三种海水经济鱼类早期发育生物学的研究

海水经济鱼类的养殖在我国已经形成第四次海水养殖浪潮，经济效益显著，有力地推动了我国海水养殖的产业结构调整和可持续发展。然而在海水养殖发展过程中也存在着诸多问题，尤其是早期发育阶段的高死亡率，严重制约了我国海水养殖产业的稳定和健康发展。 海水鱼类养殖的关键为高质量，高存活率苗种的生产和培育，由于鱼类种类繁多，生物多样性丰富，对应实际的繁育技术，尤其是新品种的开发，必须要做出相应的调整。这就要求我们必须对每一种鱼类早期发育有所了解，并将形态和组织上的数据用于指导生产。 本文通过显微观察和组织学研究，主要描述和研究了我国北方三种重要的海水经济鱼类(条斑星鲽、杂交鲆、条石鲷)的早期发育生物学，并结合实际生产进一步阐明关键期的产生原因，机理以及采用相应的对策。具体结果如下： 1.条斑星鲽：作为冷温性鲆鲽鱼类，条斑星鲽早期发育过程的特征主要有： ① 条斑星鲽受精卵无油球，卵子呈半浮性；不同步卵裂现象提前，发生在第三次卵裂；卵裂期裂球大小差异大。孵化过程较长，在水温8 ± 0.3℃，盐度33的条件下，经9 d孵化。条斑星鲽胚胎发育的不同时期对温度的敏感性不同，其中原肠期对温度比较敏感。 ②在8-10℃，盐度33的条件下，8-9 dph开口摄食。且开口时，其吻前端出现有一点状黑褐色素，构成了条斑星鲽仔鱼“开口期”的重要标志。卵黄囊于消失。在后期仔鱼末期，背鳍和臀鳍上形成特有的黑褐色条斑带。 ③杯状细胞首先出现在咽腔后部和食道前段，胃腺和幽门盲囊出现于29 dph，变态期始于30dph。在条斑星鲽早期发育过程中，观察到其直肠粘膜层细胞质出现大量嗜伊红颗粒，为仔鱼肠道上皮吸收的蛋白质。 ④首先淋巴化的免疫器官是头肾，然后是胸腺和脾脏，这与大部分硬骨鱼类不同。条斑星鲽除头肾和脾脏外，胸腺实质也形成MMCs。其中以脾脏形成MMCs最为丰富，形态多样。 2. 杂交鲆：为同属的牙鲆和夏鲆间的远缘杂交种，其发育过程的特点为： ① 在温度为15.4～16.0℃，杂交鲆胚胎从受精到孵化所需的时间为76 h左右，胚孔关闭前期，胚胎先出现视囊及克氏囊，而后形成体节。孵出前胚体在卵膜内环绕不到1周。 ② 孵化后消失。杂交鲆群体变态间隔长(34-60 dph)，且变态高峰期出现的冠状幼鳍不明显(与母本牙鲆相比)，数量为7-8根。 ③组织学观察发现，其消化系统中胃腺出现较晚，且胃腺发育过程缓慢(与母本牙鲆相比)。甲状腺滤泡增生不明显，颜色较浅，数量较少。杂交鲆在早期发育过程中，并没有出现鳔原基。 3. 条石鲷作为岩礁性的暖水性鱼类，早期发育过程也较为特殊，包括外形以及内部的器官结构。主要特点有： ① 受精卵：受精卵卵黄上具有龟裂结构，为鱼卵的分类特征之一。 ② 初孵仔鱼：初孵仔鱼背鳍膜上的黑色素，从体背面向背鳍膜边缘移动，到3dph仔鱼基本消失，此为本种仔鱼发育所特有的特点。 ③ 后期仔鱼和稚鱼：肠道肌肉层加厚明显，仔稚鱼胃肠排空率急剧上升，死亡率增加，通过改善常规的投饵方式部分解决了这个死亡高峰的问题。在幼鱼初期，牙齿融合为骨喙，为石鲷科鱼类的特征。 ④胸腺上皮分泌细胞：类似的现象同样在虹鳟鱼中发现，但是虹鳟鱼胸腺上皮分泌细胞不如条石鲷的丰富，同样也不如条石鲷的排列整齐，而是零星分布在胸腺上皮与咽腔接触的表面。除了正常的造血器官—脾脏和头肾外，肝脏、胰腺和鳔等多种组织等也出现MMCs，此现象在硬骨鱼类不多见，一般发生在软骨鱼类。

Physiologic indicators of vitamin A status.

Physiologic indicators reflect the functional consequences of vitamin A deficiency and may be particularly useful for detecting early perturbations in vitamin A status. In conjunctival impression cytology (CIC), epithelial morphology and the presence or absence of mucin spots and goblet cells allow samples, obtained by applying filter paper to the temporal conjunctiva, to be characterized as normal or typical of vitamin A-deficient keratinizing metaplasia. The validity of CIC has been established with reference to other indicators of vitamin A status, and a prevalence of > or =20% abnormal results has been suggested as indicative of a public health problem. However, interpretation of specimens requires considerable training, and nonresponsiveness to supplementation is a frequent problem, which limits the utility of CIC as a method for evaluating the impact of intervention programs. Several simplified field protocols for dark adaptation have been developed, including one in which dark adaptation is assessed by the responsiveness of the pupil to light. Night blind subjects have consistently shown abnormal results on this test, and a significant response to placebo-controlled dosing has been demonstrated for children and pregnant women. Scores have correlated significantly with serum retinol and relative dose response. Pupillary dark adaptation testing is acceptable to most children as young as 2 y old. Limitations of this technique include a time course for recovery after dosing as long as 4-6 wk, a testing time of 20 min, and the need for 1-3 d of training. Given its low cost, noninvasive nature, and lack of the need to transport samples, pupillary dark adaptation offers advantages over other techniques for assessing a population's vitamin A status.

Response of porcine intestinal in vitro organ culture tissues following exposure to Lactobacillus plantarum JC1 and Salmonella Typhimurium SL1344.

The development of novel intervention strategies for the control of zoonoses caused by bacteria such as Salmonella spp. in livestock requires appropriate experimental models to assess their suitability. Here, a novel porcine intestinal in vitro organ culture (IVOC) model utilizing cell crown (CC) technology (CCIVOC) (Scaffdex) was developed. The CCIVOC model was employed to investigate the characteristics of association of S. enterica serovar Typhimurium strain SL1344 with porcine intestinal tissue following exposure to a Lactobacillus plantarum strain. The association of bacteria to host cells was examined by light microscopy and electron microscopy (EM) after appropriate treatments and staining, while changes in the proteome of porcine jejunal tissues were investigated using quantitative label-free proteomics. Exposure of porcine intestinal mucosal tissues to L. plantarum JC1 did not reduce the numbers of S. Typhimurium bacteria associating to the tissues but was associated with significant (P < 0.005) reductions in the percentages of areas of intestinal IVOC tissues giving positive staining results for acidic mucins. Conversely, the quantity of neutrally charged mucins present within the goblet cells of the IVOC tissues increased significantly (P < 0.05). In addition, tubulin- was expressed at high levels following inoculation of jejunal IVOC tissues with L. plantarum. Although L. plantarum JC1 did not reduce the association of S. Typhimurium strain SL1344 to the jejunal IVOC tissues, detection of increased acidic mucin secretion, host cytoskeletal rearrangements, and proteins involved in the porcine immune response demonstrated that this strain of L. plantarum may contribute to protecting the pig from infections by S. Typhimurium or other pathogens.

Purified galactooligosaccharide, derived from a mixture produced by the enzymic activity of Bifidobacterium bifidum, reduces Salmonella enterica serovar Typhimurium adhesion and invasion in vitro and in vivo

The prebiotic Bimuno (R) is a mixture containing galactooligosaccharides (GOSs), produced by the galactosyltransferase activity of Bifidobacterium bifidum NCIMB 411 71 using lactose as the substrate Previous in vivo and in vitro studies demonstrating the efficacy of Bimuno (R) in reducing Salmonella enterica serovar Typhimurium (S Typhimurium) colonization did not ascertain whether or not the protective effects could be attributed to the prebiotic component GOS Here we wished to test the hypothesis that GOS, derived from Bimuno (R) may confer the direct anti-invasive and protective effects of Bimuno (R) In this study the efficacy of Bimuno (R), a basal solution of Bimuno (R) without GOS [which contained glucose, galactose, lactose, maltodextrin and gum arabic in the same relative proportions (w/w) as they are found in Bimuno (R)] and purified GOS to reduce S Typhimurium adhesion and invasion was assessed using a series of in vitro and in vivo models The novel use of three dimensionally cultured HT-29-16E cells to study prebiotics in vitro demonstrated that the presence of similar to 5 mg Bimuno (R) ml(-1) or similar to 2 5 mg GOS ml(-1) significantly reduced the invasion of S Typhimurium (SL1344nal(r)) (P<0 0001) Furthermore, similar to 2 5 mg GOS ml(-1) significantly reduced the adherence of S Typhimurium (SU 344nal(r)) (P<0 0001) It was demonstrated that cells produced using this system formed multi-layered aggregates of cells that displayed excellent formation of brush borders and tight junctions In the murine ligated deal gut loops, the presence of Bimuno (R) or GOS prevented the adherence or invasion of S Typhimurium to enterocytes, and thus reduced its associated pathology This protection appeared to correlate with significant reductions in the neutral and acidic mucins detected in goblet cells, possibly as a consequence of stimulating the cells to secrete the mucin into the lumen In all assays, Bimuno (R) without GOS conferred no such protection, indicating that the basal solution confers no protective effects against S Typhimurium Collectively, the studies presented here clearly indicate that the protective effects conferred by Bimuno (R) can be attributed to GOS

Lactulose and Lactobacillus plantarum, a potential complementary synbiotic to control postweaning colibacillosis in piglets

The potential of a prebiotic oligosaccharide lactulose, a probiotic strain of Lactobacillus plantarum, or their synbiotic combination to control postweaning colibacillosis in pigs was evaluated using an enterotoxigenic Escherichia coli (ETEC) K88 oral challenge. Seventy-two weanlings were fed four diets: a control diet (CTR), that diet supplemented with L. plantarum (2 × 10(10) CFU · day(-1)) (LPN), that diet supplemented with 10 g · kg(-1) lactulose (LAC), or a combination of the two treatments (SYN). After 7 days, the pigs were orally challenged. Six pigs per treatment were euthanized on days 6 and 10 postchallenge (PC). Inclusion of lactulose improved the average daily gain (ADG) (P < 0.05) and increased lactobacilli (P < 0.05) and the percentage of butyric acid (P < 0.02) in the colon. An increase in the ileum villous height (P < 0.05) and a reduction of the pig major acute-phase protein (Pig-MAP) in serum (P < 0.01) were observed also. The inclusion of the probiotic increased numbers of L. plantarum bacteria in the ileum and colon (P < 0.05) and in the total lactobacilli in the colon and showed a trend to reduce diarrhea (P = 0.09). The concentrations of ammonia in ileal and colonic digesta were decreased (P < 0.05), and the villous height (P < 0.01) and number of ileal goblet cells (P < 0.05) increased, at day 10 PC. A decrease in plasmatic tumor necrosis factor alpha (TNF-α) (P < 0.01) was also seen. The positive effects of the two additives were combined in the SYN treatment, resulting in a complementary synbiotic with potential to be used to control postweaning colibacillosis.

Sclerocorneal limbal stem cell autograft transplantation in dogs

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Morfologia e morfometria do oviduto de codornas Nothura maculosa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morfologia e histologia do oviduto de marrecas Ana boschas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeitos do espaço de alojamento e da idade sobre desenvolvimento, integridade e proteção da mucosa intestinal de frangos de corte

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Prevalence and histopathology of Neoechinorhynchus curemai Noronha, 1973 (Acanthocephala: Neoechinorhynchidae) in Prochilodus lineatus Valenciennes, 1836 from Volta Grande Reservoir, MG, Brazil

O presente trabalho estudou a prevalência e a histopatologia de Neoechinorhynchus curemai Noronha, 1973 (Acanthocephala: Neoechinorhynchidae) em curimbatá, Prochilodus lineatus Valenciennes, 1836. Dezoito peixes com comprimento total médio de 46,7 + 1,1 cm e peso médio de 1.674,8 + 75,6 g foram coletados com rede, bimestralmente, de dezembro de 1995 a dezembro de 1996 na usina hidrelétrica do Reservatório de Volta Grande (Cemig), Minas Gerais, Brasil. Dos peixes analisados, 15 estavam infectados com acantocéfalos no intestino (prevalência de 83,3%). A maior intensidade média ocorreu em agosto de 1996, com 66,5 (16 a 208) parasitos. A análise histopatológica revelou completa descamação do epitélio intestinal com severa hiperplasia e hipertrofia das células caliciformes. Observou-se, ainda, forte reação inflamatória na submucosa, deslocamento de feixes, associado a edemas, bem como infiltração mononuclear e eosinofílica.

Comparative evaluation of the susceptibility of cultivated fishes to the natural infection with myxosporean parasites and tissue changes in the host

O objetivo deste estudo foi avaliar a susceptibilidade de 4 importantes peixes cultivados a parasitos esporozoários. Os peixes foram coletados bimestralmente de um tanque de cultivo, durante 1 ano. Myxobolus colossomatis e Henneguya piaractus foram encontrados nos órgãos internos e brânquias, respectivamente. A incidência de ambos os parasitos foi de 97,3% em pacu (Piaractus mesopotamicus), 33,3% no híbrido tambacu (Piaractus mesopotamicus x Colossoma macropomum), 5,6% em tambaqui (Colossoma macropomum) e 0% em carpa (Cyprinus carpio). Pacu foi o peixe mais susceptível, encontrando-se parasitado 79,2% nas brânquias, 66,7% nos rins e 50% no baço. A análise histopatológica das brânquias mostrou hemorragias, reação inflamatória com células mononucleares, fibroblastos e hiperplasia das células basais e mucosas.

Piscinoodinium pillulare (Schäperclaus, 1954) Lom, 1981 (Dinoflagellida) infection in cultivated freshwater fish from the Northeast region of São Paulo State, Brazil: parasitological and pathological aspects

O Centro de Aqüicultura, Unesp, Jaboticabal, SP, Brasil, recebeu peixes para diagnose, os quais apresentavam aglomeração nas bordas dos viveiros e na entrada da água. Dos 194 casos diagnosticados, 53 apresentavam estruturas brancas circulares ou ovais, imóveis, medindo 162 mm de diâmetro, identificadas como o dinoflagelado Piscinoodinium pillulare. em 34 casos, os parasitos estavam presentes nas brânquias, em 2 casos, no corpo e em 9 casos, em ambos. Dos 53 casos observados, 31 eram o híbrido tambacu; 7, o Piaractus mesopotamicus; 6, o Colossoma macropomum; 5, o Leporinus macrocephalus; 3, o Oreochromis niloticus; e 1, o Prochilodus lineatus. Os peixes apresentaram aumento da produção de muco no corpo e nas brânquias e equimoses no pedúnculo caudal e nos opérculos. As brânquias também apresentaram palidez, congestão e petéquias. A histopatologia revelou a presença de grande número de trofontes situados entre as lamelas secundárias, fixados ou não ao epitélio. As lamelas primárias mostraram hemorragias intersticiais, severa hiperplasia do epitélio e das células caliciformes e infiltrado inflamatório. O presente trabalho é o primeiro relato de P. pillulare no Brasil e enfatiza a importância dos dinoflagelados, que causaram significativas perdas econômicas entre 1995 e 1997.

Modelos de neovascularização corneana em coelhos: análise morfológica

OBJETIVOS: Comparar em coelhos três modelos experimentais de destruição das células germinativas (CG) do limbo corneano quanto a aspectos morfológicos. MÉTODOS: Foram utilizados 54 coelhos, 108 olhos, subdivididos em 3 grupos experimentais: (G1), (G2) e (G3), formados por 18 coelhos cada, que tiveram o OE submetido às técnicas experimentais (T1), (T2) e (T3), respectivamente, e um grupo controle, formado por 54 olhos contralaterais (OD) dos coelhos do G1, G2 e G3. Quatro parâmetros morfológicos foram estudados: epitélio, resposta inflamatória, vascularização e resposta fibroblástica. RESULTADOS: Com a T1 não houve remoção da totalidade do epitélio límbico, com a T2 houve remoção da quase totalidade do epitélio límbico, com a T3 houve remoção da totalidade do epitélio límbico. A reparação da superfície corneana foi feita por epitélio de fenótipo conjuntival (conjuntivalização) com as três técnicas, havendo aparecimento de células caliciformes a partir do 14º dia, sendo a densidade maior no 28º dia. A resposta inflamatória com a T3 foi mais intensa que com a T1 e T2. A regressão foi mais rápida com a T1 e similar com a T2 e T3. Contribuiu para a turvação corneana principalmente no 14º e 28º dia, concentrando-se, principalmente, na metade estromal anterior, sendo poupada a metade posterior. Foi maior até o 28º dia, decrescendo a partir desse momento. Caracterizou-se, no início, por infiltrado com predomínio de polimorfonucleares e sofreu mudança no 56º dia para infiltrado com predomínio de mononucleares. Os neovasos apareceram a partir do 7º dia, a princípio permearam o estroma do terço médio para cima, progredindo para a superficialização com a regressão do edema estromal. A vascularização com as três técnicas, ao final do experimento, foi superficial no estroma, porém não houve a formação de tecido fibro-escleroso denso ou cicatriz propriamente dita distinta do estroma corneano. CONCLUSÕES: Ocorreu conjuntivalização e neovascularização nas três técnicas experimentais. As T2 e T3 mostraram-se adequadas como possíveis modelos de ampla remoção das CG límbicas, levando a resultados similares nos diversos parâmetros estudados. A T1 se mostrou adequada como modelo de remoção parcial do epitélio límbico.

Morphometry of the midgut epithelium of Diatraea saccharalis Fabricius, 1794 (Lepidoptera) parasitized by Cotesia flavipes Cameron, 1891 (Hymenoptera)

The morphometric study of the midgut in Diatraea saccharalis (Lepidoptera) larvae parasitized by the Cotesia flavipes (Hymenoptera) showed that there was significant increase in the columnar, goblet and regenerative cells and their nuclei; the midgut lumen diameter and the epithelial height were also increased in the parasitized larvae. The multivariate analysis showed that parasitism affected the columnar cell only in the posterior region, and the goblet cells along the midgut length (anterior and posterior regions). (c) 2006 Elsevier B.V. All rights reserved.

A morphometric study of the midgut in resistant and non-resistant Anticarsia gemmatalis (Hubner) (Lepidoptera: Noctuidae) larvae to its nucleopolyhedrovirus (AgMNPV)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)