Field emission scanning electron microscopy and transmission electron microscopy studies of the chorion, plasma membrane and syncytial layers of the gastrula-stage embryo of the zebrafish Brachydanio rerio: a consideration of the structural and functional relationships with respect to cryoprotectant penetration

The structure of the chorion and plasma membranes of gastrula‐stage zebrafish Brachydanio rerio embryos were studied using field emission scanning electron microscopy (FE‐SEM) and transmission electron microscopy (TEM). These studies confirm the outer chorion membrane complex to be 1.5–2.5 μm in thickness and to consist of three layers, electron‐dense outer and innermost layers (0.2–0.3 and 1.0–1.6 μm in thickness respectively) separated by an electron‐lucent middle layer (0.3–0.6 μm in thickness). The middle and inner layers are pierced by pore canals. A granular to farinaceous nature of the thin outer surface of the outer layer of the chorion has been revealed for the first time. The study provides original TEM images of the plasma membrane structures of gastrula‐stage embryos, and FE‐SEM and TEM images showing the plasma membrane to have three morpohologically distinct regions, being prominently ridged and folded at the surface of the blastoderm, smooth over the syncytial layer at the vegetal pole and with an intermediate region between the animal and vegetal pole where folding develops in advance of the expanding blastodermal disc of cells. FE‐SEM and TEM studies reveal details of the syncytial layer (1–4 μm thick) beneath the smooth plasma membrane at the vegetal pole, containing cytoplasmic organelles and small yolk globules. The significance of the structural detail shown in these studies is considered in the light of the difficulties experienced in cryopreservation of the embryo resulting from the inability of achieving cryoprotectant penetration of the yolk mass.     

Primary experimental infection of riverine buffaloes with Fasciola gigantica.

The clinical course of the primary experimental Fasciola gigantica infection was investigated in riverine buffalo calves of the Murrah breed. Nine male calves aged 12-15 months were randomly assigned to two groups of five (Group I) and four (Group II) animals. Each animal in Group I, was orally infected with 1000 metacercariae (mc) of F. gigantica, whereas Group II animals did not receive any infection dose and served as uninfected controls. No clinical signs of fasciolosis were observed until the sixth week post-infection (PI). Group I animals, however, developed recognised symptoms of acute fasciolosis, comprising apyrexic inappetance, anemia, poor weight gain, diarrhoea and sub-mandibular and facial oedema, respectively, from 5, 6, 8, 16 and 17 weeks PI. The signs were intermittent in nature and of variable duration. The prepatent period was of 92-97 days (mean 95.2 +/- 3.1). One of the five infected animals died on Day 147 PI. At necropsy, 36.8 +/- 11.0% of the infection dose was recovered as adult fluke population. The gross lesions were primarily biliary in nature. Group II, the uninfected controls, throughout the study period of 165 days PI, did not show any symptom and were negative for F. gigantica. The study demonstrated that the onset of adverse effects of F. gigantica on the growth and health of the infected host was mainly noted during late prepatency much before coprological prediction and diagnosis. The significance of preventive therapy against fasciolosis during prepatency has been stressed in endemic areas.     

Effect of Fasciola gigantica infection on adrenal and thyroid glands of riverine buffaloes

Effect of Fasciola gigantica infection on adrenal and thyroid glands was investigated using eight male, yearling Murrah buffaloes. The animals were randomly assigned to two groups of four buffaloes each (Group-A, infected; Group-B, non-infected control). Animals of Group-A were orally infected with 1000 F. gigantica viable metacercariae, keeping other four animals of Group-B as uninfected control. In the infected buffaloes, the clinical signs began appearing from 7th week postinfection (p.i.) and eggs were detected in the faeces between day 93 and 99 (95.5+/-1.25) postinfection (p.i.). The serum cortisol level, revealed a significant (P<0.05) rise during initial stage of the infection, followed by a continuous fall from 12th week onward. Peak cortisol level on 10th week (13.30+/-2.57ngml(-1)) was associated with eosinophilia (11.0+/-0.95%). However, non-infected controls maintained almost uniform cortisol levels (3.97+/-0.15-5.88+/-0.09ngml(-1)) throughout the period of the study. The pathological changes of adrenal glands were correlated with physiological dysfunction of the glands. The levels of T(3) and T(4) were significantly (P<0.05-0.01) low from 14th week onward and were synchronous with in situ migration, growth and development of F. gigantica. Significant reduction in the thyroid hormones was further supported by histopathological evidence of lymphocytic thyroiditis confirming hypothyroidism. A decrease in Hb, PCV, total erythrocyte counts and appearance of reticulocytes in the blood of the infected buffaloes suggested regenerative anemia, which could partly be due to hypothyroidism.