Early Stage Embryos Research Articles

Polyethylene glycol induced somatic embryogenesis and plant regeneration from cotyledons of Vigna radiata (L.) Wilczek

• Developed a high-frequency regeneration protocol via Polyethylene glycol (PEG)–induced somatic embryogenesis. • 3-day old cotyledonary explants used for the development of early-stage- somatic embryos. • Incorporation of ABA and proline was effective in the maturation of somatic embryos. • Regenerated plants are phenotypically identical to the mother plants. • Genetic fidelity analysis of in vitro regenerated Vigna radiata plants was reported newly. The results show that the in vitro regenerated plants are true-to-type with no somaclonal variation. • This protocol could be useful for genetic transformation studies as well as large-scale production strategies of true-to-type plants without any somoclonal variation. An efficient plantlet regeneration protocol of green gram [ Vigna radiata (L.) Wilczek] via somatic embryogenesis (SE) using Polyethylene glycol (PEG) is reported in this study. Embryogenic calli were induced using 3-day-old cotyledonary explants on MS medium supplemented with picloram (5 µM). Early-stage somatic embryos were generated by treating these 3-day-old cotyledonary explants or calli derived from those explants with osmolytes such as, sorbitol, mannitol and PEG, among which PEG was found to be the ideal. Early-stage embryoids were subsequently developed into torpedo and cotyledonary stage embryoids in liquid cultures supplemented with abscisic acid (5 μM), proline (130 μM). Furthermore, plantlet conversion was achieved in MS medium with 6-Benzylaminopurine (BAP) 1 μM + gibberellic acid (GA 3 ) 12 μM with a ca . 35% regeneration frequency. Plantlets were established under field conditions after the confirmation of their survival. The survival rate of plantlets post-transplantation was ca. 99%. The field-grown plants were confirmed to be phenotypically identical to the mother plant. According to RAPD profiles, in vitro regenerated plants were monomorphic and similar to the control/mother plants. The regeneration protocol developed in this study could be efficiently utilized for genetic transformation studies and large-scale production strategies of true-to-type plants without any somaclonal variation.

ROLE OF EMBRYO IGF2 IN THE ETIOLOGY OF THE LONG-TERM CONSEQUENCES OF ART IN OFFSPRING

A higher prevalence of myocardial dysfunction, blood hypertension, asthma, allergies, autism spectrum disorder, hypospadias, cryptorchidism, and cancer has been described in children conceived with assisted reproductive technology (ART) compared to those conceived spontaneously. The reasons are still unclear. Hypermethylation of the insulin-like growth factor 2/H19 differently methylated region (IGF2-H19 DMR) occurs in spermatozoa of infertile patients and can be transmitted on to offspring. In turn, this can lead to lower IGF2 expression in early-stage embryos.

DNA Damage Induction Alters the Expression of Ubiquitin and SUMO Regulators in Preimplantation Stage Pig Embryos.

DNA damage in early-stage embryos impacts development and is a risk factor for segregation of altered genomes. DNA damage response (DDR) encompasses a sophisticated network of proteins involved in sensing, signaling, and repairing damage. DDR is regulated by reversible post-translational modifications including acetylation, methylation, phosphorylation, ubiquitylation, and SUMOylation. While important regulators of these processes have been characterized in somatic cells, their roles in early-stage embryos remain broadly unknown. The objective of this study was to explore how ubiquitylation and SUMOylation are involved in the regulation of early development in porcine embryos by assessing the mRNA profile of genes encoding ubiquitination (UBs), deubiquitination (DUBs), SUMOylation (SUMOs) or deSUMOylation (deSUMOs) enzymes in oocyte and embryos at different stages of development, and to evaluate if the induction of DNA damage at different stages of embryo development would alter the mRNA abundance of these genes. Pig embryos were produced by in vitro fertilization and DNA damage was induced by ultraviolet (UV) light exposure for 10 s on days 2, 4 or 7 of development. The relative mRNA abundance of most UBs, DUBs, SUMOs, and deSUMOs was higher in oocytes and early-stage embryos than in blastocysts. Transcript levels for UBs (RNF20, RNF40, RNF114, RNF169, CUL5, DCAF2, DECAF13, and DDB1), DUBs (USP16), and SUMOs (CBX4, UBA2 and UBC9), were upregulated in early-stage embryos (D2 and/or D4) compared to oocytes and blastocysts. In response to UV-induced DNA damage, transcript levels of several UBs, DUBs, SUMOs, and deSUMOs decreased in D2 and D4 embryos, but increased in blastocysts. These findings revealed that transcript levels of genes encoding for important UBs, DUBs, SUMOs, and deSUMOs are regulated during early embryo development and are modulated in response to induced DNA damage. This study has also identified candidate genes controlling post-translational modifications that may have relevant roles in the regulation of normal embryo development, repair of damaged DNA, and preservation of genome stability in the pig embryo.

Role of the Paf1 complex in the maintenance of stem cell pluripotency and development.

Cell identity is determined by the transcriptional regulation of a cell-type-specific gene group. The Paf1 complex (Paf1C), an RNA polymerase II-associating factor, is an important transcriptional regulator that not only participates in transcription elongation and termination but also affects transcription-coupled histone modifications and chromatin organisation. Recent studies have shown that Paf1C is involved in the expression of genes required for self-renewal and pluripotency in stem cells and tumorigenesis. In this review, we focused on the role of Paf1C as a critical transcriptional regulator in cell fate decisions. Paf1C affects the pluripotency of stem cells by regulating the expression of core transcription factors such as Oct4 and Nanog. In addition, Paf1C directly binds to the promoters or distant elements of target genes, thereby maintaining the pluripotency in embryonic stem cells derived from an early stage of the mammalian embryo. Paf1C is upregulated in cancer stem cells, as compared with that in cancer cells, suggesting that Paf1C may be a target for cancer therapy. Interestingly, Paf1C is involved in multiple developmental stages in Drosophila, zebrafish, mice and even humans, thereby displaying a trend for the correlation between Paf1C and cell fate. Thus, we propose that Paf1C is a critical contributor to cell differentiation, cell specification and its characteristics and could be employed as a therapeutic target in developmental diseases.

Comparative multi-species analysis of potassium cyanide toxicity

Potassium cyanide (KCN), a highly water soluble and bioaccumulative cyanide salt, is examined to determine the toxic effects by using two green algae (Dunaliella viridis, Nannochloropsis oculata) and genetically different two sea urchin (Paracentrotus lividus, Arbacia lixula) species. To determine the toxic effects on the early developmental stages of sea urchin embryos, 72-hour embryotoxicity studies were conducted. Potassium cyanide toxicity at cellular level was also investigated and 6-hour embryos of both sea urchin species were used to determine genotoxic effects of KCN. Since plutei naturally feed on microalgae, two species of plankton were used to reveal phytotoxic effects of KCN. KCN was found to be embryo- geno- and phytotoxic. EC50's for P. lividus and A. lixula were found 7.96 and 6.52 μM. IC50's for N. oculata for 48 h and 72 h were found 23.66 and 80.45 μM. IC50's for D. viridis for 48 h and 72 h were found 14.31 and 23.36 μM.

ELF4 is critical to zygotic gene activation and epigenetic reprogramming during early embryonic development in pigs.

Zygotic gene activation (ZGA) and epigenetic reprogramming are critical in early embryonic development in mammals, and transcription factors are involved in regulating these events. However, the effects of ELF4 on porcine embryonic development remain unclear. In this study, the expression of ELF4 was detected in early porcine embryos and different tissues. By knocking down ELF4, the changes of H3K9me3 modification, DNA methylation and ZGA-related genes were analyzed. Our results showed that ELF4 was expressed at all stages of early porcine embryos fertilized in vitro (IVF), with the highest expression level at the 8-cell stage. The embryonic developmental competency and blastocyst quality decreased after ELF4 knockdown (20.70% control vs. 17.49% si-scramble vs. 2.40% si-ELF4; p < 0.001). Knockdown of ELF4 induced DNA damage at the 4-cell stage. Interfering with ELF4 resulted in abnormal increases in H3K9me3 and DNA methylation levels at the 4-cell stage and inhibited the expression of genes related to ZGA. These results suggest that ELF4 affects ZGA and embryonic development competency in porcine embryos by maintaining genome integrity and regulating dynamic changes of H3K9me3 and DNA methylation, and correctly activating ZGA-related genes to promote epigenetic reprogramming. These results provide a theoretical basis for further studies on the regulatory mechanisms of ELF4 in porcine embryos.

Searching for the Origin and the Differentiation of Haemocytes before and after Larval Settlement of the Colonial Ascidian Botryllus schlosseri: An Ultrastructural Viewpoint

The colonial ascidian Botryllus schlosseri possesses an innate immunity, which plays fundamental roles in its survival, adaptability, worldwide spread and ecological success. Three lines of differentiation pathways of circulating haemocytes are known to be present in the haemolymph, starting from undifferentiated haemoblasts: (i) the phagocytic line (hyaline amoebocytes and macrophage-like cells), (ii) the cytotoxic line (granular amoebocytes and morula cells) and (iii) the storage cell line (pigment cells and nephrocytes). Many questions remain about their origin, and thus, observations during various stages of development were undertaken in this study. Haemocytes were detected beginning from the early tailbud embryo stage. Haemoblasts were always present and morula cells were the first differentiated haemocytes detected. In both the next stage, just before hatching, and the swimming tadpole larva stage, hyaline amoebocytes and pigment cells were also recognisable. Some morula cells containing active phenoloxidase migrated from the haemolymph into the tunic after having crossed the epidermis, and this behaviour could be related to the preparation of a defensive function for spatial competition. During larval metamorphosis, macrophage-like cells appeared with their phagosomes positive to acid phosphatase activity and containing apoptotic cells from tail tissue degeneration. After metamorphosis, in the filter-feeding oozoid stage, nephrocytes involved in nitrogen catabolism finally appeared. In both the subendostylar sinus and the peripheral blind-sac vessels (ampullae), clusters of haemoblasts were recognisable, some of which showed incipient specialisations, considering the hypothesis of the presence of putative niches of haemolymph stem cells.

The Code Breaker: Jennifer Doudna, Gene Editing, and the Future of the Human Race

The Code Breaker: Jennifer Doudna, Gene Editing, and the Future of the Human Race

Transgenerational effects of gamma radiation dose and dose rate on Drosophila flies irradiated at an early embryonal stage

Ionizing radiation (IR) kills cells mainly through induction of DNA damages and the surviving cells may suffer from mutations. Transgenerational effects of IR are well documented, but the exact mechanisms underlying them are less well understood; they include induction of mutations in germ cells and epigenetic inheritance. Previously, effects in the offspring of mice and zebrafish exposed to IR have been reported. A few studies also showed indications of transgenerational effects of radiation in humans, particularly in nuclear power workers. In the present project, short- and long-term effects of low-dose-rate (LDR; 50 and 97 mGy/h) and high-dose-rate (HDR; 23.4, 47.1 and 495 Gy/h) IR in Drosophila embryos were investigated. The embryos were irradiated at different doses and dose rates and radiosensitivity at different developmental stages was investigated. Also, the survival of larvae, pupae and adults developed from embryos irradiated at an early stage (30 min after egg laying) were studied. The larval crawling and pupation height assays were applied to investigate radiation effects on larval locomotion and pupation behavior, respectively. In parallel, the offspring from 3 Gy irradiated early-stage embryos were followed up to 12 generations and abnormal phenotypes were studied. Acute exposure of embryos at different stages of development showed that the early stage embryo is the most sensitive. The effects on larval locomotion showed no significant differences between the dose rates but a significant decrease of locomotion activity above 7 Gy was observed. The results indicate that embryos exposed to the low dose rates have shorter eclosion times. At the same cumulative dose (1 up to 7 Gy), HDR is more embryotoxic than LDR. We also found a radiation-induced depigmentation on males (A5 segment of the dorsal abdomen, A5pig-) that can be transmitted up to 12 generations. The phenomenon does not follow the classical Mendelian laws of segregation.

DDX1 vesicles control calcium-dependent mitochondrial activity in mouse embryos

The DEAD box protein DDX1, previously associated with 3’-end RNA processing and DNA repair, forms large aggregates in the cytoplasm of early mouse embryos. Ddx1 knockout causes stalling of embryos at the 2-4 cell stages. Here, we identify a DDX1-containing membrane-bound calcium-containing organelle with a nucleic acid core. We show that aggregates of these organelles form ring-like structures in early-stage embryos which we have named Membrane Associated RNA-containing Vesicles. We present evidence that DDX1 is required for the formation of Membrane Associated RNA-containing Vesicles which in turn regulate the spatial distribution of calcium in embryos. We find that Ddx1 knockout in early embryos disrupts calcium distribution, and increases mitochondria membrane potential, mitochondrial activity, and reactive oxygen species. Sequencing analysis of embryos from Ddx1 heterozygote crosses reveals downregulation of a subset of RNAs involved in developmental and mitochondrial processes in the embryos with low Ddx1 RNA. We propose a role for Membrane Associated RNA-containing Vesicles in calcium-controlled mitochondrial functions that are essential for embryonic development.

Increasing vitamin D levels to improve fertilization rates in cattle.

Recently, interest in supplementing vitamin D (Vit D) to improve aspects of health, mainly in human fertility, has emerged. Still, supplementation of Vit D above the minimum required levels has yet to be explored in cattle despite evidence for Vit D receptors in reproductive tissues. The objective of this study was to establish if a dose-response relationship exists between Vit D exposure and success of in vitro production (IVP) of embryos and, if acute supplementation of Vit D improves pregnancy rates during timed artificial insemination (TAI) of dairy cows. Cumulus-oocyte complexes (COCs) were obtained from ovaries acquired from a local abattoir and cultured in five different IVP treatments from three separate collections (Control, 50, 100, 150, and 200 ng/mL of 1,25(OH)2D3; n = 20-30 COCs/group). In Experiment 2, dairy breed cows (n = 100) were synchronized for TAI with the PresynchOvsynch protocol. Cows received 150,000 IU of Vit D (n = 48) or castor oil as control (n = 53) along with gonadotropin-releasing hormone (GnRH) 24 h before TAI. Serum samples were collected before and 24 h after treatment. A small cohort of cows (n = 4) received the same treatments in two separate cycles and follicular fluid (FF) was collected after 24 h for calcidiol (25OHD) analyses. Increased concentrations of Vit D resulted in decreased rates of maturation of COC (150 and 200 ng/mL vs. control and 50 ng/mL; P = 0.01). Supplementation with 50 ng/mL resulted in greater numbers of early blastocyst and blastocyst stage embryos (P < 0.009). Pregnancy at first breeding did not differ (P = 0.13) between groups, but serum 25OHD increased in treated females after 24 h (P = 0.002). The FF 25OHD levels were reflective of serum levels, however, the observed increase in the treatment cycle (P = 0.04) was parallel to an overall increase in serum 25OHD during the entire second cycle, likely due to increased environmental sunlight exposure (March, control vs. May, treatment). A similar increase in the serum 25OHD in the lactating commercial herd maintained in covered housing was not observed, although experiments were conducted during a similar timeframe. This herd had levels of 25OHD near the low end of sufficiency according to National Research Council (NRC) guidelines. We conclude mild Vitamin D supplementation with concentrations at the higher end of NRC guidelines can improve maturation rates of recovered COCs. However, longer term supplementation may be needed to appreciate any benefits on fertility.

Altricial Bird Early-Stage Embryos Express the Molecular "Machinery" to Respond to and Modulate Maternal Thyroid Hormone Cues.

AbstractMaternal hormones, such as thyroid hormones (THs) transferred to embryos and eggs, are key signaling pathways for mediating maternal effects. To be able to respond to maternal cues, embryos must express the key molecular "machinery" of hormone pathways, such as enzymes and receptors. While altricial birds begin TH production only at or after hatching, experimental evidence suggests that their phenotype can be influenced by maternal THs deposited into the egg. However, it is not understood how or when altricial birds express genes in the TH pathway. For the first time, we measured the expression of key TH-pathway genes in altricial embryos by using two common altricial ecological model species, pied flycatcher (Ficedula hypoleuca) and blue tit (Cyanistes caeruleus). Deiodinase DIO1 gene expression could not be reliably confirmed in either species, but deiodinase enzyme genes DIO2 and DIO3 were expressed in both species. Given that DIO2 converts thyroxine to biologically active triiodothyronine and that DIO3 mostly converts triiodothyronine to inactive forms of THs, our results suggest that embryos may modulate maternal signals. TH receptors (THRA and THRB) and a monocarboxylate membrane transporter gene (SLC16A2) were also expressed, enabling TH responses. Our results suggest that altricial embryos may be able to respond to and potentially modulate maternal signals conveyed by THs in early development.

Research Progress of Totipotent Stem Cells.

Totipotent stem cells (TSCs), can develop into complete organisms, are used in biological fields such as regenerative medicine, mammalian breeding, and conservation. However, it is difficult to maintain the developmental totipotency and self-renewal capacity of cells cultured from early-stage embryos, which becomes a key factor limiting the research of TSCs. Fortunately, a breakthrough in the study of induced pluripotent stem cells returning to their totipotent state has been made, resulting in the establishment of multiple TSCs and igniting a new wave of stem cell research. Furthermore, the blastocyst-like structures can be generated by the established TSCs, which lays a foundation for synthetic embryos in vitro. In this review, we summarize the totipotent stage of early embryos, the establishment and cultivation of TSCs, and the developmental ability exploration of TSCs to promote further research of TSCs.

PLCζ can stably regulate Ca2+ fluctuations in early embryo

Phospholipase C zeta (PLCζ) is an important inducer of Ca2+ oscillations in mammalian sperm. To explore the influence of PLCζ on early embryonic Ca2+ fluctuations during sperm-egg binding, this study used PLCζ from sheep sperm to construct an early embryonic Ca2+ fluctuation model. First, sheep MII oocytes were cultivated and screened using microinjection technology. Then, a pEGFP-N1-PLCζ plasmid was constructed to activate oocytes in the test group. Ionomycin combined with 6-Dimethylaminopurine (6-DMAP) was used for the control group to explore the effects on early embryonic development and regulation of Ca2+ fluctuations during development. The results demonstrated that both the PLCζ and ionomycin combined with 6-DMAP activation methods induced sheep oocyte parthenogenetic activation and development in early embryos. In comparisons, the cleavage rate of ionomycin combined with 6-DMAP activation was significantly higher than that of PLCζ (60.9% ± 19.4% vs 76.1% ± 0.7%, respectively; p < 0.001), and the blastocyst rates were 16.2% ± 0.62% and 21.1% ± 0.92%, respectively (p < 0.05). Additionally, when comparing the distribution of Ca2+ in early embryos at different stages, Ca2+ in both treatment groups was mainly distributed in the cytoplasm, but the temporal pattern of Ca2+ fluctuations differed. PLCζ resulted in Ca2+ peaks that appeared at the cleavage and morula stages of early embryos, and Ca2+ returned to normal levels at the morula stage. However, the Ca2+ concentration after ionomycin combined with 6-DMAP activation was always much higher than that with PLCζ, and its single peak appeared later than in the PLCζ group. In summary, the PLCζ gene promoted stable regulatory effects on Ca2+ fluctuations at different stages during early embryonic development.

On genome editing in embryos and cells of the freshwater prawn Macrobrachium rosenbergii

The clustered regularly interspaced short palindromic repeats (CRISPR) technology provides the means for accurate genomic editing. It has been applied in many kinds of cells and animals for functional genomic studies and for precise selective breeding. Nonetheless, this method has not yet been applied in one of the most important – and well studied – decapod crustacean aquaculture species, the giant freshwater prawn Macrobrachium rosenbergii. We thus established two CRISPR platforms for M. rosenbergii—the first through direct injection into early-stage embryos (entire organism genome editing) and the second by electroporation of a primary embryonic cell culture. The systems were calibrated by optimizing Cas9 concentrations, delivery methods and editing efficiencies. Editing patterns utilizing multiple guides were examined through next generation sequencing. Our results showed a wide range of editing efficiencies in embryos, in some cases reaching as high as 100%. In contrast, in primary embryonic cell cultures, the highest editing efficiency obtained reached a maximum of 64%. In addition, there was a striking difference between the two platforms in terms of the pattern of deletions around the Cas9 cut site. This finding suggests distinct repair mechanisms in the two systems, which calls for further clarification. A phenotypic proof of concept was provided through the investigation of an early acting paired box protein 6 (Pax6) transcription factor, which showed clear effects on eye development in edited embryos and larvae. The current study lays down the foundations for precise functional genomic research and applications of genome editing in crustacean species for both aquaculture and sustainable biocontrol, opening opportunities for the creation of selected crustacean lines with distinct attributes.

Porcine Pluripotent Stem Cells Established from LCDM Medium with Characteristics Differ from Human and Mouse Extended Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) have unlimited self-renewal and multifunctional development potential in vitro. Porcine PSCs are highly desirable due to the conserved characteristics between pigs and humans. Extended PSCs (EPSCs) are additionally capable of differentiating into embryonic (Em) and extraembryonic (E×Em) parts. Here, we employed the LCDM culture system (consisting of human LIF, CHIR99021, (S)-(+)-dimethindene maleate, and minocycline hydrochloride), which can establish EPSCs from humans and mice, to derive and maintain stable porcine PSCs (pLCDM) from in vivo blastocysts. Transcriptome analysis revealed the unique molecular characteristics of pLCDMs compared with early-stage embryos. Meanwhile, the parallels and differences in the transcriptome features among pLCDMs, human EPSCs, and mouse EPSCs were carefully analyzed and evaluated. Most noteworthy, the trophoblast lineage differentiation tendency of pLCDMs was clarified by inducing trophoblast-like cells and trophoblast stem cells (TSCs) in vitro. Further research found that 2 of the small molecules in LCDM culture system, (S)-(+)-dimethindene maleate (DiM) and minocycline hydrochloride (MiH), probably play a crucial role in promoting trophoblast lineage differentiation potential of pLCDMs.

Transgenic mice encoding modern imaging probes: Properties and applications.

SUMMARYModern biology is increasingly reliant on optical technologies, including visualization and longitudinal monitoring of cellular processes. The major limitation here is the availability of animal models to track the molecules and cells in their natural environment in vivo. Owing to the integrity of the studied tissue and the high stability of transgene expression throughout life, transgenic mice encoding fluorescent proteins and biosensors represent unique tools for in vivo studies in norm and pathology. We review the strategies for targeting probe expression in specific tissues, cell subtypes, or cellular compartments. We describe the application of transgenic mice expressing fluorescent proteins for tracking protein expression patterns, apoptotic events, tissue differentiation and regeneration, neurogenesis, tumorigenesis, and cell fate mapping. We overview the possibilities of functional imaging of secondary messengers, neurotransmitters, and ion fluxes. Finally, we provide the rationale and perspectives for the use of transgenic imaging probes in translational research and drug discovery.

New in pathogenetic mechanisms of undeveloped pregnancy

The relevance of the study is due to the search for prognostically significant causes of non-developing pregnancy (NP) and the development of adequate prevention of identified disorders. Methylenetetrahydrofolate reductase encoded by the MTHFR gene determines the balance of folic acid derivatives and homocysteine (HC) / methionine. Polymorphic variants of folate genes can lead to excessive accumulation of HC in the blood and hypomethylation of DNA, which contributes to an increase in reproductive losses in early gestation. Carrying out periconceptional prophylaxis with high doses of folic acid (4000 mcg) reduces the level of free HC in the blood, but is effective only when it begins 3 months before conception and in the early stages of embryo and fetus development (up to 12 weeks). Based on the study, it was proposed to use folic acid at a dose of 4000 mcg in a high-risk group for the development of NP (pregnant women with hyperhomocysteinemia) 16 weeks before conception and 12 weeks after conception.

Comparison of pregnancy rate in dromedary camel between early-stage embryos and blastocyst transfer produced by somatic cell nuclear transfer using in vitro-matured oocytes.

We compared the pregnancy and live birth rates following transfer of early-stage embryos or blastocysts produced by somatic cell nuclear transfer using in vitro-matured oocytes. In total 102 ovaries were collected from dromedary camels at a local abattoir; from these 1048 cumulus-oocytes complexes (COCs) were aspirated and cultured for 42 h in a commercial maturation medium. Metaphase II oocytes were subjected to nuclear transfer. Somatic cell nuclear transfer-derived embryos were cultured in a commercial embryo medium for 2 or 7 days. Next, 71 early-stage embryos were surgically transferred to the left fallopian tube of 28 recipients and 47 blastocysts were transferred to the left uterine horn of 26 recipients. Early pregnancy was detected by serum progesterone (P4), and pregnancy was confirmed using ultrasonography on days 30 and 90 after embryo transfer. Pregnancy rate based on P4 level was 17.86% (5/28) and 11.54% (3/26) for early-stage embryo and blastocyst transfer, respectively. In the early-stage embryo group, out of five recipients, one recipient had lost the pregnancy by the first ultrasonography on day 30; two other recipients aborted at 14 and 24 weeks, and two recipients gave live births. In the blastocyst group, out of three recipients, one lost the pregnancy at an early stage and two recipients gave live births. Therefore, for dromedary camels, we recommend transvaginal blastocyst transfer from the standpoint of the pregnancy and live birth rate, ease of the transfer procedure, and comfort and safety of the recipients.

Effect of water temperature on walleye pollock (Gadus chalcogrammus) embryos, larvae and juveniles: Survival, HSP70 expression, and physiological responses

This study investigated the survival rates, physiological responses, and the induction of heat shock protein 70 (HSP70) expression in size-specific walleye pollock (Gadus chalcogrammus) at different water temperatures. Four groups consisting of walleye pollocks in various lifecycle stages—embryos, larvae, small juveniles, and large juveniles—were used. Embryos had the highest hatching rate at 5 °C, which decreased as the water temperature increased. Larval survival rate was the highest at 5 °C. The median lethal temperatures (LT50) for small and large juveniles were 19.1 and 18.9 °C for 7 d, respectively. HSP70 was cloned using the pET21b + vector and expressed in Escherichia coli to analyze HSP70 expression in walleye pollock. Polyclonal antibodies were obtained from BALB/c mice after purification on a His-Trap HP column and used for protein analysis using enzyme-linked immunosorbent assay. HSP70 mRNA and proteins were expressed in similar patterns at both 5 °C and 8 °C, with a drastic increase in HSP70 expression induction at ≥11 °C. The gill tissues of small and large juveniles had the highest HSP70 mRNA and protein levels. Physiological responses of large juveniles' plasma showed that glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, and glucose levels were highest at 14 °C, whereas total cholesterol and triglyceride levels were lowest. Plasma superoxide dismutase and cortisol levels were highest at 14 °C, whereas growth hormone and insulin-like growth factor-1 levels were highest at 8 °C. These results suggest that elevated water temperatures increased stress in the early lifecycle stages (embryos and larvae) of walleye pollock. HSP70 and stress-related indicator levels also increased with an increase in water temperature. Subsequently, the optimal water temperature for walleye pollock growth was determined to be below 8 °C.