Isso funciona foi apoiada através de subsídios da Natural Sciences and Engineering Research Council of Canada (RGPIN 238382-2011) e Canadian Institutes of Health Research (MOP115076). Heather Paul foi apoiada por uma Ciências Naturais e do Conselho de Investigação do Canadá Engenharia de Bolsas de Pós-graduação e uma bolsa Alberta inova Health Solutions. Megan Hallam foi apoiado por uma bolsa de estudos em Ciências Naturais e Engenharia do Conselho de Pesquisa de Pós-Graduação, um Scholarship Frederick Banting e Charles Best Canadá graduação, e Prêmio de Crianças Alberta Hospital Research Institute Formação em Genética, Desenvolvimento Infantil e Saúde.

Este protocolo foi aprovado pelo Comitê da Universidade de Calgary Animal Care e conformado com o Guia para o Cuidado e Uso de Animais de Laboratório. 1. Dam Separe Offspring <ol><li> Separa-se a barragem de sua prole para um mínimo de 5 min antes da ordenha 12. NOTA: A represa pode ser ordenhadas até 5-6 horas após a separação 1,6,13, no entanto períodos de separação mais do que 4 horas podem alterar a composição do leite 14. Enquanto tempo de separação não parece afetar coleção volume de leite 12, é aconselhável que um tempo de separação consistentes ser mantida durante todo o estudo. A composição do leite pode mudar ao longo da lactação 15, portanto, devem ser feitas tentativas para manter o dia da coleta de leite consistente. Máxima produção de leite é sugerida para ocorrer no dia 14 de lactação 12. </li><li> Usando uma câmara de aquecimento, garantir os filhotes são capazes de manter temperatu do corpo adequadare sem a presença da mãe durante a duração do procedimento de ordenha. NOTA: No estudo apresentado a seguir, ordenha foi realizada ao desmame, quando as barragens eram aproximadamente 22 semanas de idade e The Offspring 21 dias de idade, portanto, nenhuma câmara de aquecimento foi utilizado. </li></ol> 2. Set-up e Preparação <ol><li> Colete todos os materiais necessários para o procedimento de ordenha. NOTA: Todos os materiais pode ser encontrada na tabela de materiais e equipamento. </li><li> Coloque uma almofada de aquecimento no banco onde a ordenha terá lugar e cobrir a almofada com um banco absorvente sob-pad. </li><li> Configure o sistema anestésico. Assegure-se que o sistema tem de oxigénio e isoflurano antes do início suficiente. Fixar o anestésico máscara que irá ser utilizada para a indução da anestesia inicial para a máquina. Coloque a máscara que irá ser utilizado para a manutenção da anestesia nas proximidades se for diferente do que a máscara de anestesia inicial. </li><li> Coloque uma agulha 25 G para uma seringa de 1 ml para ocitocinainjecção utilizando uma técnica asséptica. </li><li> Ligue a almofada de aquecimento para que a temperatura do corpo materno é mantida durante o processo de ordenha. NOTA: Monitorar a temperatura da almofada de aquecimento para assegurar a almofada não se torna muito quente e causar queimaduras. Em alternativa, usar uma fonte de calor, tal como uma mesa de operações aquecida, que pode ser ajustado para uma temperatura específica. </li></ol> 3. anestesiar o Dam Usando Isoflurane <ol><li> Abra o tanque de oxigênio e virar o fluxo de 1 L (1000 cc) por min. Ligue o fluxo de isoflurano e ajustado para 5%. CUIDADO: Evite inalação direta de anestésico e evitar a acumulação de vapores anestésicos. </li><li> Anestesiar a barragem. </li><li> Mudar para a máscara de manutenção, se necessário, colocando o supino barragem no banco almofada absorvente. Confirme anesthetization por falta de pedal reflexo. </li><li> Reduzir o fluxo de isoflurano a 2-3% para manutenção da anestesia. Monitorar continuamente barragem durante todo o processo para garantir depressão da respiração não ocorre. NOTA: Uma vez sob anestesia, os olhos da represa devem ser protegidos com um lubrificante de olho estéril para evitar que os olhos sequem ou tornar-se riscado. </li></ol> 4. A oxitocina injeção <ol><li> Garantir a ocitocina (20 USP Unidades / ml) não passou da data de validade. Desinfetar o frasco de ocitocina com um álcool estéril limpar / álcool de limpeza pad. </li><li> Utilizando uma técnica asséptica, elaborar 2 UI (0,1 ml) de ocitocina para a seringa. Utilize uma nova agulha e seringa para cada barragem que vai ser ordenhadas. NOTA: A oxitocina doses variam geralmente de injecções individuais de 1 a 5 IU 1,6,12,16. Alternativamente, uma dose de 4 UI / kg de peso corporal pode ser utilizado 12. Uma dose única de 2 Ul pode ser repetida uma vez, se a ordenha for encontrada dificuldade. </li><li> Injectar a oxitocina por via intraperitoneal. Insira a agulha no quadrante inferior direito do abdômen com a agulha apontando para a cabeça, em um ângulode 15-30 °, cerca de 0,5 cm de profundidade. </li><li> Puxe o êmbolo para garantir pressão negativa antes da injeção. Se qualquer líquido (sangue, urina, conteúdo intestinal, etc.) é aspirada para a seringa, a agulha e remover tentar a injecção com uma agulha e seringa. Se nenhum fluido é aspirado injetar a oxitocina e descarte a agulha e seringa imediatamente em um recipiente de risco biológico. </li><li> Aguarde cerca de 5-15 min para a ocitocina para estimular a descida do leite. </li></ol> 5. Preparação de Sites de ordenha <ol><li> Escolha os sites / tetos de leite serão coletadas. O leite pode ser recolhida a partir de qualquer tetina 12. </li><li> Remova suavemente a pele ao redor dos tetos de ser ordenhadas com os trimmers, como a pele pode causar dificuldade na coleta de amostra devido à absorção da do leite. Seja gentil - a pele ao redor dos bicos é extremamente sensível e pode ser seca e, como tal, é suscetível a arranhões e lágrimas. </li><li> Esterilização do i tetinaNão é necessário, mas opcionalmente, limpar a tetina com água morna após a pele é removida. Preparar pelo menos dois locais como mais do que um sítio possa ser necessária para a recolha de leite. NOTA: Se a análise do leite inclui perfis de microbiana, a área de tetina pode exigir a esterilização com iodo 5. </li></ol> Coleção 6. Leite <ol><li> Espremer suavemente a base da teta, expulsando o leite manualmente para recolha. NOTA: Se a análise do leite inclui perfis de microbiana, as primeiras gotas de leite devem ser descartados. </li><li> Recolher as gotas de leite para um tubo capilar, o enchimento do tubo capilar. Tubos capilares, que podem acomodar volumes maiores (por exemplo, 50 ul) facilitar o processo. NOTA: Se houver dificuldade na coleta de leite é encontrado, uma segunda dose de oxitocina pode ser administrado. Recomenda-se a dose não deve exceder 4 IU total. </li><li> Repartir o leite a partir do tubo capilar para um tubo de microcentrífuga esterilizadotocar a extremidade do tubo que foi utilizado para desenhar a partir do leite da teta para o lado do tubo de microcentrífuga - observar o leite a ser desenhada para fora do tubo capilar por meio de acção capilar. NOTA: &#39;funda para fora &quot;do leite que não é puxado para dentro do tubo, usando uma agulha de 18 G ligada a uma seringa de 1 ml. </li><li> Monitorar continuamente a barragem para sinais de dor ou depressão respiratória e ajustar o fluxo de isoflurano em conformidade. </li><li> Continuar a recolher o leite como descrito nesta seção até que o leite suficiente tiver sido coletado para a análise de leite escolhido. Para a determinação da concentração de proteína e crematócrito descrito abaixo, recolher 0,25 ml de leite. NOTA: Use um local de ordenha diferente se a descida do leite diminui ou o local escolhido não expulsar leite suficiente. Um máximo de cerca de 2,5 ml de leite por animais pode ser recolhida 17, ou até 0,5 ml por tetina. Os autores recomendam que o tempo total sob anestesia ser limitado a aproximadamente 45-60 min, oR 45 min de ordenha. </li><li> Recolha do leite para um tubo de micro-hematócrito para medição crematócrito a partir de amostras de leite fresco, e vedar a extremidade do tubo com vedante argila. Rotular o tubo de microhematócrito com o ID barragem e armazenar a amostra de leite na posição vertical. </li><li> Quando a ordenha é completa, desligue o fluxo de isoflurano e oxigênio. Retire a máscara da barragem e continuará a acompanhar barragem até acordado. Recomenda-se que se não totalmente consciente, a barragem deve ser colocada sobre uma almofada absorvente banco durante o período de recuperação, ao invés de diretamente na cama gaiola, para evitar que a roupa de cama de ser aspirado ou coçar os olhos da represa durante a recuperação. NOTA: Não deixe a barragem autônoma até que tenha recuperado a consciência suficiente para manter recumbancy esternal. </li><li> Se não forem necessárias análises adicionais, congelar o leite à temperatura de -80 ° C. Outros têm sugerido que o leite possa ser armazenado durante até 3 horas a 4 ° C ou 5 meses a -20 ° C 18. </li></ol> 7. Medição crematócrito <ol><li> Estimar o teor de gordura do leite por meio do cálculo do crematócrito leite (a percentagem de creme na amostra de leite) 19. NOTA: As medições com leite humano demonstraram que o leite fresco ou congelado pode ser utilizado para uma medição crematócrito, no entanto leite fresco é mais altamente correlacionados (r = 0,92 versus R = 0,90) com a concentração lipídica 11. O uso de leite fresco ou congelado para medições crematócrito deve ser mantido consistente em todo o estudo, como o leite descongelado está associado a uma pequena diminuição no crematócrito valores 11. </li><li> Para o leite fresco, recolher uma amostra de leite da teta em um tubo de microhematócrito; preencher pelo menos ¾ cheio (cerca de 15-20 mL). Alternativamente, extrair leite fresco a partir da amostra recolhida para dentro do tubo capilar depois de misturar bem. Selar o fim com selante de barro. </li><li> Colocar o tubo capilar para hematócrito da fieira, com aextremidade vedada apontando para o exterior, garantindo a centrífuga é equilibrada. </li><li> Comece o spin hematócrito (120 segundos a 13.700 xg). NOTA: O tempo da rotação ou velocidade pode mudar, dependendo do modelo de centrífuga utilizada. </li><li> Remover o tubo da centrifugadora depois da rotação é completo e realizar as medições para calcular o crematócrito. Observar separação da amostra numa camada de creme e uma camada clara. </li><li> Medir e registar o comprimento total de fluido no tubo e o comprimento da gordura (creme) camada usando compassos de calibre ou uma régua. NOTA: O crematócrito é expressa como a percentagem da camada de creme na amostra de leite a 19, calculada como (comprimento da camada de nata / comprimento total da coluna de leite) x 100 (Figura 1A). Calcular os valores de concentração e de energia a partir da medição de gordura crematócrito como se segue: concentração de gordura (g / L) = (crematócrito (%) - 0,59) /0.146 (Figura 1B) 19; Valor energético (kcai / L) = 290 + (* crematócrito 66,8 (%) (Figura 1C) 19. </li></ol> 8. Proteína Concentração Determinação <ol><li> Utilizando albumina de soro bovino como padrão de proteína, determinar a concentração total de proteína do leite, utilizando um ensaio de proteína padrão, tal como ensaio de proteínas de Lowry um 6. NOTA: A diluição do leite pode ser necessária para as medições de proteínas de leite a cair dentro da curva padrão do ensaio. </li></ol>

<ol>
	<li>Izumi, H., Kosaka, N., Shimizu, T., Sekine, K., Ochiya, T., Takase, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-dependent+expression+profiles+of+microRNAs+and+mRNAs+in+rat+milk+whey.">Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.</a> PLoS ONE. 9 (2), e0088843 (2014).</li><li>Hsieh, C. C., Hern&#225;ndez-Ledesma, B., Fern&#225;ndez-Tom&#233;, S., Weinborn, V., Barile, D., de Moura Bell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milk+Proteins,+Peptides,+and+Oligosaccharides:+Effects+against+the+21st+Century+Disorders.">Milk Proteins, Peptides, and Oligosaccharides: Effects against the 21st Century Disorders.</a> BioMed Res. Int. , (2015).</li><li>Rogier, E. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+mother:+Long-term+impact+of+antibodies+in+breast+milk+on+the+gut+microbiota+and+intestinal+immune+system+of+breastfed+offspring.">Lessons from mother: Long-term impact of antibodies in breast milk on the gut microbiota and intestinal immune system of breastfed offspring.</a> Gut Microbes. 5 (5), 663-668 (2014).</li><li>Keen, C. L., L&#246;nnerdal, B., Clegg, M., Hurley, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+composition+of+rat+milk:+trace+elements,+minerals,+protein,+carbohydrate+and+fat.">Developmental changes in composition of rat milk: trace elements, minerals, protein, carbohydrate and fat.</a> J. of Nutr. 111 (2), 226-236 (1981).</li><li>Cabrera-Rubio, R., Collado, M. C., Laitinen, K., Salminen, S., Isolauri, E., Mira, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+milk+microbiome+changes+over+lactation+and+is+shaped+by+maternal+weight+and+mode+of+delivery.">The human milk microbiome changes over lactation and is shaped by maternal weight and mode of delivery.</a> Am. J. Clin. Nutr. 96 (3), 544-551 (2012).</li><li>Hallam, M. C., Barile, D., Meyrand, M., German, J. B., Reimer, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+high-protein+or+high-prebiotic-fiber+diets+affect+maternal+milk+composition+and+gut+microbiota+in+rat+dams+and+their+offspring.">Maternal high-protein or high-prebiotic-fiber diets affect maternal milk composition and gut microbiota in rat dams and their offspring.</a> Obesity. 22 (11), 2344-2351 (2014).</li><li>Munch, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+Profiling+of+microRNA+by+Next-Gen+Deep+Sequencing+Reveals+Known+and+Novel+miRNA+Species+in+the+Lipid+Fraction+of+Human+Breast+Milk.">Transcriptome Profiling of microRNA by Next-Gen Deep Sequencing Reveals Known and Novel miRNA Species in the Lipid Fraction of Human Breast Milk.</a> PLoS ONE. 8 (2), e50564 (2013).</li><li>Li, M., Sloboda, D. M., Vickers, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+obesity+and+developmental+programming+of+metabolic+disorders+in+offspring:+Evidence+from+animal+models.">Maternal obesity and developmental programming of metabolic disorders in offspring: Evidence from animal models.</a> Exp Diabetes Res. 2011, (2011).</li><li>Ellis, P. J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrifty+metabolic+programming+in+rats+is+induced+by+both+maternal+undernutrition+and+postnatal+leptin+treatment,+but+masked+in+the+presence+of+both:+implications+for+models+of+developmental+programming.">Thrifty metabolic programming in rats is induced by both maternal undernutrition and postnatal leptin treatment, but masked in the presence of both: implications for models of developmental programming.</a> BMC Genomics. 15, 49 (2014).</li><li>Gomez-Gallago, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+collect+high+volumes+of+milk+from+mice+(Mus+musculus).">A method to collect high volumes of milk from mice (Mus musculus).</a> An. Vet. Murcia. 29, 55-61 (2013).</li><li>Wang, C. D., Chu, P. S., Mellen, B. G., Shenai, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creamatocrit+and+the+nutrient+composition+of+human+milk.">Creamatocrit and the nutrient composition of human milk.</a> J. Perinatol. 19 (5), 343-346 (1999).</li><li>Rodgers, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Practical+aspects+of+milk+collection+in+the+rat.">Practical aspects of milk collection in the rat.</a> Lab. Anim. 29 (4), 450-455 (1995).</li><li>Godbole, V. Y., Grundleger, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+rat+milk+from+day+5+to+20+of+lactation+and+milk+intake+of+lean+and+preobese+zucker+pups.">Composition of rat milk from day 5 to 20 of lactation and milk intake of lean and preobese zucker pups.</a> J. Nutr. 111 (3), 480-487 (1981).</li><li>Del Prado, M., Delgado, G., Villalpando, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+lipid+intake+during+pregnancy+and+lactation+alters+milk+composition+and+production+and+litter+growth+in+rats.">Maternal lipid intake during pregnancy and lactation alters milk composition and production and litter growth in rats.</a> J. Nutr. 127 (3), 458-462 (1997).</li><li>Nicholas, K. R., Hartmann, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milk+secretion+in+the+rat:+progressive+changes+in+milk+composition+during+lactation+and+weaning+and+the+effect+of+diet.">Milk secretion in the rat: progressive changes in milk composition during lactation and weaning and the effect of diet.</a> Comp. Biochem. Physiol. A. Comp. Physiol. 98 (3-4), 533-542 (1991).</li><li>Azara, C. R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+intake+during+lactation+alters+milk+nutrient+composition+and+growth+and+mineral+status+of+rat+pups.">Ethanol intake during lactation alters milk nutrient composition and growth and mineral status of rat pups.</a> Biol. Res. 41 (3), 317-330 (2008).</li><li>Keen, C. L., L&#246;nnerdal, B., Sloan, M. V., Hurley, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+milking+procedure+on+rat+milk+composition.">Effects of milking procedure on rat milk composition.</a> Physiol. Behav. 24 (3), 613-615 (1980).</li><li>Romeu-Nadal, M., Castellote, A. I., L&#242;pez-Sabater, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cold+storage+on+vitamins+C+and+E+and+fatty+acids+in+human+milk.">Effect of cold storage on vitamins C and E and fatty acids in human milk.</a> Food Chem. 106 (1), 65-70 (2008).</li><li>Lucas, A., Gibbs, J. A., Lyster, R. L., Baum, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creamatocrit:+simple+clinical+technique+for+estimating+fat+concentration+and+energy+value+of+human+milk.">Creamatocrit: simple clinical technique for estimating fat concentration and energy value of human milk.</a> Br. Med. J. 1 (6119), 1018-1020 (1978).</li><li>Furtado, K., Andrade, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+beneficial+and+adverse+effects+of+inhalable+and+injectable+anaesthetics+in+animal+models:+a+mini-review.">Comparison of the beneficial and adverse effects of inhalable and injectable anaesthetics in animal models: a mini-review.</a> OA Anaesthetics. 1 (2), 20 (2013).</li><li>Hausman Kedem, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+advanced+maternal+age+upon+human+milk+fat+content.">The effect of advanced maternal age upon human milk fat content.</a> Breastfeed. Med. 8 (1), 116-119 (2013).</li><li>Mandel, D., Lubetzky, R., Dollberg, S., Barak, S., Mimouni, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+and+energy+contents+of+expressed+human+breast+milk+in+prolonged+lactation.">Fat and energy contents of expressed human breast milk in prolonged lactation.</a> Pediatrics. 116 (3), e432-e435 (2005).</li></ol>

Os autores não têm conflitos de interesse a declarar. Todos os experimentos com animais foram realizados em conformidade com os protocolos do CCAC aprovado.

Investigations into maternal milk components have increased as interest in early life development research rises. As the sole source of nutrition during the neonatal period, the bioactive compounds in milk are essential for ideal growth and development, especially in the context of intestinal and immune health3. The method presented here is a simple, non-invasive method of collecting milk from the lactating rat in amounts sufficient for downstream analysis, such as oligosaccharide profiling6. The me...

O leite é a única fonte de nutrição para mamíferos recém-nascidos, fornecendo energia e nutrientes para o crescimento e desenvolvimento infantil 1,2. Quando o leite é constituído principalmente por células, lípidos e proteína 1, que também contém uma pletora de compostos bioactivos que modulam o desenvolvimento vida precoce da descendência, incluindo enzimas, hidratos de carbono, hormonas, anticorpos, factores de crescimento, citocinas, exossomas, microvesículas, e pequenos RNAs tais como microARN 1,2. O papel fundamental do leite materno na criação da prole imunitário e saúde intestinal 3, juntamente com evidências de que crianças amamentadas são menos suscetíveis à doença 2, destaca a importância de identificar os constituintes do leite associados a processos de doenças no início da vida e os mecanismos moleculares envolvidos em suas ações. O rato é o desenvolvimento de um modelo popular para investigar o efeito de vários nutricional, fisiológico e intervenções químicas no iníciodesenvolvimento -life 4. A análise do leite do rato podem, portanto, proporcionar novos insights sobre a saúde materna e prole. Avanços científicos atuais fornecem agora aumentar as oportunidades para investigações aprofundadas sobre os efeitos de constituintes do leite específicos na saúde e na doença. Por exemplo, a sequenciação de leite perfis bacterianas elucidou o seu papel na colonização intestinal precoce do intestino infantil 5, análise de espectrometria de massa de oligossacarídeos do leite forneceram uma visão sobre a alteração dos perfis de oligossacarídeos do leite via dieta materna 6 e profunda seqüenciamento de microRNA secretada em os glóbulos de gordura do leite materno destaca possíveis papéis na transcrição do gene, o metabolismo e a função imunológica 7. Modelos de ratos representam um dos organismos modelo mais populares usados ​​em estudos materno 8,9. Uma vantagem é seus períodos de gestação e aleitamento curtas, durando apenas approximatEly 21 dias cada; Por conseguinte, o tempo total a partir do início da gravidez até lactação representa um curto período de tempo em que os dados valiosos podem ser gerados. Quanto maior o tamanho de ratos em comparação com os ratinhos, no contexto de recolha do leite, pode proporcionar uma vantagem significativa no que diz respeito ao volume de leite e facilidade de recolha do leite; produção de leite no rato, por exemplo, parece ser dependente do peso corporal total com os ratos mais pesados ​​que produzem mais leite 10. Aqui, uma descrição geral para a coleta manual de leite de ratas lactantes é fornecido. Este protocolo requer um equipamento mínimo, é não-invasiva e de baixo custo, e podem ser usadas para recolher os volumes adequados de leite para análises adicionais a jusante. Em breve, a barragem é anestesiados com isoflurano, a descida do leite é estimulada pela oxitocina, e de leite são recolhidas em tubos capilares por meio de extracção manual do leite. Finalmente, como dois principais componentes de leite são proteínas e gordura, uma breve description de estimar o teor de gordura do leite por meio de medidas crematócrito 11 e quantificação da concentração total de proteínas usando um ensaio de proteína padrão é apresentada.

<table border="0" cellpadding="0" cellspacing="0" width="829">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Milking</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringes</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">309602</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 G needles</td>
			<td style="width:191px;">BD-Canada</td>
			<td style="width:119px;">305196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ul Microdispenser Capillary Tubes</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">21-169D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (20 USP Units/ml)</td>
			<td style="width:191px;">Bimeda-MTC</td>
			<td style="width:119px;">1OXY015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">PPC Vet Isoflurane Inhalation Anesthetic, 250 ml</td>
			<td style="width:191px;">Fresenius Kabi</td>
			<td style="width:119px;">M60302</td>
			<td>Used on the order of a veterinarian</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile Alcohol Prep Pad</td>
			<td style="width:191px;">Dukal</td>
			<td style="width:119px;">853</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Absorbent Bench Underpad</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">82020-845</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Maxi-Therm Hyper/Hypothermia Blanket</td>
			<td style="width:191px;">Cincinnati Sub-Zero</td>
			<td style="width:119px;">274</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Rodent Anesthesia Machine with Vaporizer</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;"></td>
			<td>Subject to individual laboratory needs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:353px;">Animal Masks</td>
			<td style="width:191px;">Benson Medical Industries Inc.</td>
			<td style="width:119px;">50100/50102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge Tubes</td>
			<td style="width:191px;">Axygen</td>
			<td style="width:119px;">MCT-060-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ChroMini Professional Trimmer</td>
			<td style="width:191px;">Wahl</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Equipment - Creamatocrit</td>
			<td style="width:191px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin SafeCrit Plastic Microhematocrit Tubes (Untreated)</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">22-274-914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Critoseal Capillary Tube Sealant Tray</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">470161-478</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">StatSpin CritSpin Microhematocrit Centrifuge</td>
			<td style="width:191px;">Beckman Coulter, Inc</td>
			<td style="width:119px;">X00-004999-001</td>
			<td></td>
		</tr>
	</tbody>
</table>

milk collection in the rat using capillary tubes and estimation of milk fat content by creamatocrit

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also contains a vast number of bioactive compounds that greatly affect the development of the neonate. The analysis of milk components will help elucidate key factors that link maternal metabolism and health with offspring growth and development. The laboratory rat represents a popular model organism for maternal studies, and rat milk can be used to examine the effect of various maternal physiological, nutritional, and pharmacological interventions on milk components, which may then impact offspring health. Here a simple method of manually collecting milk from the lactating rat that can be performed by a single investigator, does not require specialized vacuum or suction equipment, and provides sufficient milk for subsequent downstream analysis is described. A method for estimating the fat content of milk by measuring the percentage of cream within the milk sample, known as the creamatocrit, is also presented. These methods can ultimately be used to increase insight into maternal-child health and to elucidate maternal factors that are involved in proper growth and development of offspring.

O leite foi recolhido como descrito no desmame de fêmeas Wistar (cerca de 22 semanas de idade, pesando 350 a 400 g) que consumiram um controlo (AIN-93G, n = 5), elevado teor de proteínas (40% de caseína p / p, n = 5) , ou alta fibra pré-biótica (21,6% p / p, 1: 1 razão de oligofrutose e inulina, n = 4) dieta durante a gravidez e lactação. A dose de oxitocina foi de 2 UI. O leite foi recolhido utilizando tubos capilares, e um tubo foi centrifugado usando um spinner hematócrito para determinar crematócrito <stro...

Watch this Scientific Journal Video about Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit at JoVE.com

Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Coleta de Leite no Rato Usando Capilar Tubos e Estimativa da gordura do leite por conteúdo crematócrito

Milk, as the sole source of nutrition for the newborn mammal, provides the necessary nutrients and energy for offspring growth and development. It also ...

milk-collection-rat-using-capillary-tubes-estimation-milk-fat-content

Research

JoVE Journal

Biology

12.3K visualizações.  University of Calgary. Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

Vídeo: Milk Collection in the Rat Using Capillary Tubes and Estimation of Milk Fat Content by Creamatocrit

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biologia

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

Imagem de receptor de estrogênio α-in Rat Arteríolas Pial usando um Microscópio Digital imunofluorescência

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

Liver fibrosis is defined by the excessive deposition of extracellular matrix by activated myofibroblasts. There are multiple precursors of hepatic myofibroblasts, including hepatic stellate cells, portal fibroblasts and bone marrow derived fibroblasts 1. Hepatic stellate cells have been the best studied, but portal fibroblasts are increasingly recognized as important contributors to the myofibroblast pool, particularly in biliary fibrosis 2. Portal fibroblasts undergo proliferation in response to biliary epithelial injury, potentially playing a key role in the early stages of biliary scarring 3-5. A method of isolating portal fibroblasts would allow in vitro study of this cell population and lead to greater understanding of the role portal fibroblasts play in biliary fibrosis.
Portal fibroblasts have been isolated using various techniques including outgrowth 6, 7 and liver perfusion with enzymatic digestion followed by size selection 8. The advantage of the digestion and size selection technique compared to the outgrowth technique is that cells can be studied without the necessity of passage in culture. Here, we describe a modified version of the original technique described by Kruglov and Dranoff 8 for isolation of portal fibroblasts from rat liver that results in a relatively pure population of primary cells.

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

A technique for isolating portal fibroblasts from rat liver is described. Livers are perfused and digested in situ with collagenase, followed by ex vivo digestion of the liver slurry and size selection of cells. This method provides a pure population of portal fibroblasts without the need for passage in culture.

Isolamento de fibroblastos de rato por Portal<em&gt; In situ</em&gt; Perfusão de fígado

Liver fibrosis is defined  by the excessive deposition of extracellular matrix by activated  myofibroblasts. There are multiple precursors of hepatic ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Imagem Funcional de gordura marrom em ratos com FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Collection, Isolation and Enrichment of Naturally Occurring Magnetotactic Bacteria from the Environment

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of Massachusetts (USA). Since then MTB have been discovered in stratified water- and sediment-columns from all over the world2. One feature common to all MTB is that they contain magnetosomes, which are intracellular, membrane-bound magnetic nanocrystals of magnetite (Fe3O4) and/or greigite (Fe3S4) or both3, 4. In the Northern hemisphere, MTB are typically attracted to the south end of a bar magnet, while in the Southern hemisphere they are usually attracted to the north end of a magnet3,5. This property can be exploited when trying to isolate MTB from environmental samples.
One of the most common ways to enrich MTB is to use a clear plastic container to collect sediment and water from a natural source, such as a freshwater pond. In the Northern hemisphere, the south end of a bar magnet is placed against the outside of the container just above the sediment at the sediment-water interface. After some time, the bacteria can be removed from the inside of the container near the magnet with a pipette and then enriched further by using a capillary racetrack6 and a magnet. Once enriched, the bacteria can be placed on a microscope slide using a hanging drop method and observed in a light microscope or deposited onto a copper grid and observed using transmission electron microscopy (TEM).
Using this method, isolated MTB may be studied microscopically to determine characteristics such as swimming behavior, type and number of flagella, cell morphology of the cells, shape of the magnetic crystals, number of magnetosomes, number of magnetosome chains in each cell, composition of the nanomineral crystals, and presence of intracellular vacuoles.

We demonstrate a method to collect magnetotactic bacteria (MTB) that can be applied to natural waters. MTB can be isolated and enriched from sediment samples using a relatively simple setup that takes advantage of the bacteria's natural magnetism. Isolated MTB can then be examined in detail using both light and electron microscopy.

Isolamento, coleta e Enriquecimento de bactérias naturais magnetotáticas do Meio Ambiente

Magnetotactic bacteria (MTB) are aquatic microorganisms that were first notably described in 19751 from sediment samples collected in salt marshes of ...

Milk Collection Methods for Mice and Reeves' Muntjac Deer

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves&#8217; muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.

Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves&#8217; muntjac deer.

Métodos de coleta de leite para Ratos e Reeves Muntjac cervos

Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen&#8217;s native ...

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It contains sugars, amino acids, proteins, RNA, lipids and other metabolites. While there is a large interest in understanding the composition and function of the phloem, the role of many of these molecules and thus, their importance in plant development and stress response has yet to be determined. One barrier to phloem analysis lies in the fact that the phloem seals itself upon wounding. As a result, the number of plants from which phloem sap can be obtained is limited. One method that allows collection of phloem exudates from several plant species without added equipment is the EDTA-facilitated phloem exudate collection described here. While it is easy to use, it does lead to the wounding of cells and care has to be taken to remove contents of damaged cells. In addition, several controls to prove purity of the exudate are necessary. Because it is an exudation rather than a direct collection of the phloem sap (not possible in many species) only relative quantification of its contents can occur. The advantage of this method over others is that it can be used in many herbaceous or woody plant species (Perilla, Arabidopsis, poplar, etc.) and requires minimal equipment and training. It leads to reasonably large amounts of exudates that can be used for subsequent analysis of proteins, sugars, lipids, RNA, viruses and metabolites. It is simple enough that it can be used in both a research as well as in a teaching laboratory.

Collection and Analysis of Arabidopsis Phloem Exudates Using the EDTA-facilitated Method

Knowledge of the composition of the phloem sap as well as the mechanism of its loading and long-distance transport is essential for the understanding of long-distance signaling in plant development and stress/pathogen response and of assimilate transport. This manuscript describes the collection of phloem exudates using the EDTA-facilitated method.

Coleta e análise de<em&gt; Arabidopsis</em&gt; Floema exsudados Usando o método EDTA facilitada

The plant phloem is essential for the long-distance transport of (photo-) assimilates as well as of signals conveying biotic or abiotic stress. It ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Análise de glicocorticóides: Non-invasive Monitoring Adrenal em eqüídeos

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Avian Semen Collection by Cloacal Massage and Isolation of DNA from Sperm

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. Birds are widely used subjects in biological research and are ideal for studies involving repeated sperm samples. However, few resources are currently available for those wishing to learn how to collect and extract DNA from avian sperm. Here we describe cloacal massage, a gentle, non-invasive manual technique for collecting avian sperm. Although this technique is established in the literature, it can be difficult to learn from the available descriptions. We also provide information for extracting DNA from avian semen using a commercial extraction kit with modifications. Cloacal massage can be easily used on any small- to medium-sized male bird in reproductive condition. Following collection, the semen can be used immediately for motility assays, or frozen for DNA extraction following the protocol described herein. This extraction protocol was refined for avian sperm and has been successfully used on samples collected from several passerine species (Passer domesticus, Spizella passerina, Haemorhous mexicanus, and Turdus migratorius) and one columbid (Columba livia).

This protocol describes cloacal massage, a non-invasive manual technique for collecting semen from birds, and DNA extraction from avian sperm using a commercial extraction kit with modifications.

Coleta de sêmen aviária por esfregaços massagem e isolamento do DNA de esperma

Collection of semen may be useful for a wide range of applications including studies involving sperm quality, sperm telomere dynamics, and epigenetics. ...

Coleta de Leite no Rato Usando Capilar Tubos e Estimativa da gordura do leite por conteúdo crematócrito

Resumo

Explore mais vídeos

Capítulos neste vídeo