Maternal zinc alleviates tert-butyl hydroperoxide-induced mitochondrial oxidative stress on embryonic development involving the activation of Nrf2/PGC-1αpathway

Background Mitochondrial dysfunction induced by excessive mitochondrial reactive oxygen species(ROS)damages embryonic development and leads to growth arrest.Objective The purpose of this study is to elucidate whether maternal zinc(Zn)exert protective effect on oxidative stress targeting mitochondrial function using an avian model.Result In ovo injected tert-butyl hydroperoxide(BHP)increases(P<0.05)hepatic mitochondrial ROS,malondialdehyde(MDA)and 8-hydroxy-2-deoxyguanosine(8-OHdG),and decreases(P<0.05)mitochondrial membrane potential(MMP),mitochondrial DNA(mtDNA)copy number and adenosine triphosphate(ATP)content,contributing to mitochondrial dysfunction.In vivo and in vitro studies revealed that Zn addition enhances(P<0.05)ATP synthesis and metallothionein 4(MT4)content and expression as well as alleviates(P<0.05)the BHP-induced mitochondrial ROS generation,oxidative damage and dysfunction,exerting a protective effect on mitochondrial function by enhancing antioxidant capacity and upregulating the mRNA and protein expressions of Nrf2 and PGC-1α.Conclusions The present study provides a new way to protect offspring against oxidative damage by maternal Zn supplementation through the process of targeting mitochondria involving the activation of Nrf2/PGC-1αsignaling.

Embryonic Development and Eclosion Season of New Species Berastagia (Lepidoptera: Pyralidae) from Taiwan

This paper describes a new species of the snout moth Berastagia tainanica sp. nov. (Lepidoptera: Pyralidae) from Taiwan. From 2009 to 2016, a biology study was conducted on population dynamics and embryonic development. Spring season is the peak of the eclosion of overwintering larvae or pupae. The average longevity of adult was 14.8 ± 6.2 days (N = 174), the average number of eggs laid was 259 ± 3 eggs/moth (N = 2), the hatching rate of eggs was 95.4% (N = 262), and the average hatching time of eggs was 99.6 ± 18.6 hours (N = 68). The average body length of males was 5.64 mm ± 0.91 mm (N = 30), and the average body length of females was 6.28 mm ± 0.84 mm (N = 30). This finding indicates that female snout moths are larger than males (Global R = 0.058, P = 0.012). The snout moth eclosion rate was 16.9 moths/100 pods in the first year (2010/2011, N = 2,224 pods) and 10.9 moths/100 pods in the second year (2014/2015, N = 6,382 pods). The pod borer rate was 31.8% (N = 707) and the seed borer rate was 41.2% (N = 3,628) in the first year, whereas the pod borer rate was 76.2% (N = 6,382) in the second year.

Embryonic development of the concave-eared torrent frog with its significance on taxonomy

We investigated the early embryonic and larval development of the concave-eared torrent frogs, Odorrana tormota （Amphibia, Anura, Ranidae）. Embryos were derived from artificial fertilization of frogs’ eggs, and the staging of development was based on morphological and physiological characteristics. Two major periods of development were designated： i） early embryonic period, from fertilization to operculum completion stage, lasted for 324 h at water temperature （WT） 18 ？23℃; ii） larval period, from operculum completion stage to tail absorbed stage, took 1207 h at WT 20 ？ 24℃. Tadpoles of the concave-eared torrent frogs showed no evidence of abdominal sucker. Absence of this key characteristic supports the view from molecular systematics that concave-eared torrent frog does not belong to the genus Amolops. Two cleavage patterns were observed in embryos at 8-cell and 16-cell stages, with Pattern I2 （latitudinal cleavage at the 8-cell stage, and meridional cleavage at the 16-cell stage with two perpendicular meridional furrows） being the predominant pattern and only 1.5% belonging to Pattern II （meridional cleavage at the 8-cell stage and latitudinal cleavage at the 16-cell stage）. The factors affecting cleavage and hatching ratios, developmental speed, and ecological adaptation were discussed.

Research on Embryonic Development of Loach

[Objective] Aim to know the whole process of embryonic development of loach. [Method] DOM + LHRH-A2 was used to induce spawning of loach,then after fertilization,the embryos were cultured into freshwater water with temperature from 24 to 26 ℃ and pH from 7.0 to 7.5. The embryonic development of loach was observed and 27 concrete morphological characteristics and development time of loach from fertilized egg to newly hatched larval period were described in detail. [Result] The embryonic development of loach could be divided into cleavage stage,blastocyst stage,gastrula stage,neurula stage and organogenesis stage. The loach embryo from fertilized egg to out membrane period was 30 h 45 min in fresh water from 24 to 26 ℃ and pH from 7.0 to 7.5. [Conclusion] It provided important reference for studying artificial propagation and genetic breeding of loach.

Localization of Neuropeptidelike Substances During Embryonic Development in Amphibian

The localization of four neuropeptidelike substances during embryonic development in amphibian was studied by using immuno cytochemical technique. The cells with positive reaction appeared firstly in the endoderm cells during early tailbud stage, and then were detected in connective tissue at the outer portion of gastrointestinal tract during tadpole stage. In the nervous system, the cells with positive reaction were observed in cranial ganglion and glial cells at the outer margin of the brain and in the inner wall of ventricles. They were also frequently observed in the epidermis during late tailbud stage. The relationship between the appearance of neuropeptides in timespatial sequences and the development of nervous system, the neural crest origin of the cells with positive reaction, and the role of epidermal conductivity in neuropeptidelike cells in epiderms were discussed.

Study of Embryonic and Postembryonic Development of the Hybrid(Ctenopharyngodon idellus ♀ ×Elopichthys bambusa♂)

[ Objective ] The purpose was to provide a reference for crossbreeding of grass carp. [ Method] We conducted hybridization between Ctenopharyngodon ideUus （ ♀ ） and Elopichthys bambusa ♂ ） by artificial methods. The process of embryo and postembryonic development were observed and recorded. [ Result ] The fertilization rate, hatching rate and survival rate of the hybrid F1 were （75.8 ± 6. 2）%, （41.9 ± 8.2）% and （9.3± 3.7）%, respectively. At the water temperature of 20.1 -21.6℃, the larvae was hatched for about 34 h and 25 rain after fertilization. The whole length of newly-hatched larvae of the hybrid Fl was （5.8 ± 0.12） mm. The larvae could feed rotifer and unicellular algae after 3 or 4 days hatching. Postembryonic development of the hybrid at the formation of scales, lasted for 65 days when the young was（74.0 ± 2.1 ） mm in whole length. [ Conclusion] The embryonic development of hybrid F1 was intermediate to their parents, the form of the hybrid F1 was similar to that of their female line of grass carp. The growth rate of larva was faster than that of grass carp and similar to that of their paternal fish.

Preliminary Observation on Embryonic Development of Leiocassis crassilabris

[Objective] This study aimed to observe the embryonic development process of Leiocassis crassilabris. [Method] Wild L. crassilabris collected from the Nanjing section of the Yangtze River was used as parent fish for intensive breeding and propagation by artificial insemination. The entire embryonic development process of L. crassilabris from insemination to hatching out of larvae fish was observed consecutively. [Result] Fertilized eggs of L. crassilabris are spherical, and the egg diameter is about 2.2-2.4 mm after water absorption; under conditions of water temperature ranging from 27 to 28 ℃, the embryonic development of L. crassilabris from insemination to hatching out of larvae fish lasted 2 496 min, with a total accumulated temperature of 1 123-1 165 h·℃. [Conclusion] This study is advantageous to better understand the characteristics of embryonic development of L. crassilabris and provides basic biological data for protection and utilization of fish resources and other related work.

Metabolism of Hydrogen Peroxide in the Course of Embryonic Development in Silkworm

Metabolism of hydrogen peroxide in the course of embryonic development ofsilkworm (variety Guang) was determined by using colorimetric analysis and oxygen electrodemethod. The results indicated that: 1) In the course of fertilization (0-4 h after egg laying), thelevel of H_2O_2 content reached its peak value at 2.5 h of the developmental course and the activity ofsuperoxidase dismutase (SOD) was at higher level while the activity of catalase (CAT) at the lowestcorrespondingly; 2) The H_2O_2 content in embryo, in which the diapause of eggs was being relievedthrough treatment with hydrochloric acid on time in the course of embryonic development, wassignificantly higher than that of diapause eggs except the lower level showed in the embryo whenthe development of it went on for 168~216 h and the activity of SOD reached, lower and higher,tWo peaks in 72 and 168 h after egg-laying, respectively, and was significantly higher in late stagewhile the activity of CAT was shown with a stable level in the period of 72-192 h after egg-laying,and, after then, a rapid rising occurred in the embryo. The level of the CAT activity in embryowas shown significantly lower than that in diapause eggs in early period and higher in late stage ofegg development; 3) In the course of formation of diapause in eggs, the level of H_2O_2contentchanged smoothly and the activity of SOD changed vigorously in early period, and kept stable statelater; and the CAT activity increased gradually; while in the course of relief of diapause under ontime-treatment with hydrochloric acid, the level of H_2O_2 was significantly higher than that indiapause eggs and the SOD activity displayed a new peak value and significantly rose in later stage,while the activity of CAT in relieving embryo from diapause was signincantly lower than that indiapause eggs. Combining the results obtained in other researches with those from ours mentionedabove, we suggest that the metabolism of H_2O_2 might be of importance in the courses of formationand relief of diapause in silkworm eggs. Maybe the esterase A4 timer hypothesis and themicropylar barrier to oxygen hypothesis could be integrated for explanation of the course offormation and relief of diapause in silkworm eggs.

Application of mRNA Differential Display to the Identification of Genes Related to Embryonic Development

mRNA differential display was established by Liang and Pardee in 1992 for the purpose of displaying the mRNA differences between two tissues. The early embryonic development in animals is primarily controlled by the maternal RNAs stored in egg. These mRNAs are being degraded as the development proceeds. In some animals, such as fish and amphibian, new transcripts do not appear until the midblastula stage (midblastula transition, MBT). In other animals, for example in mouse, the zygotic genes are expressed during very early stages of development. The mRNA programmed synthesis and degradation during embryonic development controls the cell differentiation, germlayer formation and pattern formation. All these mRNA changes could be displayed side by side as cDNA band differences by mRNA differential display and the genes corresponding to these differential mRNAs could thus be obtained.

Embryonic and Postembryonic Development of Percocypris pingi pingi( Tchang)

This study aimed to investigate the morphological characteristics of Percocypris pingi pingi （Tchang） at the embryonic and postembryonic developmental periods and to demonstrate the early developmental stage of P. pingi pingi （Tchang）. According to the results, the fully mature eggs were spherical, orange-col- ored, sticky, 2.2 mm in diameter; the maximum outer diameter reached 3.2 -3.8 mm after absorbing water. When water temperature was （18 -＋ 0.5） ~C, the first cleavage occun~ at 2h 28 min post-fertilization; organs began to form at 45 h 23 min post-fertilization; larvae were hatched at 126 h 28 min post-fertilization. Newly hatched larvae exhibited a total length of 10.4 mm, with large and compressed yolk sacs. According to the morphological characteristics, the embryonic de- velopmental process of P. pingi pingi （Tchang） could be divided into seven continuous stages, including fertilized egg stage, cleavage stage, blastula stage, early gastrula stage, neurala stage, organ formation stage and hatching stage, which were further divided into 32 phases. When water temperature ranged from 18.8 ~C to 25.0 ~C, the whole developmental process of P. pingi pingi （Tchang） from larvae to juveniles lasted 93 d. According to the morphological and ecological charac- teristics, the pestembryonic developmental process of P. pingi pingi （Tchang） could be divided into three stages, including yolk-sac larva stage, late larva stage and junenile stage. This study laid solid foundation for .

Observation on Embryonic Development of Hybrid between Ryukin(♀) and Dragon-eye(♂)

[Objective] This study aimed to observe the embryonic development of the F1 hybrid between Ryukin （♀） and Dragon-eye （♂）. [Method] Embryonic development of the FI hybrid and parents between Ryukin （♀） and Dragon-eye （♂） was observed with XTL-2400 anatomical lens continuously. The organ characteristics and structures at each development stage were observed with OLYMPUS CKX41 microscopes, and photographed and recorded with a digital camera. [Result] At a water temperature of 25℃, the hatching time of the F1 hybrid is 51.3 h, the hatching time of Ryukin embryo is 52.4 h and that of Dragon-eye embryo is 56.3 h. [Conclusion] This study provides certain theoretical and practical basis for improving morphological characteristics of the existing Dragon-eye goldfish.

Study on Artificial Propagation and Embryonic Development of Poyang Lake Catfish

Poyang Lake catfish （Silurus asotus Linnaeus） is native to Poyang Lake Basin. Artificial propagation of Poyang Lake catfish was carried out by using artificial insemination. Under water temperature of 18-26 ℃, embryonic development of Poyang Lake catfish in impounded water was observed and recorded in detail. Embryonic development of Poyang Lake catfish encountered seven stages, and it last- ed for 47.08 h from the fertilized egg stage to the stripping stage. The hatching time of Silurus meridionalis Chen was shortest, followed by that of Poyang Lake catfish, and the hatching time of Pelteobagrus vachelli was longest.

STUDY ON EMBRYONIC DEVELOPMENT AND EARLY GROWTH OF TRIPLOID AND GYNOGENETIC DIPLOID LEFT-EYED FLOUNDER, PARALICHTHYS OLIVACEUS (T. et S.)

The early effects of chromosomal manipulation of eggs and sperm on the yields of triploid and gynogenetic diploid larvae of Paralichthys olivaceus were investigated. Triploidy was achieved by cold shocking fertilized eggs at 0-2℃ for 45 minutes duration 5 minutes after fertilization, and the induced triploidy rates were 31.2%-50% and the relative hatching rates were 53.3%-99%. Gynogenetic diploids were obtained when eggs were inseminated with irradiated sperm and cold shocked at 0-2℃ for 45 minutes duration 5 minutes after fertilization. The induced gynogenetic diploid rates and the relative hatching rates were 94%-96% and 48.5%-68.5% respectively. The embryonic development of the triploid experimental group and of the gynogenetic diploid experimental group was delayed at first compared with the control group. But from the gastrula stage, it was not delayed anymore. There were no significant differences in the growth of the triploid experimental group larvae and the control group larvae, and in the growth of the gynogenetic diploid experimental group larvae and the control group larvae according to Student’s t test (α=0.05). The relationship between the early growth of the triploid experimental group larvae and that of gynogenetic diploid experimental group larvae was also studied.

Effects of Salinity on Embryonic Development, Survival, and Growth of Crassostrea hongkongensis

This study examined the effects of salinity on embryonic development, survival, and growth of the Hong Kong oyster Crassostrea hongkongensis. The embryos, larvae, and juveniles of C. hongkongensis were held in artificial seawater at three different salinities(low, 15; medium, 23; and high, 30) to determine the optimum hatchery and nursery conditions for mass production of the seeds. Results showed that the percentage production of straight-hinged larvae from fertilized eggs was significantly lower at the high salinity than at the low- and medium-salinities(P < 0.05). The survival rates of larvae and juveniles differed significantly among the three salinity trials, with the highest survival rate observed at the low salinity(P < 0.05). The shell height of larvae was significantly larger at the low salinity than at the high and medium salinities from days 9 to 15(P < 0.05), whereas that of juveniles was significantly larger at the low salinity than at the high and medium salinities on day 70(P < 0.05). These results indicate that the larvae and juveniles of C. hongkongensis are tolerant to a wide range of salinities(15 to 30), but show better growth and survival at relatively low salinities. Thus, it is recommended to use relatively low salinities in hatchery and nursery systems for improved yields of C. hongkongensis.

Study on Involvement of the GTPase RABE1b in Embryonic Development of Arabidopsis thaliana

[Objective] This study aimed to study the genes affecting division mode in the early embryonic development of Arabidopsis thaliana.[Method] Two T-DNA insertion mutants were isolated from the mutant library of A.thaliana inserted by T-DNA,and the embryonic development was reflected by phenotypes.[Result] Genomic PCR-WALKING analyses demonstrated that T-DNA insertion sites were in 5＇ non-coding region and promoter region of gene At4g20360 which encodes Rab GTPase RABE1b,and the two mutants were named as Atrabe1b-1 and Atrabe1b-2,respectively.Microscopic analysis on mutants revealed that there was an abnormal cell division pattern in early globular stage of embryonic development.In addition,RT-PCR analysis showed that gene At4g20360 was constitutively expressed in A.thaliana.[Conclusion] Gene At4g20360 affected the division pattern of early embryonic development in A.thaliana,and encoded protein GTPase RABE1b which may play an important role in cell division during the embryonic development of A.thaliana.

Effect of in ovo zinc injection on the embryonic development, tissue zinc contents, antioxidation, and related gene expressions of broiler breeder eggs

Two experiments were conducted to investigate the effect of in ovo zinc （Zn） injection on the embryonic development, tissue Zn contents, antioxidation and related gene expressions of fertilized eggs of Arbor Acres broiler breeders. Experiment 1 was conducted to determine an optimal embryonic age for early in ovo injection. A total of 720 fertilized eggs with similar weights were randomly allotted to 4 treatments with 6 replicates per treatment and 30 eggs per replicate in a completely randomized design. The eggs were injected with 0.1 mL sterilized water at 3, 6 and 9 embryonic days of incubation （E3, E6 and E9） or non-injection （the control）, respectively. The results from experiment 1 showed that E3 and E6 injections increased （P〈0.05） the embryonic mortalities, and decreased （P〈0.05） hatchabilities compared to the non-injected control, but no differences （P〉0.05） between E9 injection and the non-injected control were observed in either embryonic mortality or hatchability. The findings suggest that the E9 is the optimal embryonic age for early in ovo injection. In experiment 2, a total of 672 fertilized eggs with similar weights were randomly allocated to 7 treatments with 6 replicates per treatment and 16 eggs per replicate in a completely randomized design. The eggs were injected with 0 （the negative control）, 50, 100, 150, 200, or 250 μg Zn/egg as reagent grade ZnSO4.7H20 in a 0.1-mL solution, or non-injection （the positive control）, respectively at E9-10. The results from the experiment 2 demonstrated that no differences （P〉0.05） among 50 and 100 μg Zn/egg groups and the negative control were observed in the embryonic mortality and hatchability, however, the injection of 200 μg Zn/egg increased （P〈0.05） the embryonic mortality, and injections of 150 and 200 μg Zn/egg decreased （P〈0.05） hatchabilities compared with the controls. The embryonic tibia Zn contents at E20 were increased （P〈0.05） by injections of 150, 200 and 250μg Zn/egg. Zinc injection did not affect （P〉0.05） malonaldehyde （MDA） contents, copper- and Zn- containing superoxide dismutase （CuZnSOD） activities and mRNA expression levels in the liver and heart of chick embryos at E15 and E20. Compared with the negative control, injections of 50, 150 and 200 μg Zn/egg up-regulated （P〈0.05） themetallothionein （MT） mRNA expression levels in the embryonic liver at E20. These results indicated that in ovo Zn injections increased Zn contents in the embryonic tibia and MT mRNA expression levels in the embryonic liver at E20, however, injections of 150-200 μg Zn/egg were harmful to the embryonic development.

Study on Embryonic Development, Larva,Juvenile and Young Fish of Epinephelus fuscoguttatus

This study aimed to study the development characteristics of Epinephelus fuscoguttatus at the embryo, larval, juvenile and young fish stages, thereby improving the breeding efficiency of E. fuscoguttatus. [Method] The morphological characteristics and embryonic and post embryonic development of E. fuscoguttatus, including the occurrence and development of nervous system, digestive system and circulatory system, were observed and analyzed using eggs self-fertilized with artificially induced spawning in the breeding base of Hainan Province. [Result] The fertilized egg of E. fuscoguttatus is spherical, colorless transparent with single oil ball, and its diameter ranges from 0.83 to 0.94 mm. Based on the development process, the embryonic development of E. fuscoguttatus was divided into six stages, namely fertilized egg stage, cleavage stage, blastula stage, gastrula stage, idiosome formation stage and hatching stage. The entire embryonic development of E. fuscoguttatus required 20 h 47 min in the sea water with temperature of 28.4-31.9℃ salinity of 31-32 and pH of 8.4, and the total cumulative temperature was 455.361 ℃·h. The post embryonic development of E. fuscoguttatus was divided into larval stage, juvenile stage and young fish stage based on the features of the yolk-sac, second dorsal fin spine, pelvic fin spine, and body color. Accordingly, the larval stage was further classified into pre-larva and post-larva, with or without yolk-sac, respectively. The extension and shrinkage of the first pelvic fin spine and the second dorsal fin spine as well as the spinules on aforementioned spines were the most significant changes during the post embryonic development of E. fuscoguttatus. [Conclusion] This study will provide some basic data for industrial breeding of E. fuscoguttatus.

Embryonic development of Pelteobagrus fulvidraco(Richardson,1846)

For production enhancement and procedure upgrade, the developmental phases of labora-tory-reared eggs of catfish Pelteobagrus fulvidraco were investigated. Twenty mature females and 10 males were collected from Dadongmen wholesale fisheries market in Wuhan City on May 8, 2003. Zy-gotes were stripped from mature fish after hormone-induced ovulation, fertilized, and incubated through whole embryonic development. The fertilized eggs were stocked in density of 100 eggs/L in white square tanks of 10 L. Incubation water was dechlorinated tap water with continuous aeration. The tanks were lit directly with 60 W fluorescent bulbs with a 12 light:12 dark photoperiod. Water temperature, dissolved oxygen and pH were 29.0±0.5℃, 6.7±0.4 mg/L and 7.4±0.2, respectively. The results showed that the eggs of P. fulvidraco were yellow, sticky and contained much yolk. The mean diameter of fertilized eggs was 2.03 mm. At the water temperature of 29.0±0.5℃, the ontogenesis spent about 33 h after fertilization. From fertilization to hatching, the embryonic development can be divided into 30–40 phases, which var-ies in the emphasis and direction of development. The detailed embryonic movement was also described.

Effect of zeaxanthin on porcine embryonic development during in vitro maturation

Zeaxanthin is a common carotenoid, which is a powerful antioxidant that protects against damage caused by reactive oxygen species. The aim of the present study was to investigate the effects of zeaxanthin supplementation on in vitro maturation of porcine embryo development. We investigated nuclear maturation, intracellular glutathione （GSH）, and reactive oxygen species （ROS） levels during in vitro maturation, and subsequent embryonic development following parthenogenetic activation and in vitro fertilization OVF）. The oocytes were maturated and used at the metaphase II stage. After 42 hours of in vitro maturation, the zeaxanthin-treated group （0.5 μmol/L） showed significant increases in nuclear maturation （89.6%） than the control group （83.4%） （P 〈 0.05）. The intracellular GSH levels increased significantly （P 〈 0.05） as zeaxanthin concentrations increased; ROS generation levels decreased with increased zeaxanthin concentrations, but there were no significant differences. There were no significant differences in subsequent embryonic development, cleavage rate, blastocyst stage rate, and total blastocyst cell numbers following parthenogenetic activation and IVF when in vitro maturation media was supplemented with zeaxanthin. These results suggest that treatment with zeaxanthin during in vitro maturation improved the nuclear maturation of porcine oocytes by increasing the intracellular GSH level, thereby slightly decreasing the intracellular ROS level.

DNA methylation-mediated expression of zinc finger protein 615 affects embryonic development in Bombyx mori

Cell division and differentiation after egg fertilization are critical steps in the development of embryos from single cells to multicellular individuals and are regulated by DNA methylation via its effects on gene expression.However,the mechanisms by which DNA methylation regulates these processes in insects remain unclear.Here,we studied the impacts of DNA methylation on early embryonic development in Bombyx mori.Genome methylation and transcriptome analysis of early embryos showed that DNA methylation events mainly occurred in the 5'region of protein metabolism-related genes.The transcription factor gene zinc finger protein 615(ZnF615)was methylated by DNA methyltransferase 1(Dnmt1)to be up-regulated and bind to protein metabolism-related genes.Dnmt1 RNA interference(RNAi)revealed that DNA methylation mainly regulated the expression of nonmethylated nutrient metabolism-related genes through ZnF615.The same sites in the ZnF615 gene were methylated in ovaries and embryos.Knockout of ZnF615 using CRISPR/Cas9 gene editing decreased the hatching rate and egg number to levels similar to that of Dnmt1 knockout.Analysis of the ZnF615 methylation rate revealed that the DNA methylation pattern in the parent ovary was maintained and doubled in the offspring embryo.Thus,Dnmt1-mediated intragenic DNA methylation of the transcription factor ZnF615 enhances its expression to ensure ovarian and embryonic development.