/min (DD 31) and 136 beats/min (DD 34) decreased over time by 44–48 beats/minute. No significant differences were observed between groups. All narcotic gases led to distinct movement reduction after mean 8 min. Embryos exposed to desflurane 6% showed residual movements. Isoflurane 6% and sevoflurane 8% produced motion-free time intervals of mean 70 min after discontinuation of narcotic gas exposure. Only one embryo death occurred after narcotic gas exposure with desflurane 6%. This study shows that isoflurane, desflurane and sevoflurane are suitable for ostrich embryo immobilization, which is a prerequisite for motion-artifact free imaging. Application of isoflurane 6% and sevoflurane 8% is a) safe as no embryonal deaths occurred after exposure and b) effective as immobilization was observed for approx. 70 min after the end of narcotic gas exposure. These results should be interpreted with caution regarding transferability to other avian species as differences in embryo size and incubation duration exist.

The impact of lead, cadmium, and mercury on the reproduction of mountain hares (Lepus timidus L., 1758) in the north of Krasnoyarsk Krai

Reproduction is key to the survival and development of a species. Anthropogenic activities release significant amounts of toxic pollutants into the environment. In this study, we aimed to determine effects of heavy metals on some reproductive parameters of the mountain hare. Female mountain hares (n = 41) were hunted in the reference and industrially polluted areas of Krasnoyarsk Krai during four seasons. Their skeletal muscles, liver, and kidneys were subjected to atomic absorption spectrometry to determine concentrations of lead, cadmium, and mercury. The contents of lead, cadmium, and mercury were significantly higher in the hares from the contaminated areas compared to the reference sites. According to the results, the exposure to lead, cadmium, and mercury had an impact on the reproductive potential of the female mountain hares. In particular, we established correlations between numbers of embryos and corpora lutea and contents of lead in the kidneys and liver, as well as cadmium in the kidneys. The number of corpora lutea and embryonic losses in the female hares from the contaminated areas were higher than those in the hared from reference areas. However, the numbers of embryos did not differ significantly between the compared areas. Our study showed that about 40% of the liver samples and 100% of the muscle tissue samples obtained from the hares in the impact zone contained high concentrations of lead and cadmium. Therefore, hunting in these industrially polluted areas may pose a toxic hazard to the indigenous peoples living there. Further research is needed to assess potential and actual fertility, offspring survival, and other important parameters of mountain hare populations exposed to different levels of chemical pollution.

Is prolonged luteal phase a problem in lactating Holstein cows?

In this study, the main objective was to assess if long luteal phases could have other causes than pregnancy losses. We enrolled Holstein dairy cows ≥50 d in milk (DIM) from a commercial herd in Brazil from October 2016 to August 2017. All cows received an estradiol-based synchronization protocol, and, on the day of insemination (d 0), were randomly assigned either an artificial insemination (AI) or a placebo insemination (PBO) in a 3:1 ratio. An ultrasound was used to assess the presence of a CL on d17, 24, and 31, which, combined to the information from patches for the detection of estrus, was used to determine the length of the luteal phase following AI or PBO. Pregnancy was assessed by ultrasound on d 31 and cows that were pregnant were excluded from the analyses. The length of the estrous cycles was categorized as short (<17 d), normal (17-23 d), long (24-30 d), and very long (≥31 d). We compared the proportion of cows in each category between the AI and PBO groups using a cumulative ordinal mixed model. We define prolonged luteal phase as estrous cycles ≥24 d and tested its association with potential risk factors (parity, season, DIM, uterine size and position score, milk production, body condition score, and the presence of a corpus luteum (CL) at enrollment to the synchronization protocol) using mixed logistic regression models. Results are presented as odds ratio (OR) and 95% credible intervals (BCI). Data from 876 inseminations (AI: n = 616, PBO: n = 260) was collected. Overall, 12% of estrous cycles were short, 31% were normal, 19% were long, and 38% were very long. There was no difference in the odds of being in longer estrous cycle categories for the AI compared with the PBO group (OR = 0.92, 95% BCI = 0.76-1.10). Season and presence of a CL at enrollment were associated with prolonged luteal phase. In the AI group, there was a possible effect of early pregnancy losses on the lifespan of the CL, but not the PBO group, which led us to conclude that long and very long estrous cycles were not all caused by the embryonic loss. In fact, the high prevalence of cows with an extended CL lifespan in the present study suggests this could be an under- or miss-reported characteristic of high-producing lactating Holstein cows. This finding may have important repercussions in the understanding of the CL function physiology of lactating Holstein cows.

Subacute exposure to DEHP leads to impaired decidual reaction and exacerbates the risk of early miscarriage in mice.

To investigate the effect of subacute exposure of Di (2-ethylhexyl) phthalate (DEHP) on endometrial decidualization and early pregnancy miscarriage in mice. CD1 mice were orally administrated with 300 (low-dose group), 1000 (medium-dose group), or 3000 mg·kg－1·d－1 DEHP (1/10 LD50, high-dose group) for 28 days, respectively. An early natural pregnancy model and an artificially induced decidualization model were established. The uterine tissues were collected on D7 of natural pregnancy and D8 of artificially induced decidualization, respectively. The effects of a subacute exposure to DEHP on the decidualization of mice were detected by HE staining, Masson staining, TUNEL assay, and Western blotting. A model of spontaneous abortion was constructed in mice after subacute exposure to 300 mg·kg－1·d－1 DEHP, and the effect of impaired decidualization on pregnancy was investigated by observing the pregnancy outcome on the 10th day of gestation. Compared with the control group, the conception rate was significantly decreased in the high-dose DEHP subacute exposure group (P<0.05). HE staining showed that, compared with the control group, the decidual stromal cells in the low- and medium-dose exposure groups were disorganized, the nuclei of the cells were irregular, the cytoplasmic staining was uneven, and the number of polymorphonuclear cells was significantly reduced. Masson staining showed that compared with the control group, the collagen fibers in the decidua region of the DEHP low-dose group and the medium-dose group were more distributed, more abundant and more disorderly. TUNEL assay showed increased apoptosis in the decidua area compared to the control group. Western blotting showed that the expression of BMP2, a marker molecule for endometrial decidualization, was significantly reduced (P<0.05 or P<0.01). The abortion rate and embryo resorption rate were increased, and the number of embryos, uterine wet weight, uterine area and placenta wet weight were decreased in DEHP low-dose group compared to the control group stimulated by mifepristone, an abortifacient drug (P<0.05 or P<0.01). Subacute exposure to DEHP leads to impaired endometrial decidualization during early pregnancy and exacerbates the risk of adverse pregnancy outcomes in mice.