Cloning of a quail homologue of hatching enzyme: its conserved function and additional function in egg envelope digestion

The aim of the present study was to reveal molecular entities participating in the digestion of the egg envelope in the Japanese quail, Coturnix japonica. We isolated a 1,510-bp cDNA from extraembryonic tissues of developing embryos and designated it quail hatching enzyme (QHE) cDNA. The QHE cDNA was found to code a protein molecule comprising an astacin protease domain in the N-terminal half and a complement subcomponents C1r/C1s, Uegf, Bmp1 (CUB) domain in the C-terminal half. A phylogenetic analysis showed that QHE belonged to the hatching enzyme group and was distinct from other proteases in the astacin family. Northern blotting and in situ hybridization demonstrated that expression of the QHE mRNA occurred twice during the development: first in ectodermal cells of the yolk sac on days 0–5, then in those of the albumen sac on days 8–13. Zymography revealed that proteolytic activity in extracts of days 3–4 and 9–12 embryos appeared at the position of 40 kDa. Immunoblotting tests showed that anti-QHE antiserum stained a 40-kDa molecule in extracts of day 3 area vitellina. Anti-QHE antibody stained the ectodermal cells of the area opaca on days 0–1, those of the area vitellina of the yolk sac on days 2–5, and those of the albumen sac on days 9–12. The temporal and spatial expression pattern of QHE mRNA was closely associated with digestion of the vitelline membrane occurring on days 1–4, and with that of the egg white on days 9–12.     

Exogenous administration of octanoic acid accelerates octanoylated ghrelin production in the proventriculus of neonatal chicks

Ghrelin is modified by fatty acid at the third serine residue. In this study, derivation of fatty acid for acylation of ghrelin was investigated using a hatchling chicken model. We first studied ghrelin gene expression and production in the neonatal chick proventriculus and then investigated the effect of exogenous octanoic acid (OA) administration on acylated ghrelin production. In a free-feeding condition on day 2.5 after hatching, the density of ghrelin mRNA-expressing (ghrelin-ex) cells was greater than that of ghrelin-immunopositive (ghrelin-ip) cells, but no difference was found between those densities in adult chickens. Intraperitoneal or oral administration of OA for a few days significantly increased the density of ghrelin-ip cells without any changes in ghrelin-ex cells and elevated only octanoylated ghrelin levels in the proventriculus. The results indicate that fatty acid absorbed from food is directly utilized in acylated ghrelin production in the chicken.     

非洲爪蟾孵化酶分子对卵黄膜作用方式的研究

非洲爪蟾的孵化液对卵黄膜和二甲基酷蛋白具有降妥活性。用非洲爪蟾孵化酶的特异性抗ＧＳＴ－ＵＶ．２抗体进行Ｗｅｓｔｅｒｎ杂交的结果表明,孵化液中出现一种分子量为６０ｋＤ的大组分,有时也会出现一种分子量为４０ｋＤ的小组分。     

Successful sperm cryopreservation of the brown-marbled grouper,Epinephelus fuscoguttatus using propylene glycol as cryoprotectant

This study developed the cryopreservation of brown-marbled grouper spermatozoa for practical application. We examined 32 cryodiluents, developed from four types of cryoprotectants [propylene glycol (PG), dimethyl-sulphoxide (Me₂SO), dimethyl-acetamide (DMA) and ethylene glycol (EG)] at four concentrations of 5, 10, 15 and 20% in combination with two extenders [Fetal bovine serum (FBS) and artificial seminal plasma (ASP). Cooling rates were examined by adjusting the height of straws (2.5–12.5 cm) from the liquid nitrogen (LN) vapor and cooled for 5 min before immersion into LN. DNA laddering was used to detect DNA damage in cryopreserved sperm. In fertilization trials, 0.5 g of eggs was mixed with cryopreserved sperm stored for 30 days in LN. The best motility of post-thaw sperm was achieved using 15% PG + 85% FBS (76.7 ± 8.8%); 10% PG + 90% FBS was also effective as cryodiluent. Generally, FBS gave better post-thaw motility compared to ASP. The optimum cooling rate was at 17.6 °C min⁻¹ obtained by freezing at the height of 7.5 cm surface of LN. The results obtained showed that cryopreserved sperm of brown-marbled grouper suffered slight DNA fragmentation, which resulted in significantly lower motility. However, the fertilization (90.9 ± 0.5%), hatching (64.5 ± 4.1%) and deformity rates (3.8 ± 0.2%) obtained from cryopreserved sperm showed no significant difference with fresh sperm. These findings show that the developed protocol for cryopreservation of brown-marbled grouper sperm was viable and will be useful for successful breeding and seed production of brown-marbled grouper.     

Elongated microvilli support the sea urchin embryo concentrically within the perivitelline space until hatching

Summary The early sea urchin embryo is supported in a concentric position within the perivitelline space by elongated microvilli which are attached to the fertilization envelope by extracellular matrix fibers. This “attachment complex,” of microvillus tip: extracellular matrix fibers: fertilization envelope, was revealed by two methods: the use of pronase or calcium-free sea water to dissolve the extracellular matrix fibers, thus causing the eggs to lose their concentric location, and the visualization of the “attachment complex” using video-enhanced differential interference contrast microscopy and transmission electron microscope images. The presence of the “attachment complex” helps in understanding two types of early developmental events: (1) the apparently continual change in microvillus length during cleavage stages which retains the embryos in their concentric position and (2) the hatching process.     

歪水蚤两种生理型卵的季节变化及其温度效应

实验室观测表明,3种歪水蚤的卵皆覆以半透明外鞘,仅特氏歪水蚤生产一定数量的不孵的无鞘卵。特氏歪水蚤夏卵与滞育卵的外鞘形态不同,前者较窄(≤15μm)且折皱均匀,后者多宽而折皱不均匀,随其外鞘加宽,活力明显提高。在钳形歪水蚤,两种卵的形态无明显差异;而右突歪水蚤尚未发现能够生产滞育卵。歪水蚤卵型的复杂多变,可能与其能够生产两种不同生理型卵的遗传特性正相适应。在短至12小时和6天的驯养期内,右突歪水蚤所产卵的卵径和鞘宽均分别与温度呈显著负相关及正相关,并随季节而异;这进一步表明动物对本海区温度季节性变化的适应。     

A review of toxicity testing protocols and endpoints with Artemia spp.

Artemia spp. is an historically popular biological model still requiring an official internationally based standardization. Several endpoints are currently available. Short-term acute endpoints include biomarker (acetylcholinesterase; heat stress proteins; lipid peroxidation; thiobarbituric acid reactive substances; thioredoxin reductase; glutathione-peroxidase; glutathione S-transferase; glutathione reductase; aldehyde dehydrogenase; and adenylpyrophosphatase and Fluotox), hatching (dry biomass, morphological disorders and size), behavioral (swimming speed and path length), teratogenicity (growth), and immobilization (meaning mortality after 5–30 s observation). Long-term chronic tests focus on growth, reproduction and survival or mortality after 7–28 d exposure from larval to adulthood stage. We analyzed each test looking at its endpoint, toxicant and experimental design including replicates, exposure time, number of exposed cysts or organisms and their relative life stage, exposure conditions during hatching and testing (salinity, pH, light intensity, aeration dilution media, and food supply), type of testing chambers, and quality assurance and quality control criteria. Similarities and differences between the identified approaches were highlighted. Results evidenced that hatching 24 h short-term and 14 d long-term mortality are the most promising Artemia spp. protocols that should go forward with international standardization.     

Effect of brood size and hatching sequence on prefledging mortality of Sandwich terns: why lay two eggs?

The mortality of Sandwich tern Sterna sandvicensis chicks held in enclosures was studied in colonies on Griend, in the Dutch Wadden Sea, from 1992 to 1999, and on Hirsholm, in the Danish Kattegat, in 1997. Survival of chicks until fledging was 73% for chicks hatching from first-laid eggs or single-egg clutches and 59–64% for partially hatched two-egg clutches, whereas 6% of second hatchlings survived until fledging. Less than 2% of all two-chick broods actually fledged two chicks. Because 18% of the two-egg clutches only hatched one egg, 7% of fledglings of two-egg clutches originated from a second-laid egg. In nests where both eggs hatched, the number of chicks was usually reduced soon after hatching. Within five days of hatching more than 50% of the second hatchlings died of starvation or were preyed upon. It seems that overproduction commonly occurs in Sandwich terns and that investment in a surplus egg mainly serves as an insurance mechanism. On Griend and Hirsholm, chick productivity of two-egg clutches was somewhat higher than for one-egg clutches. Undernourishment was an important cause of death, either directly by starvation or by selective predation of chicks in poor condition. This, in combination with earlier, studies suggests that Sandwich tern parents on Griend are exposed to severe food stress.     

Optimal hatching conditions of Zophobas atratus (Coleoptera: Tenebrionidae) eggs under various culture conditions

Supermealworm, Zophobas atratus (Coleoptera: Tenebrionidae) is a tropical species used for human consumption and as an ingredient in animal feed. We investigated the hatching characteristics of Z. atratus eggs under varying conditions to optimize artificial rearing conditions. Overall, the average egg weight, width, and length decreased by 42.3%, 3.7%, and 10.1%, respectively, on the 6th day post-oviposition. We varied either temperature, photoperiod, illuminance, or humidity, while the other conditions remained constant. Of the four experimental temperatures—25 °C, 27 °C, 30 °C, and 33 °C–30 °C was associated with the highest hatching rate (91.0%) during days 5–7. Although other experimental conditions did not have a significant effect on hatchability, we were able to elucidate the ideal conditions for improved hatchability. For photoperiod, hatchability was the highest (84%) at 12L/12D, followed by 81.0% at 15L/9D, and 70% at 9L/15D. Among the illuminance conditions tested—600, 1800, and 3000 lx—hatchability was the highest at 1800 lx (82.3% on days 4–6). Among the relative humidity (RH) levels tested—50%, 65%, and 80%—hatchability was the highest at 65% RH (88.5% on days 5–6). Thus, the ideal conditions for maximum hatchability were 30 °C, 12L/12D, 65% RH, and 1800 lx. Under these combined conditions, the hatching rate increased by 93.6%, and the hatching time was reduced by two days.     

1-苯基-2-硫脲对斑马鱼胚胎发育与黑色素生成的影响

目的1-苯基-2-硫脲（PTU）可抑制斑马鱼胚胎黑色素的产生,保持斑马鱼透明,便于形态观察和信号检测。本文研究了PTU对斑马鱼胚胎发育的影响和抑制斑马鱼胚胎黑色素生成,保持斑马鱼透明性的最佳浓度。方法用不同浓度PTU处理23hpf（受精后,hourspostfertilization,hpf）斑马鱼胚胎,作用57h后观察80hpf斑马鱼的形态学、生理学改变,计算死亡率和孵化率,测量心率和静脉窦-动脉球之间的距离。结果浓度为0．197mmo]／L、0．296mmol／LPTU可以有效抑制黑色素生成,保持斑马鱼整体透明,对斑马鱼心血管系统结构和生理功能无影响,且不影响斑马鱼正常孵化过程。随着PTU浓度的增加,斑马鱼死亡率增加,孵化率下降,出现心包水肿,心脏畸形等改变,心率下降,静脉窦-动脉球之间的距离增大。结论浓度不高于0．296mmol／L的PTU溶液能有效抑制斑马鱼黑色素生成,对斑马鱼心血管毒性研究无影响。