These experiments were funded by the NIH grant #RAI093634A. We express immense gratitude to Jenny Powers, DVM, PhD, for her assistance in muntjac milking.

1. Rato ordenha Protocolo Os métodos descritos neste protocolo foram aprovados pelo IACUC. 1. Separação da Barragem de Offspring <ol><li> Selecione a barragem a ser ordenhadas. Escolha uma barragem com uma ninhada de 4 ou mais filhotes que é de 8-12 dias após o parto para fornecer leite máxima coleção, embora coleção é possível a qualquer momento ponto pós parto até 21 dias. </li><li> Separe a barragem de sua ninhada, pelo menos 2 horas antes da ordenha. Nota: Caso um desejo de leite mais de uma barragem em um determinado período de tempo, é aceitável para abrigar as barragens separados na mesma gaiola com alimentação adequada e abastecimento de água. </li></ol> 2. Administração de ocitocina, anestesia e Eye Lubrificante <ol><li> Administrar 2 UI / kg de oxitocina por via intraperitoneal (IP). A ocitocina é um hormônio que atua sobre as glândulas mamárias das fêmeas lactantes para estimular a liberação de leite. A ocitocina pode ser adquirido from um veterinário. </li><li> Administrar uma mistura de anestésicos contendo 80-100 mg / kg de cetamina, em combinação com 5-10 mg / kg IP de xilazina. Por exemplo, um ml de caldo de mistura de anestésico 10 pode ser preparado antecipadamente por ratinhos pesando cerca de 30 g e que incluem 0,1 ml de xilazina, 8,9 ml de H 2 O, e 1,0 ml de cetamina. 0,25 ml de anestésico total será sedar um rato deste tamanho por aproximadamente 20-30 min, que deverá fornecer o tempo adequado para a coleta de leite. Notas: Para determinar que o animal foi anestesiado com sucesso, coloque-a suavemente em seu estômago em uma superfície sólida; ela não deve se mover seus pés ou a tentativa de subir para os pés dela se ela está totalmente anestesiado. No caso de um rato não ser completamente anestesiados com a dose inicial, o pesquisador pode administrar um outro ¼ ou C da dose original da mistura de anestésico, dependendo do tamanho do animal e / ou o nível de sedação que o rato já atingiu . No entanto, isso poderia increase a possibilidade de uma reacção adversa à anestesia, e pode também resultar no rato sendo mais profundamente anestesiados e / ou anestesiados por um longo período de tempo. Nota: Se a ordenha mais de uma barragem, que é necessário escalonar oxitocina e anestésicos injectáveis ​​devido à meia-vida curta de oxitocina e tempos de sedação de cetamina e xilazina. </li><li> Aplique uma pequena quantidade de lubrificante olho para o canto de cada olho e se espalhar para evitar que os olhos do rato de secar sob anestesia. </li></ol> 3. Coleta de leite A coleta do leite é mais facilmente executada com dois pesquisadores: um pesquisador para segurar o rato anestesiado ao expressar manualmente o leite (referido como R1), e um pesquisador para recolher o leite (R2). Este método também pode ser realizado com uma pessoa, se o rato é fixada sobre uma superfície plana, para evitar danos. <ol><li> R2 deve ser equipado com as almofadas da preparação do álcool estéril, um pip P-200Etman, uma ponteira limpa para cada rato, e um recipiente para armazenar o leite, como um tubo Eppendorf de 1,5 ml ou uma cryovial 1,2 ml (ver Tabela de Materiais). </li><li> Como a ocitocina começa a fazer efeito, a descida do leite se tornará visível na área mamária do rato (ver Figura 1A). R1: Retire uma compressa embebida em álcool preparação estéril e limpe a área mamária do mouse para limpar a área antes da ordenha. Expressar manualmente leite da teta, utilizando o polegar e dedo indicador para massajar suavemente e apertar o tecido mamário de um movimento para cima até que uma gota visível de leite começa a formar-se na base da teta. </li><li> Pressione o êmbolo Pipetman P-200 à sua primeira parada para liberar o ar para fora da ponteira e se preparar para a coleta de leite. </li><li> Posicione a ponta da pipeta no topo da gota de leite e retire o leite na ponta da pipeta, liberando lentamente a pressão sobre o êmbolo. Tome cuidado para não colocar a ponta muito perto do teto ou da pele (verFigura 1B). Não é necessário para libertar completamente o êmbolo, como esta vai puxar o leite todo o caminho para o topo da ponta da pipeta e fará com que seja difícil para expelir o leite muitas vezes viscoso para dentro do recipiente de retenção. </li><li> Expelir o leite no recipiente, utilizando o polegar para pressionar o êmbolo após a primeira paragem (ver Figura 1C). Para facilitar a expulsão de pequenos volumes, pressionar a ponta da pipeta contra o lado do tubo, de modo que o líquido não se perde. </li><li> Continue a expressar manualmente leite de cada teta, movendo-se em um ou outro sentido horário ou anti-horário, de modo a não pular nenhuma tetas. É ao mesmo tempo aceitável e útil para voltar e expressar continuamente leite da mesma teta deveria produzir mais leite do que o outro. </li></ol> 4. Reversão anestésica na Barragem <ol><li> Na maioria dos casos, é comum que a represa para começar a começar a acordar durante o processo de ordenha. Se este não for o caso, colocar a barragem em um cage indiretamente, debaixo de uma fonte de calor, como um cobertor circulando aquecido, até que ela começa a acordar. Nota: Como a barragem começa a acordar, manter observação atenta de sua atividade até que ela é capaz de andar sobre a sua gaiola com ela própria. </li><li> Uma vez que a barragem está se movendo sobre a gaiola sem luta, colocá-la de volta em sua gaiola original com sua ninhada. Sem efeitos nocivos têm sido observados em relação à capacidade dos filhotes para alimentar após a coleta do leite. </li></ol> 2. Muntjac ordenha Protocolo Os métodos descritos neste protocolo foram aprovados pelo IACUC. 1. Separação da Silva de Offspring <ol><li> Selecione o doe para serem ordenhadas. Veado muntjac Reeves &#39;geralmente dão à luz a um jovem corça 2. Leite a corça do dia depois do parto para permitir a corça oportunidade para ganhar acesso a nutrientes essenciais no colostro 8. Nota: O fulvo geralmente se esconde e não reaparecer durante o procedure; separação do binômio mãe-filho não é necessário. </li></ol> 2. Administração de ocitocina, anestesia e Eye Lubrificante <ol><li> Administrar 10 UI / kg de ocitocina por via intramuscular (IM). A ocitocina é um hormônio que atua sobre as glândulas mamárias das fêmeas lactantes para estimular o let-down do leite. A ocitocina pode ser adquirido a partir de um veterinário. </li><li> Administrar o anestésico BAM IM 11 butorfanol contendo 0,45 mg / kg, azaperona 0,25 mg / kg, medetomadine 0,07 mg / kg. Uma dose da mistura de anestésico BAM vai sedar um muntjac por aproximadamente 20-30 min, que fornece tempo suficiente para coleta de leite completa. Outra opção para o anestésico é midazolam a 1-2 mg / kg, o que irá sedar um muntíaco durante aproximadamente 10-15 min. Enquanto isto proporciona o tempo adequado para a recolha de leite, este anestésico é metabolizada de forma mais rápida do que BAM e o animal pode tentar escapar no final do período de tempo mais curto. Nota: resposta mínima à dor, Reflexos menores ao tocar as orelhas, seguido pela capacidade de R1 para segurar o MJ sem esforço indica que o animal foi anestesiado com sucesso. Nota: No caso de um muntíaco não ser completamente anestesiados com a dose inicial, o pesquisador pode administrar um outro ¼ ou C da dose original da mistura de anestésico, dependendo do tamanho do animal e / ou o nível de sedação, que já tenha sido conseguida. No entanto, isto pode aumentar a probabilidade de uma reacção adversa à anestesia, e pode também resultar na muntíaco ser mais profundamente anestesiados e / ou anestesiados por um longo período de tempo. </li><li> Aplique uma pequena quantidade de lubrificante no canto de cada olho e se espalhar para evitar que os olhos do muntjac de secar sob anestesia. </li></ol> 3. Coleta de leite A coleta do leite é mais facilmente executada com dois pesquisadores; um pesquisador para segurar o muntjac anestesiado ema volta ao expressar manualmente o leite (Referido como R1), e um segundo pesquisador para manter a cabeça do muntjac em uma posição confortável e segura e para recolher o leite (R2). <ol><li> Dependendo do anestésico utilizado, a ordenha deve ser realizada ou 1) em suas canetas para fornecer um lugar seguro para eles que eles deveriam acordar durante o procedimento (induzido por midazolam) 2) ou em uma ante-sala sem cama para manter a limpeza (BAM- induzida). </li><li> R1 e R2 deve ser equipado com uma gaze embebida com etanol e vários tubos de 15 ml de polipropileno cónicos para segurar o leite. </li><li> Como a ocitocina começa a fazer efeito, a descida do leite se tornará visível na área mamária do muntjac. R1: Higienizar as mãos e os dedos enluvados, bem como todo o úbere e cada teto individual do animal com a gaze embebida em álcool. R2: Segure a cabeça do muntjac em uma posição segura e confortável para permitir a facilidade de respiração e ajudar a prevenir a regurgitação rúmen. Segure os cl tubo de coleta<html> ose para a tetina sendo ordenhada sem tocá-lo para evitar a contaminação potencial. </li><li> Expresse o leite de uma teta, usando o polegar eo dedo indicador para apertar suavemente o tecido mamário em um movimento ascendente até o teto é ingurgitadas com leite, e rolar suavemente o leite para fora do teto e no tubo que R2 está segurando (ver Figura 2 ). </li><li> Nota: O leite sai em um córrego, por isso é importante para R2 para ter o tubo de coleta posicionada de modo que pouco ou nenhum leite será desperdiçado. </li><li> O úbere da muntjac é dividido em quadrantes. R1: Continue a extrair o leite de cada quadrante até que o leite já não flui facilmente. Se o leite não pode ser expresso a partir de uma teta, expresso a partir de todas as outras tetinas antes de voltar para o primeiro. É ao mesmo tempo aceitável e útil para voltar e expressar continuamente leite da mesma teta deveria produzir mais leite do que o outro. </li></ol> 4. Reversão anestésica no DOE t &quot;&gt; É comum que doe induzido por midazolam para começar a acordar durante o processo de ordenha. Na maioria dos casos, a fêmea vai tentar escapar antes de todo o leite tem sido expressa. contenção Menor é usado em um esforço para aumentar o leite produção, porém a segurança eo conforto de ambos os pesquisadores e os doe são igualmente importantes. <ol><li> Administrar atipamezole em 2,5 mg / mg medetomadine por via subcutânea para a reversão rápida do BAM anestesiados doe. Nota: Com os dois tipos de anestesia, é necessário manter uma estreita observação do doe até que ela é capaz de andar por conta própria, sem tropeçar ou cair, e é capaz de comer e beber sem medo de aspiração ou asfixia. Sem efeitos nocivos têm sido observados em relação à capacidade do fulvo para amamentar após a coleta do leite. </li></ol>

<ol>
	<li>Dueling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Muntiacus reevesi. Animal Diversity Web. , (2004).</li><li>Pei, K., Taber, R., O'Gara, B., Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breeding+cycle+of+the+Formosan+Reeves+muntjac+(Muntiacus+reevesi+micrurus)+in+northern+Taiwan,+Republic+of+China.">Breeding cycle of the Formosan Reeves muntjac (Muntiacus reevesi micrurus) in northern Taiwan, Republic of China.</a> Mammalia. 59 (2), 223-228 (1995).</li><li>Browning, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+of+prions+from+mule+deer+and+elk+with+chronic+wasting+disease+to+transgenic+mice+expressing+cervid+PrP.">Transmission of prions from mule deer and elk with chronic wasting disease to transgenic mice expressing cervid PrP.</a> J. Virol. 78 (23), 13345-13350 (2004).</li><li>Van de perre, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+antibody+via+mother's+milk.">Transfer of antibody via mother's milk.</a> Vaccine. 21 (24), 3374-3376 (2003).</li><li>Maden, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+modified+mice+produce+human+milk+protein.">Genetically modified mice produce human milk protein.</a> BioNews. , (2009).</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>Depeters, E. J., Hovey, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+collecting+milk+from+mice.">Methods for collecting milk from mice.</a> J. Mammary Gland Biol. Neoplasia. 14 (4), 397-400 (2009).</li><li>Knight, C. H., Maltz, E., Docherty, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milk+yield+and+composition+in+mice:+effects+of+litter+size+and+lactation.">Milk yield and composition in mice: effects of litter size and lactation.</a> 84 (1), 127-133 (1986).</li><li>Hedrich, H. J., Bullock, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+laboratory+mouse.">The laboratory mouse.</a> , 735-736 (2004).</li><li>Nishimori, K., Young, L. J., Guo, Q., Wang, Z., Insel, T. R., Matzuk, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxytocin+is+required+for+nursing+but+is+not+essential+for+parturition+or+reproductive+behavior.">Oxytocin is required for nursing but is not essential for parturition or reproductive behavior.</a> Proc. Natl. Acad. Sci. USA. 93 (21), 11699-11704 (1996).</li><li>Miller, B. F., Osborn, D. A., Lance, W. R., Howze, M. M., Warren, R. J., Miller, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Butorphanol-azaperone-medetomidine+for+immobilization+of+captive+white-tailed+deer.">Butorphanol-azaperone-medetomidine for immobilization of captive white-tailed deer.</a> 45 (2), 457-467 (2009).</li></ol>

The authors have nothing to disclose.

Mouse Existem vários factores a ter em consideração aquando da recolha de leite de um rato, incluindo 1) a quantidade de tempo reservado para a coleta, 2) a dose de ocitocina administrada, 3) quantos filhotes da barragem é atualmente de enfermagem e 4) a quantidade de tempo que se passou desde o parto, no momento da coleta. Usando estudos anteriores como diretrizes, partimos para otimizar as condições de produção de leite. Pesquisas anter...

Os modelos animais fornecem informações sobre a patologia da doença que não pode ser adquirida através da análise in vitro. Para obter os resultados mais eficazes, é importante o uso de um modelo animal, que está intimamente relacionado com a doença e da espécie de interesse. Por exemplo, o muntjac Reeves (Muntiacus reevesi), um pequeno cervo asiáticos 1,2 e camundongos transgênicos que expressam a proteína príon cervídeos (CerTgPrP) 3 são modelos animais úteis para as espécies de cervídeos. Ambas as espécies são polyestrous, permitindo reprodução durante todo o ano e, portanto, uma fonte consistente de tecidos e fluidos relacionados com a gravidez para estudar mecanismos específicos em biologia cervídeos. Estudos de leite tem uma vasta gama de aplicações, que são mais simples (e barata) realizados em modelos animais do que em seres humanos. Pesquisadores poderia investigar leite e colostro como uma fonte potencial de 1) a transmissão de doenças infecciosas, 2) imunoglobulinas transferidos de mãe para filho no desenvolvimento da imunidade passiva 4 e 3) a lactoferrina, uma proteína encontrada no leite materno humano envolvido na imunidade passiva que os investigadores estão actualmente a tentar produzir comercialmente 5. Coleta de uma quantidade substancial de leite de pequenos animais pode revelar-se uma tarefa difícil. Rogers propôs uma abordagem para coletar o leite de ratos 6, que foi posteriormente utilizados em camundongos. DePeters e Hovey propostos dois métodos para a recolha de leite, um utilizando um vácuo gerado produzido manualmente por um bolbo de pipeta de borracha ligada a uma pipeta de Pasteur, e uma segunda que exige a construção de uma unidade de ordenha, que é então ligado a uma fonte de vácuo (tal como uma torneira) para colher o leite do mouse 7. Aqui propomos um método de custo simples, de baixo para a recolha de leite de ratos e Reeves &#39;veado muntjac, que requer equipamentos de laboratório apenas prontamente disponíveis e habilidades técnicas básicas. Nosso método produz volumes suficientes de leite para diversasaplicações.

<table border="0" cellpadding="0" cellspacing="0" width="1009">
	<tbody>
		<tr height="28">
			<td height="28" style="height:28px;width:360px;">Materials-Mouse</td>
			<td style="width:297px;"></td>
			<td style="width:236px;"></td>
			<td style="width:116px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Reagent</td>
			<td>Vendor</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ketamine (KetaVed 100 mg/mL) (dose: 80-100 mg/kg)</td>
			<td>MWI/Vedco</td>
			<td>011075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine (TranquiVed 100 mg/ml) (dose:5-10 mg/kg)</td>
			<td>MWI/Vedco</td>
			<td>NADA 139236</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eye lubricant</td>
			<td>MWI/Apexa</td>
			<td>701013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (2 IU/kg)</td>
			<td>Vet One</td>
			<td>501013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">Materials- Muntjac</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Material/Reagent</td>
			<td>Vendor</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Midazolam (1-2 mg/kg)</td>
			<td>Amerisource Bergen Drug Corp</td>
			<td>924326</td>
			<td>optional: researcher can either use Midazolam on its own or BAM combination as an anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butorphanol tartrate (Torbugesic) (0.45 mg/kg)</td>
			<td>Fort Dodge Animal Health</td>
			<td>NADA 135-780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Azaperone tartrate (0.25 mg/kg)</td>
			<td>ZooPharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Medetomadine HCl (0.07 mg/kg)</td>
			<td>ZooPharm</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Antipamezol hydrochloride (Antisedan) (dose: 0.25 mg/kg)</td>
			<td>Orion Corporation</td>
			<td>NADA 141-033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oxytocin (10 IU/kg)</td>
			<td>Vet One</td>
			<td>501013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">Equipment- Mouse</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Equipment</td>
			<td>Vendor</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">P-200 Pipetman</td>
			<td>Gilson</td>
			<td>F123601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 &#956;L pipette tips</td>
			<td>Light Labs</td>
			<td>B-2004-SH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 ml syringes</td>
			<td>MWI</td>
			<td>005427</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">27 gauge needles</td>
			<td>MWI</td>
			<td>002031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;">Equipment- Muntjac</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name of Equipment</td>
			<td>Vendor</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 ml conical tubes</td>
			<td>VWR</td>
			<td>21008-918</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">22 gauge needles</td>
			<td>MWI</td>
			<td>873</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3 ml luer lock syringes</td>
			<td>MWI</td>
			<td>001377</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Mouse A partir de nossas experiências, nós determinamos que é possível recolher cerca de 100-400 mL de leite de um rato de laboratório, dependendo de diversas variáveis. Estas variáveis ​​incluem: 1) a quantidade de tempo reservado para a coleta, 2) a dose de ocitocina administrada, 3) quantos filhotes da barragem é atualmente de enfermagem e 4) o período de tempo após o parto. Os nossos estudos mostraram que o rendimento mais elevado é obtido quando os reserv...

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Milk Collection Methods for Mice and Reeves&#039; Muntjac Deer

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 ...

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Research

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Biology

Milk Collection Methods for Mice and Reeves' Muntjac Deer

19.7K visualizações.  Colorado State University. 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’ muntjac deer. 

Vídeo: Milk Collection Methods for Mice and Reeves' Muntjac Deer

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae produção de ascósporos e Coleta de Experimentos de Microarray.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

Biologia

Seawater Sampling and Collection

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses. For today's demonstration samples were collected from the deck of the HMS John Strickland operating in Saanich Inlet. An A-frame derrick, with a multi-purpose winch and cable system, is used in combination with Niskin or Go-Flo water sampling bottles. Conductivity, Temperature, and Depth (CTD) sensors are also used to sample the underlying water mass. To minimize outgassing, trace gas samples are collected first.  Then, nutrients, water chemistry, and cell counts are determined. Finally, waters are collected for biomass filtration. The set-up and collection time for a single cast is ~1.5 hours at a maximum depth of 215 meters. Therefore, a total of 6 hours is generally needed to complete the collection series described here.

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass concentration, nucleic acid purification, cell abundance, nutrient and trace gas analyses.

A amostragem da água do mar e Cobrança

This video documents methods for collecting coastal marine water samples and processing them for various downstream applications including biomass ...

Diagnostic Necropsy and Selected Tissue and Sample Collection in Rats and Mice

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal colony. Proper collection of tissues for further histological processing can impact the quality of testing results. This article describes the conduct of a basic gross examination including identification of heart, liver, lungs, kidneys, and spleen, as well as how to collect those organs. Additionally four of the more difficult tissue/sample collection techniques are demonstrated. Lung collection and perfusion can be particularly challenging as the tissue needs to be properly inflated with a fixative in order for inside of the tissue to fix properly and to enable thorough histologic evaluation. This article demonstrates the step by step technique to remove the lung and inflate it with fixative in order to achieve optimal fixation of the tissue within 24 hours. Brain collection can be similarly challenging as the tissue is soft and easily damaged. This article demonstrates the step by step technique to expose and remove the brain from the skull with minimal damage to the tissue. The mesenteric lymph node is a good sample type in which to detect many common infectious agents as enteric viruses persist longer in the lymph node than they are shed in feces. This article demonstrates the step by step procedure for locating and aseptically removing the mesenteric lymph node. Finally, identification of infectious agents of the respiratory tract may be performed by bacterial culture or PCR testing of nasal and/or bronchial fluid aspirates taken at necropsy. This procedure describes obtaining and preparing the respiratory aspirate sample for bacterial culture and PCR testing.

This article describes the procedures for conducting a basic postmortem examination of a mouse or rat, and the collection of basic organs, as well as more challenging sample types from for histological, microbiological, and PCR evaluation.

Necropsia de diagnóstico e coleta de tecido e amostra selecionada em ratos e camundongos

There are multiple sample types that may be collected from a euthanized animal in order to help diagnose or discover infectious agents in an animal ...

Basic Caenorhabditis elegans Methods: Synchronization and Observation

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has since been used extensively as a model organism 1. C. elegans possesses key attributes such as simplicity, transparency and short life cycle that have made it a suitable experimental system for fundamental biological studies for many years 2. Discoveries in this nematode have broad implications because many cellular and molecular processes that control animal development are evolutionary conserved 3.
C. elegans life cycle goes through an embryonic stage and four larval stages before animals reach adulthood. Development can take 2 to 4 days depending on the temperature. In each of the stages several characteristic traits can be observed. The knowledge of its complete cell lineage 4,5 together with the deep annotation of its genome turn this nematode into a great model in fields as diverse as the neurobiology 6, aging 7,8, stem cell biology 9 and germ line biology 10. 
An additional feature that makes C. elegans an attractive model to work with is the possibility of obtaining populations of worms synchronized at a specific stage through a relatively easy protocol. The ease of maintaining and propagating this nematode added to the possibility of synchronization provide a powerful tool to obtain large amounts of worms, which can be used for a wide variety of small or high-throughput experiments such as RNAi screens, microarrays, massive sequencing, immunoblot or in situ hybridization, among others. 
Because of its transparency, C. elegans structures can be distinguished under the microscope using Differential Interference Contrast microscopy, also known as Nomarski microscopy. The use of a fluorescent DNA binder, DAPI (4',6-diamidino-2-phenylindole), for instance, can lead to the specific identification and localization of individual cells, as well as subcellular structures/defects associated to them.

Basic Caenorhabditis elegans Methods: Synchronization and Observation

The easiness of maintaining and propagating the nematode C. elegans make it a nice model organism to work with. The possibility of synchronizing worms allows the work with a significant amount of subjects at the same developmental stage, what facilitates the study of one particular process in many animals.

Básico<em&gt; Caenorhabditis elegans</em&gt; Métodos: Sincronização e observação

Research into the molecular and developmental biology of the nematode Caenorhabditis elegans was begun in the early seventies by Sydney Brenner and it has ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

Métodos para a pele ferimento e Ensaios para Responses ferida no<em&gt; C. elegans</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

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.

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 ...

Methods for the Isolation, Culture, and Functional Characterization of Sinoatrial Node Myocytes from Adult Mice

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). These pacemaker APs reflect the coordinated activity of numerous membrane currents and intracellular calcium cycling. However the precise mechanisms that drive spontaneous pacemaker activity in SAMs remain elusive. Acutely isolated SAMs are an essential preparation for experiments to dissect the molecular basis of cardiac pacemaking. However, the indistinct anatomy, complex microdissection, and finicky enzymatic digestion conditions have prevented widespread use of acutely isolated SAMs. In addition, methods were not available until recently to permit longer-term culture of SAMs for protein expression studies. Here we provide a step-by-step protocol and video demonstration for the isolation of SAMs from adult mice. A method is also demonstrated for maintaining adult mouse SAMs in vitro and for expression of exogenous proteins via adenoviral infection. Acutely isolated and cultured SAMs prepared via these methods are suitable for a variety of electrophysiological and imaging studies.

Methods are demonstrated for the isolation of sinoatrial node myocytes (SAMs) from adult mice for patch clamp electrophysiology or imaging studies. Isolated cells can be used directly or can be maintained in culture to permit expression of proteins of interest, such as genetically encoded reporters.

Métodos para o isolamento, Cultura e caracterização funcional de SINOATRIAL Nó miócitos de ratos adultos

Sinoatrial node myocytes (SAMs) act as the natural pacemakers of the heart, initiating each heart beat by generating spontaneous action potentials (APs). ...

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in mammals. However, humans cannot oxidize cytidine monophosphate-N-acetylneuraminic acid to cytidine monophosphate-N-glycolylneuraminic acid due to a primary exon deletion of the CMAH gene. To understand the effects and implications of the lack of CMAH activity in more detail, a Cmah knock-out model in mice is of keen interest in basic and applied research. The analysis method to determine the phenotype of this mouse model is herein described in detail, and is based on the detection of both N-acetylneuraminic acid and N-glycolylenuraminic acid in the liver and milk of wild-type and Cmah knock-out mice. Endogenous sialic acids are released and derivatized with o-phenylenediamine to generate fluorogenic derivatives, which can be subsequently analyzed by HPLC. The presented protocol can be also applied for the analysis of milk and tissue samples from various other origins, and may be of use to investigate the nutritional and health effects of N-glycolylneuraminic acid.

Determination of Sialic Acids in Liver and Milk Samples of Wild-type and CMAH Knock-out Mice.

We describe a HPLC-based method for the determination of N-acetylneuraminic acid and N-glycolylenuraminic acid in mouse liver and milk.

Determinação de ácidos sialic em amostras de fígado e leite de tipo selvagem e<em&gt; CMAH</em&gt; Ratinhos de eliminação.

CMAH (cytidine monophosphate-N-acetylneuraminic acid hydroxylase) is responsible for the oxidation of cytidine monophosphate-N-acetylneuraminic acids in ...

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish species is that it possesses interfertile cave and river-dwelling populations. This enables the genetic mapping of heritable traits that were fixed during adaptation to the different environments of these populations. While this species can be maintained and bred in the lab, it is challenging to both obtain embryos during the daytime and create hybrid embryos between strains. In vitro fertilization (IVF) has been used with a variety of different model organisms to successfully and repeatedly breed animals in the lab. In this protocol, we show how, by acclimatizing A. mexicanus to different light cycles coupled with changes in water temperature, we can shift breeding cycles to a chosen time of the day. Subsequently, we show how to identify suitable parental fish, collect healthy gametes from males and females, and produce viable offspring using IVF. This enables related procedures such as the injection of genetic constructs or developmental analysis to occur during normal working hours. Furthermore, this technique can be used to create hybrids between the cave and surface-dwelling populations, and thereby enable the study of the genetic basis of phenotypic adaptations to different environments.

Gamete Collection and In Vitro Fertilization of Astyanax mexicanus

In vitro fertilization is a commonly used technique with a variety of model organisms to maintain lab populations and produce synchronized embryos for downstream applications. Here, we present a protocol that implements this technique for different populations of the Mexican tetra fish, Astyanax mexicanus.

Coleção gamete e fertilização in vitro de Astyanax mexicanus

Astyanax mexicanus is emerging as a model organism for a variety of research fields in biological science. Part of the recent success of this teleost fish ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

Coleção de tecidos de morcegos para análises de Omics e cultura de células primárias

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...