Embryonic Death Research Articles

121 Unraveling the biology of bovine conceptus elongation

Abstract The establishment of pregnancy is a well-regulated process that involves the development of a viable blastocyst, conceptus elongation, maternal recognition of pregnancy, implantation, and the onset of placentation. Pregnancy loss is a major factor limiting reproductive efficiency in livestock species. In cattle, most embryonic losses occur during the first month of gestation. The genetic and epigenetic regulation of embryonic and placental development in ruminants is still poorly understood. To investigate the transcriptome profile of the bovine conceptus during elongation, Angus heifers (n = 8) were superovulated and bred using semen from a proven high-fertility Bos indicus sire. Heifers were flushed on d 14 of gestation using a standard nonsurgical flushing technique. Recovered conceptuses were individually photographed and snap-frozen for total RNA extraction and bulk mRNA sequencing. Recovered conceptuses were classified based on size into ovoid (0.2 – 5mm; n = 6), tubular (<15 mm; n = 7), or filamentous (>16 mm; n = 7). Differentially expressed genes (DEG: FDR < 0.05) were determined using DESeq2 analysis and were functionally annotated using DAVID. The most pronounced transcriptomic changes were identified between the ovoid and tubular stages, with 5,937 DEGs (2,913 upregulated and 3,024 downregulated) and affected pathways were primarily involved in ribosome assembly and rRNA processing. There were 3,966 DEGs (2,069 upregulated and 1,897 downregulated) between the ovoid and filamentous stages, and the most significant pathways were involved in protein translation, embryonic development, and cell cycle. The transition between tubular to filamentous presented 1,435 DEGs (775 upregulated and 660 downregulated) and significant pathways were involved in protein degradation, cell division, cell cycle, protein stabilization, and mRNA splicing. Our findings indicate that the greatest alterations in the transcriptome occur precisely at the initiation of elongation, suggesting that the bovine conceptus undergoes major structural changes at the outset of this process. This major shift in gene expression is presumed to be necessary to support the subsequent exponential growth phase of elongation, required for maternal recognition of pregnancy.

First report of the egg fertility of the hawksbill turtle (Eretmochelys imbricata) (Testudines: Cheloniidae) in Melaka, Malaysia

The unhatched eggs of the hawksbill turtle (Eretmochelys imbricata) are often found in the hatchery in Melaka, Malaysia, raising concerns if the eggs remain unfertilized. Examining unhatched eggs during post-emergence nest excavation could lead to eggs being mistakenly identified as unfertilized eggs due to the difficulty in differentiating between unfertilized eggs and early embryonic mortality. It is essential to determine the egg fertility of hawksbill turtles in Melaka before concluding that hatching failure is due to infertility. Egg fertility was determined using the white spot method conducted at the beginning of egg incubation on 50 clutches. Mean egg fertility was 85.9%, but the mean hatching success was significantly lower at 57.6% (mean emergence success was 56.8%). The results showed that egg fertility did not reflect hatching success and infertility was unlikely the cause of hatching failure. The huge number of undeveloped eggs may have been due to early embryonic death that went undetected at the end of incubation.

Sexual dimorphic miRNA-mediated response of bovine elongated embryos to the maternal microenvironment.

A skewed male-to-female ratio in cattle is believed to be due to the biased embryo losses during pregnancy. The changes in biochemical secretion such as miRNAs by the embryo due to altered maternal environment could cause a sex biased selective implantation resulting in a skewed male to female ratio at birth. Nevertheless, it is still not clear whether the male and female embryos could modify their miRNA expression patterns differently in response to altered physiological developmental conditions. Therefore, this study was focused on identifying sex specific miRNA expression patterns induced in the embryo during the elongation period in response to the maternal environment. For this, in vitro produced day female and male embryos were transferred to Holsteins Frisian cows and heifers. The elongated female and male embryos were then recovered at day 13 of the gestation period. Total RNA including the miRNAs was isolated from each group of elongated embryo samples were subjected to the next generation miRNA sequencing. Sequence alignment, identification and quantification of miRNAs were done using the miRDeep2 software package and differential miRNA expression analyses were performed using the edgeR bioconductor package. The recovery rate of viable elongating embryos at day 13 of the gestation period was 26.6%. In cows, 2.8 more viable elongating male embryos were recovered than female embryos, while in heifers the sex ratio of the recovered elongating embryos was close to one (1.05). The miRNA analysis showed that 254 miRNAs were detected in both male and female elongated embryos developed either in cows or heifers, of which 14 miRNAs including bta-miR-10b, bta-miR-148a, bta-miR-26a, and bta-miR-30d were highly expressed. Moreover, the expression level of 32 miRNAs including bta-let-7c, bta-let-7b, bta-let-7g, bta-let-7d and bta-let-7e was significantly different between the male and female embryos developed in cows, but the expression level of only 4 miRNAs (bta-miR-10, bta-mR-100, bta-miR-155 and bta-miR-6119-5p) was different between the male and female embryos that were developed in heifers. Furthermore, 19 miRNAs including those involved in cellular energy homeostasis pathways were differentially expressed between the male embryos developed in cows and heifers, but no significantly differentially expressed miRNAs were detected between the female embryos of cows and heifers. Thus, this study revealed that the sex ratio skewed towards males in embryos developed in cows was accompanied by increased embryonic sexual dimorphic miRNA expression divergence in embryos developed in cows compared to those developed in heifers. Moreover, male embryos are more sensitive to respond to the maternal reproductive microenvironment by modulating their miRNA expression.

Blastocyst telomere length predicts successful implantation after frozen-thawed embryo transfer.

Do embryos with longer telomere length (TL) at the blastocyst stage have a higher capacity to survive after frozen-thawed embryo transfer (FET)? Digitally estimated TL using low-pass whole genome sequencing (WGS) data from the preimplantation genetic testing for aneuploidy (PGT-A) process demonstrates that blastocyst TL is the most essential factor associated with likelihood of implantation. The lifetime TL is established in the early cleavage cycles following fertilization through a recombination-based lengthening mechanism and starts erosion beyond the blastocyst stage. In addition, a telomerase-mediated slow erosion of TL in human fetuses has been observed from a gestational age of 6-11 weeks. Finally, an abnormal shortening of telomeres is likely involved in embryo loss during early development. Blastocyst samples were obtained from patients who underwent PGT-A and FET in an IVF center from March 2015 to May 2018. Digitally estimated mitochondrial copy number (mtCN) and TL were used to study associations with the implantation potential of each embryo. In total, 965 blastocysts from 232 cycles (164 patients) were available to investigate the biological and clinical relevance of TL. A WGS-based workflow was applied to determine the ploidy of each embryo. Data from low-pass WGS-PGT-A were used to estimate the mtCN and TL for each embryo. Single-variant and multi-variant logistic regression, decision tree, and random forest models were applied to study various factors in association with the implantation potential of each embryo. Of the 965 blastocysts originally available, only 216 underwent FET. While mtCN from the transferred embryos is significantly associated with the ploidy call of each embryo, mtCN has no role in impacting IVF outcomes after an embryo transfer in these women. The results indicate that mtCN is a marker of embryo aneuploidy. On the other hand, digitally estimated TL is the most prominent univariant factor and showed a significant positive association with pregnancy outcomes (P < 0.01, odds ratio 79.1). We combined several maternal and embryo parameters to study the joint effects on successful implantation. The machine learning models, namely decision tree and random forest, were trained and yielded classification accuracy of 0.82 and 0.91, respectively. Taken together, these results support the vital role of TL in governing implantation potential, perhaps through the ability to control embryo survival after transfer. The small sample size limits our study as only 216 blastocysts were transferred. The number was further reduced to 153 blastocysts, where pregnancy outcomes could be accurately traced. The other limitation of this study is that all data were collected from a single IVF center. The uniform and controlled operation of IVF cycles in a single center may cause selection bias. We present novel findings to show that digitally estimated TL at the blastocyst stage is a predictor of pregnancy capacity after a FET cycle. As elective single-embryo transfer has become the mainstream direction in reproductive medicine, prioritizing embryos based on their implantation potential is crucial for clinical infertility treatment in order to reduce twin pregnancy rate and the time to pregnancy in an IVF center. The AI-powered, random forest prediction model established in this study thus provides a way to improve clinical practice and optimize the chances for people with fertility problems to achieve parenthood. This study was supported by a grant from the National Science and Technology Council, Taiwan (MOST 108-2321-B-006-013 -). There were no competing interests. N/A.

Arsenic disturbs neural tube closure involving AMPK/PKB-mTORC1-mediated autophagy in mice

Arsenic exposure is a significant risk factor for folate-resistant neural tube defects (NTDs), but the potential mechanism is unclear. In this study, a mouse model of arsenic-induced NTDs was established to investigate how arsenic affects early neurogenesis leading to malformations. The results showed that in utero exposure to arsenic caused a decline in the normal embryos, an elevated embryo resorption, and a higher incidence of malformed embryos. Cranial and spinal deformities were the main malformation phenotypes observed. Meanwhile, arsenic-induced NTDs were accompanied by an oxidant/antioxidant imbalance manifested by elevated levels of reactive oxygen species (ROS) and decreased antioxidant activities. In addition, changes in the expression of autophagy-related genes and proteins (ULK1, Atg5, LC3B, p62) as well as an increase in autophagosomes were observed in arsenic-induced aberrant brain vesicles. Also, the components of the upstream pathway regulating autophagy (AMPK, PKB, mTOR, Raptor) were altered accordingly after arsenic exposure. Collectively, our findings propose a mechanism for arsenic-induced NTDs involving AMPK/PKB-mTORC1-mediated autophagy. Blocking autophagic cell death due to excessive autophagy provides a novel strategy for the prevention of folate-resistant NTDs, especially for arsenic-exposed populations.

Advancements in Genetic Biomarkers and Exogenous Antioxidant Supplementation for Safeguarding Mammalian Cells against Heat-Induced Oxidative Stress and Apoptosis.

Heat stress represents a pervasive global concern with far-reaching implications for the reproductive efficiency of both animal and human populations. An extensive body of published research on heat stress effects utilizes controlled experimental environments to expose cells and tissues to heat stress and its disruptive influence on the physiological aspects of reproductive phenotypic traits, encompassing parameters such as sperm quality, sperm motility, viability, and overall competence. Beyond these immediate effects, heat stress has been linked to embryo losses, compromised oocyte development, and even infertility across diverse species. One of the primary mechanisms underlying these adverse reproductive outcomes is the elevation of reactive oxygen species (ROS) levels precipitating oxidative stress and apoptosis within mammalian reproductive cells. Oxidative stress and apoptosis are recognized as pivotal biological factors through which heat stress exerts its disruptive impact on both male and female reproductive cells. In a concerted effort to mitigate the detrimental consequences of heat stress, supplementation with antioxidants, both in natural and synthetic forms, has been explored as a potential intervention strategy. Furthermore, reproductive cells possess inherent self-protective mechanisms that come into play during episodes of heat stress, aiding in their survival. This comprehensive review delves into the multifaceted effects of heat stress on reproductive phenotypic traits and elucidates the intricate molecular mechanisms underpinning oxidative stress and apoptosis in reproductive cells, which compromise their normal function. Additionally, we provide a succinct overview of potential antioxidant interventions and highlight the genetic biomarkers within reproductive cells that possess self-protective capabilities, collectively offering promising avenues for ameliorating the negative impact of heat stress by restraining apoptosis and oxidative stress.

A metabolic signature for NADSYN1-dependent congenital NAD deficiency disorder.

Nicotinamide adenine dinucleotide (NAD) is essential for embryonic development. To date, biallelic loss-of-function variants in 3 genes encoding nonredundant enzymes of the NAD de novo synthesis pathway - KYNU, HAAO, and NADSYN1 - have been identified in humans with congenital malformations defined as congenital NAD deficiency disorder (CNDD). Here, we identified 13 further individuals with biallelic NADSYN1 variants predicted to be damaging, and phenotypes ranging from multiple severe malformations to the complete absence of malformation. Enzymatic assessment of variant deleteriousness in vitro revealed protein domain-specific perturbation, complemented by protein structure modeling in silico. We reproduced NADSYN1-dependent CNDD in mice and assessed various maternal NAD precursor supplementation strategies to prevent adverse pregnancy outcomes. While for Nadsyn1+/- mothers, any B3 vitamer was suitable to raise NAD, preventing embryo loss and malformation, Nadsyn1-/- mothers required supplementation with amidated NAD precursors (nicotinamide or nicotinamide mononucleotide) bypassing their metabolic block. The circulatory NAD metabolome in mice and humans before and after NAD precursor supplementation revealed a consistent metabolic signature with utility for patient identification. Our data collectively improve clinical diagnostics of NADSYN1-dependent CNDD, provide guidance for the therapeutic prevention of CNDD, and suggest an ongoing need to maintain NAD levels via amidated NAD precursor supplementation after birth.

Success rates and factors influencing pregnancy outcome after 464 transvaginal ultrasound-guided twin reductions in the mare.

Transvaginal ultrasound-guided aspiration (TUA) is used for post-fixation twin reduction in mares. However, there is limited information regarding factors that influence pregnancy outcome after TUA. To evaluate the effect of day of gestation on which TUA is performed, aspiration volume, puncture of the conceptus, medication administered before and after TUA, embryo location, mare age and parity and operator experience on pregnancy and foaling rates after TUA. Retrospective case series. Data were collected from case records of 464 TUAs performed by 14 operators in 422 mares diagnosed pregnant with dizygotic twins in two different facilities between 2010 and 2019. Pregnancy status was determined by ultrasonography at 5-7 days and 3-4 weeks after the TUA was performed. Subsequent pregnancy and foaling results were obtained by follow-up communication. The effects of mare, gestation- and TUA-related variables on pregnancy and foaling rates were analysed by the chi-square-test for homogeneity and Fisher's exact test and logistic regression. TUA was performed between 21 and 82 days of gestation in unilaterally (267/359 [74.4%]) and bilaterally fixed (92/359 [25.6%]) twin pregnancies. A singleton pregnancy (218/381 [57.2%]), persistent twin pregnancy (60/381 [15.8%]), or the loss of both conceptuses (103/381 [27%]) was confirmed 5-7 days after TUA was performed. At 3-4 weeks post TUA 50.3% (163/324) of mares were diagnosed with a single viable pregnancy and 40.1% (127/317) went on to deliver a live single foal. TUA performed early in gestation (D 25-35) resulted in the birth of a live singleton foal in 49.3% (74/150) of mares. Missing retrospective data despite extensive follow-up. This is the first large scale study to demonstrate that acceptable pregnancy and foaling rates can be achieved in mares diagnosed with twins when TUA is performed early in gestation (<40 days).

MUC1 regulation in the left and right uterine horns and conceptus trophectoderm during the peri-implantation period of dromedary camel

Pregnancy maintenance in dromedary camels poses significant challenges, including early embryonic loss in the left uterine horn (LH) and unsuccessful pregnancy in the right uterine horn (RH), suggesting a potential asynchrony between conceptus signaling and uterine receptivity. The transition of the uterine epithelium from a pre-receptive to a receptive state requires a delicate balance of adhesion-promoting and anti-adhesion molecules. Mucin-1 (MUC1) acts as an anti-adhesive molecule on the uterine luminal (LE) and glandular (GE) epithelium. Downregulation of MUC1 is believed to be crucial for successful embryo attachment in various mammals. This study aimed to investigate the temporospatial expression of MUC1 in the LH and RH on Days 8, 10, and 12 pregnant dromedaries and their conceptuses. Quantitative real-time polymerase chain reaction (qrt-PCR), Western blot analysis, immunohistochemistry, and immunofluorescence techniques were employed to assess MUC1 expression at the mRNA and protein levels. The results demonstrated a reduction in MUC1 mRNA expression on Day 8, then increased on Day 10, followed by a decrease on Day 12 in LH. While the RH exhibited progressive increases, peaking on Day 12. However, MUC1 expression constantly exhibited higher levels in RH than in LH in all days. Two bands were detected at 150-kDa and 180-kDa, with the highest intensity observed on Day 10. Spatially, MUC1 was localized in the apical, cytoplasmic, and lumen of uterine glands only. MUC1 was barely detectable on Day 8 but gradually increased on Days 10 and 12 in both horns. Likewise, the RH exhibited higher MUC1 signals than the LH on Days 10 and 12. In the conceptuses, MUC1 mRNA increased on Day 8, peaked on Day 10, and declined on Day 12. Notably, MUC1 protein was detected in both the trophectoderm and endoderm, with high expression observed on Day 10 and reduced by Day 12. In conclusion, the decrease in MUC1 expression on Day 8 in the LH may be associated with maternal recognition of pregnancy (MRP), and the increase on Day 10 may related to embryo protection and movement, while the subsequent decrease on Day 12 could be linked to the embryo attachment and preparation for the implantation. Conversely, the increase of MUC1 in the RH implies a role in the anti-adhesion mechanism. These findings contribute to understanding MUC1's involvement in reproductive processes and provide insights into the complex mechanisms underlying successful pregnancy establishment and maintenance in dromedary camels.

Chromosome Division in Early Embryos-Is Everything under Control? And Is the Cell Size Important?

Chromosome segregation in female germ cells and early embryonic blastomeres is known to be highly prone to errors. The resulting aneuploidy is therefore the most frequent cause of termination of early development and embryo loss in mammals. And in specific cases, when the aneuploidy is actually compatible with embryonic and fetal development, it leads to severe developmental disorders. The main surveillance mechanism, which is essential for the fidelity of chromosome segregation, is the Spindle Assembly Checkpoint (SAC). And although all eukaryotic cells carry genes required for SAC, it is not clear whether this pathway is active in all cell types, including blastomeres of early embryos. In this review, we will summarize and discuss the recent progress in our understanding of the mechanisms controlling chromosome segregation and how they might work in embryos and mammalian embryos in particular. Our conclusion from the current literature is that the early mammalian embryos show limited capabilities to react to chromosome segregation defects, which might, at least partially, explain the widespread problem of aneuploidy during the early development in mammals.

Bacterial community and genome analysis of cytoplasmic incompatibility-inducing Wolbachia in American serpentine leafminer, Liriomyza trifolii.

Liriomyza trifolii, an agricultural pest, is occasionally infected by Wolbachia. A Wolbachia strain present in Liriomyza trifolii is associated with cytoplasmic incompatibility (CI) effects, leading to the death of embryos resulting from incompatible crosses between antibiotic-treated or naturally Wolbachia-free strain females and Wolbachia-infected males. In this study, high-throughput sequencing of hypervariable rRNA genes was employed to characterize the bacterial community in Wolbachia-infected L. trifolii without antibiotic treatment. The analysis revealed that Wolbachia dominates the bacterial community in L. trifolii, with minor presence of Acinetobacter, Pseudomonas, and Limnobacter. To elucidate the genetic basis of the CI phenotype, metagenomic sequencing was also conducted to assemble the genome of the Wolbachia strain. The draft-genome of the Wolbachia strain wLtri was 1.35 Mbp with 34% GC content and contained 1,487 predicted genes. Notably, within the wLtri genome, there are three distinct types of cytoplasmic incompatibility factor (cif) genes: Type I, Type III, and Type V cifA;B. These genes are likely responsible for inducing the strong cytoplasmic incompatibility observed in L. trifolii.

Transcriptomic analysis of bovine endometrial epithelial cells in response to interferon tau and hormone stimulation.

The embryonic loss during early stage of gestation is one of the major causes of infertility for domestic ruminants, causing huge economic losses to pasture. Maternal recognition of pregnancy and implantation are the crucial process for determining the successful establishment and development of pregnancy in cattle. The research on molecular mechanisms of pregnancy recognition will facilitate illustrating the complex process of pregnancy establishment and help to improve pregnancy outcomes. In this study, we performed transcriptomic analysis of primary bovine endometrial epithelial cells (BEND) with or without IFNT and hormones intervention through RNA sequencing. We eventually identified 608 differentially expressed genes (DEGs) including 409 up-regulated genes and 199 down-regulated genes in IFNT and hormones-treated group compared with control group. Gene Ontology (GO) enrichment analysis demonstrated that the majority of DEGs were implicated in immune system process, response to external stimulus, response to cytokine, regulation of response to stress. Results from KEGG analysis showed a significant enrichment of NOD-like receptor signaling pathway, antigen processing and presentation, necroptosis, oxidative phosphorylation, RIG-I-like receptor signaling pathway. Additionally, a set of promising candidate genes, including (USP18, STAT1, PSMB8, IFIH1, MX2, IFI44, DHX58, CASP8, DRAM1, CXCR4), were characterized by constructing an integrated interaction network. Specifically, the mRNA expression of HOXA11, PTGS1 and PTGS2 were remarkably suppressed by silencing DRAM1 under IFNT and hormone administration, thus speculating that DRAM1 might play a crucial role in early pregnancy by regulating endometrial function. The results of this study depicted a relatively comprehensive transcriptional profiles of BEND in response to IFNT and hormones, which contributes to a better understanding of gene interaction network and underlying regulatory mechanisms in endometrium of ruminants during early pregnancy.

Effect of Footrot on embryo losses in Polwarth sheep

Footrot is caused by the association of Dichelobacter nodosus and Fusobacterium necrophorum. The disease is characterised by severe lameness associated with initial inflammation at the coronary band, followed by progressive separation of the hoof capsule from the underlying tissue. This determines uneasiness in the animal and loss of body condition. Embryo losses are affected by several factors that interact with each other. Among them, the body condition of the sheep is an important factor. Therefore, this study aimed to evaluate the embryo losses associated with footrot in Polwarth sheep. In autumn, 130 ewes were synchronized with intravaginal sponges for 14 days. According to the footrot score diagnosed, three groups were made: Score 0) Healthy ;1) with minor injuries and 3) with more significant injuries without becoming serious (Scores 4 and 5). Embryo loss (EM) was measured from 18 days post-service to day 50, followed up every 5 days, by transrectal ultrasonography. Subsequently, the presence, number, and size of embryos or foetuses were recorded, as well as the presence of dead embryos or foetuses. The loss of body condition status was determined to have higher embryonic losses in group 3 (72,0%) than in the others (&lt; 25,0).

Removal of pomt1 in zebrafish leads to loss of α-dystroglycan glycosylation and dystroglycanopathy phenotypes.

Biallelic mutations in Protein O-mannosyltransferase 1 (POMT1) are among the most common causes of a severe group of congenital muscular dystrophies (CMDs) known as dystroglycanopathies. POMT1 is a glycosyltransferase responsible for the attachment of a functional glycan mediating interactions between the transmembrane glycoprotein dystroglycan and its binding partners in the extracellular matrix (ECM). Disruptions in these cell-ECM interactions lead to multiple developmental defects causing brain and eye malformations in addition to CMD. Removing Pomt1 in the mouse leads to early embryonic death due to the essential role of dystroglycan during placental formation in rodents. Here, we characterized and validated a model of pomt1 loss of function in the zebrafish showing that developmental defects found in individuals affected by dystroglycanopathies can be recapitulated in the fish. We also discovered that pomt1 mRNA provided by the mother in the oocyte supports dystroglycan glycosylation during the first few weeks of development. Muscle disease, retinal synapse formation deficits, and axon guidance defects can only be uncovered during the first week post fertilization by generating knock-out embryos from knock-out mothers. Conversely, maternal pomt1 from heterozygous mothers was sufficient to sustain muscle, eye, and brain development only leading to loss of photoreceptor synapses at 30days post fertilization. Our findings show that it is important to define the contribution of maternal mRNA while developing zebrafish models of dystroglycanopathies and that offspring generated from heterozygous and knock-out mothers can be used to differentiate the role of dystroglycan glycosylation in tissue formation and maintenance.

Changes in Immune Response during Pig Gestation with a Focus on Cytokines.

Pigs have the highest percentage of embryonic death not associated with specific diseases of all livestock species, at 20-45%. During gestation processes, a series of complex alterations can arise, including embryonic migration and elongation, maternal immunological recognition of pregnancy, and embryonic competition for implantation sites and subsequent nutrition requirements and development. Immune cells and cytokines act as mediators between other molecules in highly complex interactions between various cell types. However, other non-immune cells, such as trophoblast cells, are important in immune pregnancy regulation. Numerous studies have shed light on the crucial roles of several cytokines that regulate the inflammatory processes that characterize the interface between the fetus and the mother throughout normal porcine gestation, but most of these reports are limited to the implantational and peri-implantational periods. Increase in some proinflammatory cytokines have been found in other gestational periods, such as placental remodeling. Porcine immune changes during delivery have not been studied as deeply as in other species. This review details some of the immune system cells actively involved in the fetomaternal interface during porcine gestation, as well as the principal cells, cytokines, and molecules, such as antibodies, that play crucial roles in sow pregnancy, both in early and mid-to-late gestation.

Bovine Genital Leptospirosis: An Update of This Important Reproductive Disease.

Bovine leptospirosis is an important disease that affects the reproductive sphere. Due to its high relevance for the bovine production chain in a worldwide scenario, a better understanding of the disease is crucial to reduce its negative impacts. The main agents are strains from the Sejroe serogroup, such as Hardjo and Guaricura, which lead to renal and genital infection. The genital colonization causes a chronic, silent, and subclinical reproductive syndrome, called Bovine Genital Leptospirosis (BGL). Embryonic death, estrus repetition, subfertility, and abortions are the main signs of BGL condition in females. However, although leptospires have been identified in semen, the manifestation of BGL in bulls remains to be clarified. The recommended diagnosis of BGL includes a serologic screening of the herds using the microscopic agglutination test followed by PCR of genital samples (cervicovaginal mucus, uterine fragment, or semen), especially from animals with reproductive failures. After the identification of carriers, control is carried out considering three steps: antimicrobial treatment of the carriers, environmental and reproductive management, and herd vaccination. Systematic testing, quarantine of newly arrived animals, and usage of antimicrobials in semen diluents or embryo culture media are other sanitary approaches that are encouraged to improve the control of the syndrome. Herein we discuss protocols for an efficient diagnosis and preventive procedures of BGL, which are fundamental to reducing the negative impact of the disease on cattle reproduction and its consequent economic hazards.

Liver and kidney damage in quail embryos hatched from eggs sprayed once with chlorpyrifos during artificial incubation.

Birds have enough conditions to be used as bioindicators for the presence of environmental contaminants. Notably, chlorpyrifos (CPF) remains extensively employed in Argentina, particularly in fruit plantations and livestock settings This study aimed to assess the potential impacts of CPF on common quail (Coturnix coturnix) embryos through external eggshell application during key embryonic stages (days 1, 4, and 14). Concentrations equivalent to those used in fruit applications, 5× and 10 × (38.4, 192, and 384µg/egg), were employed. A 48% commercial formulation of CPF (Pirfos - Icona®) was utilized. An increase in embryonic deaths, as well as a statistical association between the degree of liver or kidney lesions and CPF concentrations was observed. The results suggest that CPF can induce embryotoxic effects with a single exposure to quail embryos and compromise the viability of the organisms. The study suggests a potential exposure risk for embryos through contact with the insecticide on the eggshell's exterior.

Decidual stromal cells-derived exosomes incurred insufficient migration and invasion of trophoblast by disturbing of β-TrCP-mediated snail ubiquitination and degradation in unexplained recurrent spontaneous abortion

BackgroundExosomes released from decidual stromal cells (DSC-exos) play a crucial role in facilitating the epithelial–mesenchymal transition (EMT) of trophoblasts and insufficient trophoblasts EMT are associated with URSA (unexplained recurrent spontaneous abortion). However, the mechanisms underlying DSC-exos inducing EMT is not completely understood.MethodsDSC-exos of normal pregnant women (N-DSC-exos) and URSA patients (URSA-DSC-exos) were extracted and characterized. Characterization of the isolated DSC-exos was performed using with TEM (transmission electron microscopy), NTA (nanoparticle tracking analysis), and WB (western blot) techniques. Subsequently, these DSC-exos were co-cultured with trophoblasts cell lines (HTR-8/SVneo). The influence of both N-DSC-exos and URSA-DSC-exos on trophoblasts proliferation, invasion and migration, as well as on the expression of EMT-related proteins, was evaluated through a series of assays including CCK8 assays, wound healing assays, transwell assays, and western blot, respectively. Then rescue experiments were performed by β-TrCP knockdown or β-TrCP overexpressing trophoblasts with snail-siRNA transfection or β-TrCP overexpressing Lentivirus infection, respectively. Finally, animal experiments were employed to explore the effect of N-DSC-exos on embryo absorption in mice.ResultsWe found increased β-TrCP expression in the villus of URSA patients when compared to the normal pregnant women, alongside reduction in the levels of both snail and N-cadherin within URSA patients. N-DSC-exos can promote the EMT of the trophoblast by inhibiting β-TrCP-mediated ubiquitination and degradation of transcription factor snail. Moreover the capacity to promote EMT was found to be more potent in N-DSC-exos than URSA-DSC-exos. Down-regulation of snail or overexpression of β-TrCP can reverse the effects of N-DSC-exos on trophoblast. Finally, in vivo experiment suggested that N-DSC-exos significantly reduced the embryo resorption rate of spontaneous abortion mouse model.ConclusionsOur findings indicate that URSA-DSC-exos caused insufficient migration and invasion of trophoblast because of disturbing of β-TrCP-mediated ubiquitination and degradation of EMT transcription factor snail. Elucidating the underlying mechanism of this dysregulation may shed light on the novel pathways through which DSC-exos influence trophoblast function, thereby contributing to our understanding of their role in URSA.

Arabic Chicken from Marriage with Artificial Insemination (IB) Using a Manual Hatching Machine On Fertility And Hatchability

Arab Chicken Marriage with Artificial Insemination (IB) Using Manual Incubator on Fertility and Hatchability. This study aims to determine the level of fertility and hatchability of Arabic chicken mating with artificial insemination in a manual incubator. This research was carried out at Mr. Saiful Karang Rejo's farm, Tulung Agung, from September 15 to October 15, 2022. The number of eggs used were 72 eggs from Arab chicken mating with a male to female ratio of 50: 500. The chickens are housed individually, the feed given is a mixture of corn, bran, and concentrate. IB is carried out 3 times a week, semen collection is carried out by massage (sequence) and accommodated in a glass then IB is carried out on Arab females. Using Chi Square analysis, then followed by a contingency test (a condition filled with uncertainty between fertile or not, the effect of fertility expectations) , Correlation (the relationship between two variables that are quantitative) and Regression (influence between 2 or more variables). The parameters observed included fertility and egg hatchability. From the results of the study, the following conclusions were obtained: the highest percentage of fertility was obtained in stages 1 and 2 with a value of 73.61%, while the lowest percentage was at stage 3 with a value of 66.66%. Meanwhile, the highest hatchability percentage was at stage 1 with a value of 83.01%, and the lowest percentage was at stage 3 with a value of 79.16%. Cleanliness in the chicken semen reservoir and egg selection, such as the cleanliness of egg shells, eggs that are too small or too large need to be considered. The temperature and humidity of the incubator must be constant to avoid embryo death and hatching failure.

Uncovering Phenotypes in Mutant Mice by Determining Embryo, Organ, Tissue, and Cell Developmental Potential.

The death of an embryo during gestation does not necessarily preclude the study of the mutant embryo or the developmental potential of its individual cells, tissues, or organs. Whole-embryo in vitro culture prior to the time of death will allow real-time observation of living embryos and direct comparisons with controls. Organ anlage can be removed from embryos and cultured in vitro beyond the time of death of the whole embryo. In both whole embryos and organ anlage culture, fluorescent protein reporters may be used productively to follow cell types or specific gene expression changes. Some cells, such as hematopoietic cells, and organ anlage, may be suitable for transplantation to wild-type hosts for further analysis of their potential. Additionally, cell lines, including embryonic stem (ES) cells, trophoblast stem (TS) cells, extraembryonic endoderm (XEN) stem cells, and epiblast-derived stem cells (EpiSC), can be derived from mutant embryos to reveal the potential of the mutant cells outside the context of the whole organism. Mutant stem cells or even whole mutant embryos can be used to test potential in chimeras or in teratomas.