Egg Injection Research Articles

Efficacy of in ovo delivered resveratrol (Trans 3, 4, 5-trihydroxystilbene) on growth, carcass weights, and blood metabolites of broiler chicks

This research aimed to determine the impact of in ovo resveratrol (RV) (Trans.3, 4, 5-trihydroxystilbene) injection on performance, carcass weights, and blood constituents in Cobb chicks. A total of 300 fertile broiler breeder eggs were divided into five treatments as 60 eggs in each group, with 6 replicates (10 eggs each). The groups were as follows: (1) negative control (without additive), (2) positive control (saline injection), (3) 1.5 mg RV, 4) 3.0 mg RV, and (5) 4.5 mg in ovo RV injections. Significant changes were found in the amount of forage consumed per day, and the feed conversion ratio between treatment groups. RV injection significantly altered the parameters of the complete lipid profile, including total cholesterol, triglycerides, low-density lipoproteins, and very low-density lipoproteins, in plasma. RV injection significantly affected blood biochemical parameters, including total protein, albumin, and globulin in broiler chicken plasma. Liver and kidney functions were influenced by the injection of RV eggs into broiler chickens. The antioxidant enzymes also changed significantly after RV injection. Moreover, in egg injection from RV the immunogenicity (IgG and IgM) in broiler chickens were significantly altered. In conclusion, in ovo injection with RV significantly improved liver and kidney functions, antioxidant activity, and immune function in Cobb broilers.

The Effect of intra egg injection of different levels of Thyme essential oil nanoemulsion on reproductive and blood parameters in Japanese quail

زمینه مطالعاتی: تزریق عوامل محرک رشد به تخم نطفه‌دار پرندگان به منظور بهبود فراسنجه‌های تولیدمثلی و عملکردی مورد توجه می‌باشد. هدف: این تحقیق به منظور بررسی تأثیر تزریق سطوح مختلف نانوامولسیون اسانس آویشن به درون تخم بلدرچین ژاپنی بر فراسنجه‏های تولیدمثلی و خونی انجام شد. روش کار: آزمایش با استفاده از 600 عدد تخم بلدرچین ژاپنی در قالب طرح کاملا تصادفی با 5 تیمار، 4 تکرار و 30 عدد تخم بلدرچین در هر تکرار انجام شد. گروه‏های آزمایشی شامل: گروه شاهد منفی (بدون تزریق)، شاهد مثبت (تزریق درون‏تخمی مخلوط آب و امولسیفایر) و تزریق درون‏تخمی اسانس آویشن نانوامولسیون ‏شده در سطوح 50، 100 و150 پی‌پی‌ام بود. تزریق محلول‎های آزمایشی به درون کیسه هوایی تخم‌ها در قبل از انکوباسیون آن‌ها انجام شد. پس از پایان انکوباسیون تخم‎ها در داخل دستگاه جوجه‌کشی، درصد هچ و تلفات رویانی در هر کدام از گروه‏های تیماری بررسی شد. سپس جوجه‌های هچ شده برای ارزیابی فراسنجه‌های خونی و خصوصیات دستگاه‌ تناسلی به مدت 8 هفته در بستر پرورش داده شدند. نتایج: نه تنها تیمارهای آزمایشی موجب بهبود درصد هچ کلی، هچ تخم‌های بارور و کاهش تلفات رویانی تخم‌ها نشدند (05/0 P). در پایان دوره پرورش، وزن و طول لوله‌های رحمی، وزن تخمدان، وزن و قطر فولیکول‏های F1 تا F3 در پرنده‌های ماده و نیز غلظت تستوسترون در پرنده‌های نر، تحت تأثیر تیمارهای آزمایشی قرار نگرفت (05/0 P). تری‌گلیسرید کل سرم در گروه تیماری دریافت‏کننده ppm 100 اسانس نانوامولسیونی کمتر از گروه‎های شاهد بود (05/0>P)، ولی میانگین مقادیر سایر فراسنجه‏های خونی در سایر گروه‏های آزمایشی نسبت به گروه‌های شاهد معنی‏دار نبود. نتیجه‌گیری نهایی: به‏طور کلی، تزریق نانوامولسیون اسانس آویشن به کیسه هوای تخم بلدرچین ژاپنی به منظور بهبود عملکرد تولیدمثلی در این پرنده توصیه نمی‌شود.

Group 2 Innate Lymphoid Cells Exhibit Tissue-Specific Dynamic Behaviour During Type 2 Immune Responses.

Group 2 innate lymphoid cells (ILC2s) are early effectors of mucosal type 2 immunity, producing cytokines such as interleukin (IL)-13 to mediate responses to helminth infection and allergen-induced inflammation. ILC2s are also present in lymph nodes (LNs) and can express molecules required for antigen presentation, but to date there are limited data on their dynamic behaviour. We used a CD2/IL-13 dual fluorescent reporter mouse for in vivo imaging of ILC2s and Th2 T cells in real time following a type 2 priming helminth infection or egg injection. After helminth challenge, we found that ILC2s were the main source of IL-13 in lymphoid organs (Peyer’s patches and peripheral LNs), and were located in T cell areas. Intravital imaging demonstrated an increase in IL-13+ ILC2 size and movement following helminth infection, but reduced duration of interactions with T cells compared with those in homeostasis. In contrast, in the intestinal mucosa, we observed an increase in ILC2-T cell interactions post-infection, including some of prolonged duration, as well as increased IL-13+ ILC2 movement. These data suggest that ILC2 activation enhances cell motility, with the potential to increase the area of distribution of cytokines to optimise the early generation of type 2 responses. The prolonged ILC2 interactions with T cells within the intestinal mucosa are consistent with the conclusion that contact-based T cell activation may occur within inflamed tissues rather than lymphoid organs. Our findings have important implications for our understanding of the in vivo biology of ILC2s and the way in which these cells facilitate adaptive immune responses.

Effects of two Bisphenol A replacement compounds, 1,7-bis (4-hydroxyphenylthio)-3,5-dioxaheptane and Bisphenol AF, on development and mRNA expression in chicken embryos

Concerns about the estrogenic properties of Bisphenol A (BPA) have led to increased efforts to find BPA replacements. 1,7-bis(4-Hydroxyphenylthio)-3,5-dioxaheptane (DD-70) and 4,4′-(hexafluoroisopropylidene) diphenol (bisphenol AF, BPAF) are two potential chemical substitutes for BPA; however, toxicity data for these chemicals in avian species are limited. To determine effects on avian embryonic viability, development, and hepatic mRNA expression at two distinct developmental periods (mid-incubation [day 11] and term [day 20]), two egg injection studies were performed. Test chemicals were injected into the air cell of unincubated, fertilized chicken eggs at concentrations ranging from 0–88.2 µg/g for DD-70 and 0–114 µg/g egg for BPAF. Embryonic concentrations of DD-70 and BPAF decreased at mid-incubation and term compared to injected concentrations suggesting embryonic metabolism. Exposure to DD-70 (40.9 and 88.2 µg/g) and BPAF (114 µg/g) significantly decreased embryonic viability at mid-incubation. Exposure to DD-70 (88.2 µg/g) decreased embryo mass and increased gallbladder mass, while 114 µg/g BPAF resulted in increased gallbladder mass in term embryos. Expression of hepatic genes related to xenobiotic metabolism, lipid homeostasis, and response to estrogen were altered at both developmental stages. Given the importance of identifying suitable BPA replacements, the present study provides novel, whole animal avian toxicological data for two replacement compounds, DD-70 and BPAF. Data availabilityData, associated metadata, and calculation tools are available from the corresponding author (doug.crump@canada.ca).

Low sperm to egg ratio required for successful in vitro fertilization in a pair-spawning teleost, Senegalese sole (Solea senegalensis).

Cultured Senegalese sole (Solea senegalensis) breeders fail to spawn fertilized eggs. The implantation of large-scale in vitro fertilization protocols, to solve this problem, has been frustrated by low production of poor quality sperm. Cultured females were induced to ovulate with a 5 µg kg−1 single injection of gonadotropin-releasing hormone agonist (GnRHa) and viable eggs (82.6 ± 9.2% fertilization) were stripped 41:57 ± 1:46 h after the injection. Sperm was collected from cultured males, diluted in modified Leibovitz and used fresh to fertilize the eggs. Males were not treated with hormones. A nonlinear regression, an exponential rise to a maximum (R = 0.93, p < 0.0001) described the number of motile spermatozoa required to fertilize a viable egg and 1617 motile spermatozoa were sufficient to fertilize 99 ± 12% (±95% CI) of viable eggs. Similar, spermatozoa egg−1 ratios of 592 ± 611 motile spermatozoa egg−1 were used in large-scale in vitro fertilizations (190 512 ± 38 471 eggs). The sperm from a single male (145 ± 50 µl or 8.0 ± 6.8 × 108 spermatozoa) was used to fertilize the eggs. The mean hatching rate was 70 ± 14% to provide 131 540 ± 34 448 larvae per fertilization. The viability of unfertilized eggs stored at room temperature decreased gradually, and the sooner eggs were fertilized after stripping, the higher the viability of the eggs. The collection of sperm directly into a syringe containing modified Leibovitz significantly increased the percentage of motile spermatozoa (33.4 ± 12.2%) compared with other collection methods. The spz egg−1 ratios for Senegalese sole were at the lower end of ratios required for fish. Senegalese sole have a pair-spawning reproductive behaviour characterized by gamete fertilization in close proximity with no sperm competition. The provision of a large-scale in vitro fertilization protocol (200 µl of sperm per 100 ml of eggs) will enable the industry to operate sustainably and implement breeding programmes to improve production.

Ingestion of Poisonous Eggs of the Invasive Apple Snail Pomacea canaliculata Adversely Affects Bullfrog Lithobathes catesbeianus Intestine Morphophysiology

The poisonous eggs of Pomacea canaliculata hardly have any predators. This invasive snail, listed among 100 of the worst invasive species, is a serious crop pest and a vector of human parasitic diseases. Females lay eggs in pink-reddish masses, the colour presumably a warning of their chemical defenses. Egg ingestion alters rodent gastrointestinal tracts and is lethal if injected, but its effect on other taxa is unknown. Here we explored the toxic effects of Pomacea canaliculata eggs on the bullfrog Lithobathes catesbeianus (Anura, Ranidae).Juvenile bullfrogs were gavaged with egg extracts and their digestive tract analyzed after 24 h and 48 h using histological, immunohistochemical and lectin-histochemical techniques. Toxicity was also evaluated by intraperitoneal injection of egg extract.Egg extract ingestion adversely affected the small intestine of the bullfrogs. Short term (24 h) effects included large, reversible changes of the intestinal wall, villi morphology, and changes in the glycosylation patterns of enterocytes. The mucosal area increased and infiltration of inflammatory cells, mainly eosinophils and macrophages, were observed together with a weak hemorrhage. Most of these changes reversed after 48 h. In addition, intraperitoneal injection of egg extract was nontoxic to bullfrog juveniles and no lethality or behavioral changes were observed, a remarkable difference from the effect on rodents.Overall, these results indicate that toxins of apple snail eggs reversibly modify gut morphology, which may alter bullfrog physiology, limiting their ability to absorb egg nutrients. This study extends the known targets of the apple snail egg defenses against predation to amphibians.

Verification Of Molecular And Conventional Techniques Used In The Diagnosis Of Equine Herpes Virus In Some Egyptian Governorates

Equid herpesviruses (EHVs) affect equine health and can cause significant economic losses to the equine industry worldwide. In the current study, the circulation of two infectious equid herpesviruses (EHV-1 and EHV-4) among different horse populations in some farms was monitored. In the present study, 50 samples of nasal secretions and tissue homogenates from neurological disease cases, abortion, neonatal foal deaths, and 36 serum samples. Samples of swabs and organs inoculated in embryonated chiken egg and Madin darby bovine kidney cell line. 29samples were positive in egg injection but no detected CPE in cell line for three passages. DNA was extracted and subjected to conventional PCR to detect the two herpesviruses' presence using specific primers. Three isolates of EHV-1 and four were detected. One EHV-1 and two EHV-4 were subjected to phylogenetic analysis. Phylogenetic analysis confirmed the existence of the isolated EHV-1 and EHV-4. They were more closely related to other previously isolated EHV-1 and EHV-4 from Egypt and other countries. Antibodies against EHV-1 and EHV-4 were tested using ELISA. The results showed that EHV-1 and EHV-4 are endemic and can be a continuous threat for horses in the absence of vaccination programs and frequent virus reactivation.

CRISPR/Cas9-mediated genome editing of Schistosoma mansoni acetylcholinesterase.

CRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. We report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. Eggs recovered from livers of experimentally infected mice were transfected by electroporation with a CRISPR/Cas9-vector encoding gRNA X5 or X7 combining with/ without a ssODN donor. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, an significantly enhanced Th2 response involving IL-4, -5, -10, and-13 was detected in lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, -13, and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.

Effects of acute stressors experienced by five strains of layer breeders on measures of stress and fear in their offspring

Stressors experienced by layer breeders during egg production can lead to changes in the egg hormone content, potentially impacting their offspring, the commercial layers. Genetic differences might also affect the offspring's susceptibility to maternal experiences. In this study, we tested if maternal stress affects measures of stress and fear in five strains of layer breeders: commercial brown 1 & 2, commercial white 1 & 2 and a pure line White Leghorn. Each strain was equally separated into two groups: “Maternal Stress” (MS), where hens were subjected to a series of 8 consecutive days of acute psychological stressors, and “Control,” which received routine husbandry. Additional eggs from Control were injected either with corticosterone diluted in a vehicle solution (“CORT”) or just “Vehicle.” Stress- and fear-responses of the offspring were measured in a plasma corticosterone test and a combined human approach and novel object test. While the stress treatments did not affect the measured endpoints in the offspring, significant strain differences were found. The offspring of the white strains showed a higher physiological response compared to brown strains and the White 2 offspring was the least fearful strain in the human approach test. Our study found that neither the acute psychological stressors experienced by layer breeders nor the egg injections of corticosterone affected the parameters tested in their offspring. Post hoc power analyses suggest that the lack of treatment effects might be due to a small sample size (type II error). Although studies on larger flocks of layers are still needed, our results provide an initial understanding of an important subject, as in poultry production, layer breeders are often subjected to short-term stressors. In addition, our results suggest the dissociation between the physiological and behavioural parameters of stress response in laying hens, showing that increased concentrations of plasma corticosterone in response to stress might not be directly associated with high levels of fear.

Incubation temperature and PCB-126 exposure interactively impair shorebird embryo and post-hatch development

In oviparous wildlife, many critical physiological and behavioural components are strongly influenced by the embryonic and early post-hatch developmental environment. As such, early life stages in these species are highly vulnerable to both natural and anthropogenic stressors. For example, in birds, incubation temperature may influence the rate of egg development while also affecting contaminant metabolism and absorption in body tissues, resulting in potentially multiplicative impacts on embryonic and posthatch development. We tested the hypothesis that cumulative effects of early contaminant exposure and temperature stress can negatively affect avian development and may have interactive effects that are more detrimental than either stressor individually. Using a controlled egg injection and incubation study on killdeer (Charadrius vociferous), eggs were exposed to a known endocrine disruptor, 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126) and incubated at either low (36 °C), intermediate (37.5 °C), or high (39 °C) temperatures. Our results indicated that eggs incubated at low temperature had earlier detection of heartbeat, longer incubation length, lower growth rate post-hatch, and higher post-hatch mortality, compared to eggs incubated under intermediate temperatures. Higher incubation temperatures resulted in shorter incubation length, earlier detection of heart rate and faster righting time. As predicted, embryo and chick mortality were greater in the PCB-dosed birds incubated at intermediate and high temperatures. Incidence of distended yolk sacs (%) also increased with PCB exposure in all temperature groups, with the largest increase in the high temperature group. Overall, our results show that low incubation temperature can cause greater adverse effects than PCB-126 exposure alone, but that negative effects of PCB-126 exposure are exacerbated by high incubation temperatures. These findings suggest that in natural settings, shorebird embryos may be more susceptible to contaminant exposure when incubated at temperatures either below or above the apparent optimum.

Reprograming of epigenetic mechanisms controlling host insect immunity and development in response to egg-laying by a parasitoid wasp.

Parasitoids are insects that use other insects as hosts. They sabotage host cellular and humoral defences to promote the survival of their offspring by injecting viruses and venoms along with their eggs. Many pathogens and parasites disrupt host epigenetic mechanisms to overcome immune system defences, and we hypothesized that parasitoids may use the same strategy. We used the ichneumon wasp Pimpla turionellae as a model idiobiont parasitoid to test this hypothesis, with pupae of the greater wax moth Galleria mellonella as the host. We found that parasitoid infestation involves the suppression of host immunity-related effector genes and the modulation of host genes involved in developmental hormone signalling. The transcriptional reprogramming of host genes following the injection of parasitoid eggs was associated with changes in host epigenetic mechanisms. The introduction of parasitoids resulted in a transient decrease in host global DNA methylation and the modulation of acetylation ratios for specific histones. Genes encoding regulators of histone acetylation and deacetylation were mostly downregulated in the parasitized pupae, suggesting that parasitoids can suppress host transcription. We also detected a strong parasitoid-specific effect on host microRNAs regulating gene expression at the post-transcriptional level. Our data therefore support the hypothesis that parasitoids may favour the survival of their offspring by interfering with host epigenetic mechanisms to suppress the immune system and disrupt development.

Uptake, Deposition, and Metabolism of Triphenyl Phosphate in Embryonated Eggs and Chicks of Japanese Quail (Coturnix japonica).

The toxicokinetics of triphenyl phosphate (TPHP) in vivo including the uptake, deposition, and biotransformation into the metabolite diphenyl phosphate (DPHP) is presently reported in embryonated eggs and chicks of Japanese quail. Quail were dosed with TPHP at 3 concentrations by air cell egg injection on embryonic day 0, followed by daily oral dosing after chicks hatched (5 d). Vehicle-only exposed controls were also used. In dosed eggs, only 33% of the TPHP remained 2 d after injection (no hepatic development); after 10 d (post-hepatogenesis), only 2% remained. The estimated TPHP half-lives in the eggs ranged from 1.1 to 1.8 d for the 3 dosed groups. In all exposed eggs and chicks, DPHP significantly increased with dose (0.001 < p < 0.044). It appears that DPHP is an important metabolite in quail, making up 41 to 74% of all metabolites formed in embryonated eggs. In chicks, at medium and high doses, DPHP concentrations significantly exceeded those of TPHP (p ≤ 0.007), making up 67 and 76% of the total burden, respectively. Our findings suggest that rapid TPHP metabolism occurred in chicks and embryonated quail eggs but that this may vary with the age of the embryonated egg and the stage of embryo development, which should be considered when evaluating concentrations of TPHP and DPHP measured in eggs of wild birds. Environ Toxicol Chem 2020;39:565-573. © 2019 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry © 2019 SETAC.

Vision-Based Sensor for Three-Dimensional Vibrational Motion Detection in Biological Cell Injection.

Intracytoplasmic sperm injection (ICSI) is an infertility treatment where a single sperm is immobilised and injected into the egg using a glass injection pipette. Minimising vibration in three orthogonal axes is essential to have precise injector motion and full control during the egg injection procedure. Vibration displacement sensing using physical sensors in ICSI operation is challenging since the sensor interfacing is not practically feasible. This study proposes a non-invasive technique to measure the three-dimensional vibrational motion of the injection pipette by a single microscope camera during egg injection. The contrast-limited adaptive histogram equalization (CHALE) method and blob analyses technique were employed to measure the vibration displacement in axial and lateral axes, while the actual dimension of the focal axis was directly measured using the Brenner gradient algorithm as a focus measurement algorithm. The proposed algorithm operates between the magnifications range of 4× to 40× with a resolution of half a pixel. Experiments using the proposed vision-based algorithm were conducted to measure and verify the vibration displacement in axial and lateral axes at various magnifications. The results were compared against manual procedures and the differences in measurements were up to 2% among all magnifications. Additionally, the effect of injection speed on lateral vibration displacement was measured experimentally and was used to determine the values for egg deformation, force fluctuation, and penetration force. It was shown that increases in injection speed significantly increases the lateral vibration displacement of the injection pipette by as much as 54%. It has been demonstrated successfully that visual sensing has played a key role in identifying the limitation of the egg injection speed created by lateral vibration displacement of the injection pipette tip.

Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function in Zebrafish.

Inconsistent activity limits the use of CRISPR-Cas9 in zebrafish. We show supernumerary guanine nucleotides at the 5' ends of single guide RNAs (sgRNAs) account for diminished CRISPR-Cas9 activity in zebrafish embryos. Genomic sequences can be targeted consistently with extremely high efficiency using Cas9 ribonucleoproteins (RNPs) containing either a sgRNA molecule or a synthetic crRNA:tracrRNA duplex that perfectly matches the protospacer target site. Following injection of zebrafish eggs with such RNPs, virtually every copy of a targeted locus harbors an induced indel mutation. Loss of gene function is often complete, as F0 embryos closely resemble true null mutants without detectable non-specific effects. Mosaicism is sufficiently low in F0 embryos that cell non-autonomous gene functions can be probed effectively and redundant activities of genes can be uncovered when two genes are targeted simultaneously. Finally, heritable deletion mutations of at least 50 kbp can be readily induced using pairs of duplex guide RNPs targeted to a single chromosome.

Continuous exposure to mercury during embryogenesis and chick development affects later survival and reproduction of zebra finch (Taeniopygia guttata).

Methylmercury (MeHg) is a global environmental contaminant that bioaccumulates and has multiple toxic modes of action. Aquatic species have traditionally been the focus of wildlife toxicological research on mercury, but terrestrial organisms, including passerine birds, can be exposed to similarly elevated levels of MeHg. In this study we exposed a model passerine, the zebra finch (Taeniopygia guttata), to MeHg in ovo, as chicks only, or with a combined 'in ovo + chick' treatment. We isolated exposure to specific developmental stages through the use of egg injections (3.2 µg Hg/g egg) and controlled oral dosing of chicks (0.24 µg Hg/g bw/day from day 1 to day 30). In ovo exposure to MeHg reduced hatching success, but there was no effect of MeHg on chick growth. We found that in ovo only or chick only exposure did not have long-term effects, but there was some evidence for longer-term effects of combined 'in ovo + chick' exposure on post-fledging survival and potentially sex-biased survival which resulted in very few 'in ovo + chick' exposed females surviving to breed. These females also had lower overall breeding productivity that was mainly due to lower hatching success of their offspring, not lower chick-rearing success. We found no effect of treatment on clutch size or latency to laying among females that did lay eggs. Our study suggests that combined embryonic and nestling MeHg exposure has compounding latent effects on productivity, likely through a mechanism that influences the ability of females to lay fertile eggs that hatch.

CRISPR/Cas9 Genome Editing Introduction and Optimization in the Non-model Insect Pyrrhocoris apterus.

The CRISPR/Cas9 technique is widely used in experimentation with human cell lines as well as with other model systems, such as mice Mus musculus, zebrafish Danio reiro, and the fruit fly Drosophila melanogaster. However, publications describing the use of CRISPR/Cas9 for genome editing in non-model organisms, including non-model insects, are scarce. The introduction of this relatively new method presents many problems even for experienced researchers, especially with the lack of procedures to tackle issues concerning the efficiency of mutant generation. Here we present a protocol for efficient genome editing in the non-model insect species Pyrrhocoris apterus. We collected data from several independent trials that targeted several genes using the CRISPR/Cas9 system and determined that several crucial optimization steps led to a remarkably increased efficiency of mutant production. The main steps are as follows: the timing of embryo injection, the use of the heteroduplex mobility assay as a screening method, in vivo testing of sgRNA efficiency, and G0 germline mosaicism screening. The timing and the method of egg injections used here need to be optimized for other species, but other here-described optimization solutions can be applied immediately for genome editing in other insect species.

An integrated protocol for targeted mutagenesis with CRISPR-Cas9 system in the pea aphid

CRISPR-Cas9 technology is a very efficient functional analysis tool and has been developed in several insects to edit their genome through injection of eggs with guide RNAs targeting coding sequences of genes of interest. However, its implementation in aphids is more challenging. Aphids are major pests of crops worldwide that alternate during their life cycle between clonality and sexual reproduction. The production of eggs after mating of sexual individuals is a single yearly event and is necessarily triggered by a photoperiod decrease. Fertilized eggs then experience an obligate 3-month diapause period before hatching as new clonal colonies. Taking into consideration these particularities, we developed in the pea aphid Acyrthosiphon pisum a step-by-step protocol of targeted mutagenesis based on the microinjection within fertilized eggs of CRISPR-Cas9 components designed for the editing of a cuticular protein gene (stylin-01). This protocol includes the following steps: i) the photoperiod-triggered induction of sexual morphs (2 months), ii) the mating and egg collection step (2 weeks), iii) egg microinjection and melanization, iv) the 3-month obligate diapause, v) the hatching of new lineages from injected eggs (2 weeks) and vi) the maintenance of stable lineages (2 weeks). Overall, this 7-month long procedure was applied to three different crosses in order to estimate the impact of the choice of the genetic combination on egg production dynamics by females as well as hatching rates after diapause. Mutation rates within eggs before diapause were estimated at 70–80%. The hatching rate of injected eggs following diapause ranged from 1 to 11% depending on the cross and finally a total of 17 stable lineages were obtained and maintained clonally. Out of these, 6 lineages were mutated at the defined sgRNAs target sites within stylin-01 coding sequence, either at the two alleles (2 lineages) or at one allele (4 lineages). The final germline transmission rate of the mutations was thus around 35%. Our protocol of an efficient targeted mutagenesis opens the avenue for functional studies through genome editing in aphids.

Effects of in ovo injection of carbohydrates, β-hydroxy-β-methylbutyrate, and vitamins on ostrich organ weight, bone characteristics, and small intestinal morphology

A total of 144 ostrich eggs (24 per group) were injected with 4 mL of physiological saline solution [positive control (PC)], carbohydrates (CHO — 20% maltose, 2.5% sucrose, and 2.5% dextrin), β-hydroxy-β-methylbutyrate (HMB), vitamin B6 solution, and vitamin D3 (vit D3) solution at 38 d of incubation. Injection of 4 mL physiological saline decreased the hatchability compared with negative control (NC) group (not injected eggs) (68.5% vs. 71.7%; P &lt; 0.05). There was also lower hatchability and more number of embryonic deaths before piping in vitamin-injected group (72.2%). Heart, gizzard, pancreas, spleen and thymus, ileum, cecum, and rectum relative weight were not significantly different among treatments at hatch (P &gt; 0.05). Carbohydrates group had higher duodenum and jejunum relative weight, whereas NC and PC groups had the least duodenum, jejunum relative weight (P &lt; 0.05). Vit D3 injection increased bone strength, fresh and dry bone relative weight (P &lt; 0.01). Chicks from HMB and CHO group had the highest levels of glycogen in the livers, whereas PC, NC, and vit D3 exhibited very low levels of glycogen in their liver (P = 0.014). Vitamins in ovo injected groups had the least duodenum villus height and also vit D3 group had the least jejunum villus height (P &lt; 0.01). In conclusion, the use of CHO for the in ovo injection of ostrich hatching eggs would be practical in industry due to improvement in hatchability, bone characteristics, small intestine villus height and capacity, and also liver glycogen sources.

Schistosoma haematobium cercarial infection alters subsequent systemic immune responses to eggs but has minimal impact on immune responses to egg injection of the bladder.

Mouse bladder wall injection with Schistosoma haematobium eggs has been used to overcome limitations in animal models of urogenital schistosomiasis. However, the effect of the absence of cercarial infection on immune responses to eggs in this model is unknown. We hypothesized that cercarial infection would alter local bladder and systemic immune responses to eggs in this model. Mice were infected or not infected with Shaematobium cercariae, and then, their bladder walls injected with Shaematobium eggs or vehicle 5weeks following cercarial infection. Three weeks later, mice were bled, sacrificed, perfused and their bladders harvested. Parasitological parameters and gross bladder pathology were not changed in egg-injected bladders by cercarial exposure. Figure S1 shows no changes in either granulomas or fibrosis. The only bladder cytokine upregulated in egg-injected bladders by cercarial exposure (vs no exposure) was leptin. Cercarial exposure, compared to no exposure, resulted in increased serum, IL-1α, IL-13 and TGF-β in bladder egg-injected mice. Cercarial exposure altered systemic responses of several cytokines in bladder egg-injected mice, but surprisingly, only modified leptin expression in bladder tissue. This suggests that depending on the specific application, cercarial exposure may not be strictly necessary to model local immune responses in the bladder wall egg injection mouse model of urogenital schistosomiasis.

INFLUENCE OF IN-OVO INJECTION OF SELENIUM NANOPARTICLES AND SELENOMETHIONINE ON GROWTH PERFORMANCE AND PHYSIOLOGICAL PARAMETERS OF BROILER CHICKS

This study was conducted to investigate the effect of in ovo injection of broiler eggs with different levels and forms of selenium on growth performance and some blood parameters of post hatched chicks. A total of 300 fertilized eggs obtained from a commercial Arbor Acres broiler breeder flock, were used in the present experiment. They were divided randomly into six treatment groups, 50 eggs each. The first one was kept as a control– non injected group, the second group (sham) injected with phosphate buffered saline (PBS), the 3rd and 4th groups were injected with selenium nanoparticles (SeNPs) at 10 and 20 ug/egg, while the 5th and 6th groups were injected with organic selenium (Se-Methionine) at 50 and 100 ug/egg , respectively. All eggs were incubated in a forced draught incubator at the recommended temperature, relative humidity and turning patterns .The in ovo injection procedure was done at the 16th day of incubation. The criteria of response includes: live body weight, weight gain, feed consumption, feed conversion ratio, total protein, albumin, globulin, A/G ratio, cholesterol, triglyceride, HDL and glucose. The results showed that the supplementation of Se did not significantly affect growth performance traits. Supplementation of nano-Se increased plasma concentrations of total protein, albumin and HDL, and decrease glucose, however, no significant differences in globulin, A/G ratio, cholesterol and triglyceride levels. It can be concluded that the use of 20 ug /egg of Nano-Se caused an improvement of growth performance and feed conversion ratio without negative effects on blood constituents.