In Vitro Regeneration of Tea (Camellia sinensis (L). O. Kuntze) By Somatic Embryogenesis from Immature Cotyledon Tissues

Tea (Camellia sinensis) is the world's most popular beverage plant, as well as an important plantation crop with high commercial value. It has been maintained for centuries through conventional vegetative propagation. Tea clonal propagation in vitro has the advantage of producing a large number of elite plants. If an efficient in vitro regeneration technology is available, this technique could be exploited for selection of tea plants for desired trait. The selected plants could be later on multiplied through in vitro or ex vitro techniques. The study aimed to induced somatic embryogenesis from immature embryo explants to genetic variaton. Different concentrations of phenylboronic acid with benzyladenine and phenylboronic acid with kinetin were tested in MS medium with 30 g/L sucrose and 8 g/L agar. MS medium without any plant growth regulators was used as control group. Considering the embryo survival rate, 1.5 mg/ L-1 phenylboronic acid + 1 mg/ L-1 kinetin produced highest result as 87.3% while lowest was in control group as 36.7%. The highest plant regeneration rate was found in 1,5 mg/ L-1 phenylboronic acid + 1 mg/ L-1 kinetin and 1.5 mg/ L-1 phenylboronic acid + 1 mg/ L-1 benzyladenine medium respectively as 58.3% and 55.6%. Kinetin treatment with increasing phenylboronic acid concentrations gave the best results in terms of somatic embryo survival rate. Also, kinetin treatment produced better results when compared to benzyladenine concentrations.

Effects of Vitrified Cryopreservation Duration on the IVF and Neonatal Outcomes

Objective: To evaluate the impact of cryopreservation storage duration on embryo viability, implantation competence, pregnancy outcome and neonatal outcomes.Design: Retrospective study.Setting: Center for Reproductive Medicine，The Third Affiliated Hospital of Guangzhou Medical University.Patient(s): In vitro fertilization patients who had vitrified cryopreserved embryos and following the first frozen embryo transfer cycles from January 2004 to August 2019. A total of 31143 patients met the inclusion criteria and were grouped according to the storage time (20926 patients in Group 1 with storage time <3 months, 6472 patients in Group 2 with storage time between 3 and 6 months, 2237 patients in Group 3 with storage time between 6 and 12 months and 746 in Group 4 with storage time between 12 and 24 months, 762 patients in Group 5 with storage time >24 months).Intervention(s): None.Main Outcome Measure(s): In the total FET cycles, the embryo survival rate was decreased significantly with the increase of cryopreservation time, and the highest rate was 98. 63 % in the 1-3 months group, and the lowest was 71.13% in the >=731 days group (P <0. 01). The HCG positive rate (57.85%) and clinical pregnancy rate (55. 26%) in the 1-3 months group were the highest (P<0. 01). The >=731 group had the lowest sex ratio of 0.96. There were no significant differences in neonatal birth weight, neonatal height and congenital anomalies among the groups (P>0. 05).Result(s): Length of storage time had a significant effect on post-thaw survival and outcomes for IVF cycles. Conclusion(s): With the prolongation of cryopreservation time, the embryonic survival rate and pregnancy rate were decreased significantly. Short-term cryopreservation (<=3 months) can obtain higher clinical pregnancy rate. Therefore, although long-term hryopreservation of the embryo has no effect on the health of the new baby, but hryopreserved embryos should be recovery as soon as possible if condition allows.

Effects of In-Channel Structure on Chinook Salmon Spawning Habitat and Embryo Production

Adult salmonids are frequently observed building redds adjacent to in-channel structure, including boulders and large woody debris. These areas are thought to be preferentially selected for a variety of reasons, including energy and/or predation refugia for spawners, and increased hyporheic exchange for incubating embryos. This research sought to quantify in-channel structure effects on local hydraulics and hyporheic flow and provide a mechanistic link between these changes and the survival, development, and growth of Chinook salmon Oncorhynchus tshawytscha embryos. Data were collected in an eight-kilometer reach, on the regulated lower Mokelumne River, in the California Central Valley. Nine paired sites, consisting of an area containing in-channel structure paired with an adjacent area lacking in-channel structure, were evaluated. Results indicated that in-channel structure disrupts surface water velocity patterns, creating pressure differences that significantly increase vertical hydraulic gradients within the subsurface. Overall, in-channel structure did not significantly increase survival, development, and growth of Chinook salmon embryos. However, at several low gradient downstream sites containing in-channel structure, embryo survival, development, and growth were significantly higher relative to paired sites lacking such features. Preliminary data indicate that adding or maintaining in-channel structure, including woody material, in suboptimal spawning reaches improves the incubation environment for salmonid embryos in regulated reaches of a lowland stream. More research examining temporal variation and a full range of incubation depths is needed to further assess these findings.

Roles of WNT6 in Sheep Endometrial Epithelial Cell Cycle Progression and Uterine Glands Organogenesis

The uterus, as part of the female reproductive tract, is essential for embryo survival and in the maintenance of multiple pregnancies in domestic animals. This study was conducted to investigate the effects of WNT6 on Hu sheep endometrial epithelial cells (EECs) and uterine glands (UGs) in Hu sheep, with high prolificacy rates. In the present study, Hu sheep with different fecundity, over three consecutive pregnancies, were divided into two groups: high prolificacy rate group (HP, litter size = 3) and low prolificacy rate group (LP, litter size = 1). A comparative analysis of the endometrial morphology was performed by immunofluorescence. RNA-seq was used to analyze the gene’s expression in endometrium of HP and LP Hu sheep, providing a candidate gene, which was investigated in EECs and organoid culture. Firstly, higher density of UGs was found in the HP Hu sheep groups (p < 0.05). The RNA-seq data revealed the importance of the WNT signaling pathway and WNT6 gene in Hu sheep endometrium. Functionally, WNT6 could promote the cell cycle progression of EECs via WNT/β-catenin signal and enhance UGs organogenesis. Taken together, WNT6 is a crucial regulator for sheep endometrial development; this finding may offer a new insight into understanding the regulatory mechanism of sheep prolificacy.

Vitrification Effects on the Transcriptome of in vivo-Derived Porcine Morulae

Despite the reported promising farrowing rates after non-surgical and surgical transfers of vitrified porcine morulae and blastocysts produced in vivo (range: 70–75%), the pregnancy loss is 5–15 fold higher with vitrified than with fresh embryos. The present study aimed to investigate whether vitrification affects the transcriptome of porcine morulae, using microarrays and RT-qPCR validation. Morulae were obtained surgically from weaned sows (n = 13) on day 6 (day 0 = estrus onset). A total of 60 morulae were vitrified (treatment group). After 1 week of storage, the vitrified morulae were warmed. Vitrified-warmed and non-vitrified fresh morulae (control; n = 40) were cultured for 24 h to assess embryo survival by stereomicroscopy after. A total of 30 vitrified/warmed embryos that were deemed viable and 30 fresh control embryos (three pools of 10 for each experimental group) were selected for microarray analysis. Gene expression was assessed with a GeneChip® Porcine Genome Array (Affymetrix). An ANOVA analysis p-unadjusted &lt;0.05 and a fold change cut-off of ±1.5 were set to identify differentially expressed genes (DEGs). Data analysis and biological interpretation were performed using the Partek Genomic Suite 7.0 software. The survival rate of morulae after vitrification and warming (92.0 ± 8.3%) was similar to that of the control (100%). A total of 233 DEGs were identified in vitrified morulae (38 upregulated and 195 downregulated), compared to the control group. Nine pathways were significantly modified. Go-enrichment analysis revealed that DEGs were mainly related to the Biological Process functional group. Up-regulated DEGs were involved in glycosaminoglycan degradation, metabolic pathways and tryptophan metabolism KEGG pathways. The pathways related to the down-regulated DEGs were glycolysis/gluconeogenesis, protein export and fatty acid elongation. The disruption of metabolic pathways in morulae could be related to impaired embryo quality and developmental potential, despite the relatively high survival rates after warming observed in vitro. In conclusion, vitrification altered the gene expression pattern of porcine morulae produced in vivo, generating alterations in the transcriptome that may interfere with subsequent embryo development and pregnancy after embryo transfer.

Direct and Latent Effects of Pathogen Exposure Across Native and Invasive Amphibian Life Stages

Emerging infectious diseases are one of the multiple factors contributing to the current “biodiversity crisis”. As part of the worldwide biodiversity crisis, amphibian populations are declining globally. Chytridiomycosis, an emerging infectious disease, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is a major cause of amphibian population declines. This fungus primarily affects keratinized structures in larval, juvenile, and adult amphibians as well as heart function. However, we know little about how Bd can impact embryos as well as potential latent effects of Bd exposure over ontogeny. Using two different Bd strains and multiple exposure times, we examined the effects of Bd exposure in Pacific chorus frog (Pseudacris regilla), Western toad (Anaxyrus boreas) and American bullfrog (Lithobates catesbeianus) life stages. Using a factorial experimental design, embryos of these three species were exposed to Bd at early and late embryonic stages, with some individuals re-exposed after hatching. Embryonic Bd exposure resulted in differential survival as a function of host species, Bd strain and timing of exposure. P. regilla experienced embryonic mortality when exposed during later developmental stages to one Bd strain. There were no differences across the treatments in embryonic mortality of A. boreas and embryonic mortality of L. catesbeianus occurred in all Bd exposure treatments. We detected latent effects in A. boreas and L. catesbeianus larvae, as mortality increased when individuals had been exposed to any of the Bd strains during the embryonic stage. We also detected direct effects on larval mortality in all three anuran species as a function of Bd strain, and when individuals were double exposed (late in the embryonic stage and again as larvae). Our results suggest that exposure to Bd can directly affect embryo survival and has direct and latent effects on larvae survival of both native and invasive species. However, these impacts were highly context dependent, with timing of exposure and Bd strain influencing the severity of the effects.

In ovo inhibition of avian pox virus replication by mangosteen rind and red ginger ethanolic extracts

Background and Aim: Avian pox is a contagious disease caused by the avian pox virus (APV). Mangostin and γ-mangostin in mangosteen rind (MR) and gingerol in red ginger (RG) exhibit antiviral activity. In this study, we evaluated the effect of MR and RG ethanolic extracts on APV based on pock lesions on the chorioallantoic membrane (CAM) of specific pathogen-free (SPF) embryonated chicken eggs (ECEs). Materials and Methods: Three APVs from chicken isolates (C1, C2, and C3), one APV from a pigeon isolate (P), 1.5% and 3% MR ethanolic extract, 5% and 10% RG ethanolic extract, and a combination of 1.5% MR and 5% RG at 0.1 mL/ egg were inoculated in ovo (7th day incubation, chorioallantoic route) in SPF ECEs. A control group inoculated in ovo with APV alone was also established. Each treatment consisted of three replicates. Parameters including embryo survival, CAM lesions, and average number of pock lesions were determined. Results: In ovo inoculation of MR and RG ethanolic extracts was not harmful to the ECEs and did not induce CAM lesions. The average number of pock lesions in the control group (C1, C2, C3, and P) was 35, 14, 10, and 17, respectively, whereas in all treatment groups, the number was 0, except in the 5% RG group of C1, which had a value of 10. Conclusion: In ovo inoculation of 1.5% and 3% MR, 5% and 10% RG, and the combination of 1.5% MR plus 5% RG ethanolic extract s at 0.1 mL/egg inhibit APV by reducing the number of pock lesions on the CAM of the ECE.

Alterations of miR-16, miR-let-7a and their target genes expression in human blastocysts following vitrification and re-vitrification

Embryo cryopreservation is a widely used technique in infertility management and today is an essential part of assisted reproductive technology (ART). In some cases, re-vitrification can be applied to good quality supernumerary warmed embryos that have not been transferred in the present cycle. However, there is no study about re-vitrification impact on microRNA and gene expression in human embryos. The purpose of this study is to evaluate miR-16, miR-let7a and target genes expression in in vitro produced human blastocysts following re-vitrification.Day3 embryos obtained from ICSI cycles of fertile couples referring for family balancing program were biopsied and cultured individually. On the fourth day (post-ICSI) male ones (choices of their parents) were transferred and the females (good quality embryos) were donated for research. Donated embryos were cultured to blastocyst stage and assigned to three groups: fresh, vitrified and re-vitrification. Embryos were vitrified on Cryotech carriers. Then blastocysts of three groups were individually assessed for expression of miR-16, miR-let7a and target genes.The results showed that re-vitrification of human blastocysts did not affect the ability to re-expand in culture. In addition, significant decrease was observed in miR-16 and miR-let7a expression in re-vitrified group compared to fresh (p < 0.05). A significant upregulation of the target genes ITGβ3 and BCL-2 in re-vitrified and vitrified embryos was observed compared to the fresh group (p < 0.05). The expression of BAX as a pro-apoptotic gene showed a significant decrease in re-vitrification group comparing with the fresh one (P < 0.05).The results of this research indicated that re-vitrification of embryos changes the expression of miR-16, miR-let-7a and their target genes. These alterations include increased expression of BCl-2 and ITGβ3 genes which play important roles in embryo survival and implantation, respectively. Clinical proof of these effects requires further research.

Enhancers of host immune tolerance to bacterial infection discovered using linked computational and experimental approaches

Current therapeutic strategies against bacterial infections focus on reduction of pathogen load through antibiotics; however, stimulation of host tolerance to infection might offer an alternative approach. Here we used computational transcriptomics and a Xenopus embryo infection model to rapidly discover infection response pathways, identify potential tolerance inducer drugs, and validate their ability to induce broad tolerance. Xenopus embryos exhibit natural tolerance to A. baumanii, K. pneumoniae, S. aureus, and S. pneumoniae bacteria, whereas A. hydrophila and P. aeruginosa produce infection that leads to death. Transcriptional profiling led to definition of a 20-gene signature that allows for discrimination between tolerant and susceptible states, as well as identification of active and passive tolerance responses based on the degree of engagement of gene transcription modulation. Upregulation of metal ion transport and hypoxia pathways reminiscent of responses observed in primate and mouse infection models were identified as tolerance mediators, and drug screening in the susceptible A. hydrophila infection model confirmed that a metal chelator (deferoxamine) and HIF-1α agonist (1,4-DPCA) increase embryo survival despite high pathogen load. These data demonstrate the value of combining the Xenopus embryo infection model with multi-omics analyses for mechanistic discovery and drug repurposing to induce host tolerance to bacterial infection.