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. Protocolo de ordeño Ratón Los métodos descritos en este protocolo fue aprobado por el IACUC. 1. Separación de la presa de Offspring <ol><li> Seleccione la presa para ser ordeñadas. Elija una presa con una camada de 4 o más crías que es 8-12 días post parto para proporcionar leche máxima de recogida, aunque la colección es posible en cualquier punto después del parto hasta 21 días. </li><li> Separe la presa de su camada por lo menos 2 horas antes del ordeño. Nota: En caso de un deseo a la leche más de una presa en un determinado período de tiempo, es aceptable para albergar las presas separadas en la misma jaula con una alimentación adecuada y el suministro de agua. </li></ol> 2. La administración de oxitocina, Anestesia y lubricantes para ojos <ol><li> Administrar 2 UI / kg de oxitocina por vía intraperitoneal (IP). La oxitocina es una hormona que actúa sobre las glándulas mamarias de hembras lactantes para estimular la liberación de la leche. La oxitocina puede ser adquirido from un veterinario. </li><li> Administrar una mezcla anestésica que contiene 80-100 mg / kg de ketamina en combinación con 5-10 mg / kg IP xilazina. Por ejemplo, una mezcla de 10 ml de solución madre anestesia se podría preparar con antelación para los ratones con un peso aproximado de 30 g e incluiría 0,1 ml de xilazina, 8,9 ml de H2O y 1,0 ml de ketamina. 0.25 anestésica ml se sedar a un ratón de este tamaño por aproximadamente 20 a 30 min, que debe proporcionar el tiempo adecuado para la recogida de leche. Notas: Para determinar que el animal ha sido anestesiado con éxito, coloque suavemente su boca abajo sobre una superficie sólida; ella no debe estar en movimiento sus pies o intentar ponerse de pie si ella está totalmente anestesiado. En el caso de un ratón no está totalmente anestesiado con la dosis original, el investigador puede administrar otra de ¼ o ½ de la dosis original de la mezcla anestésica dependiendo del tamaño del ratón y / o el nivel de sedación que el ratón ya ha alcanzado . Sin embargo, esto podría iaumentar la cant posibilidad de una reacción adversa a la anestesia, y también puede resultar en el ratón está más profundamente anestesiados y / o anestesiado durante un período de tiempo más largo. Nota: Si el ordeño más de una presa, es necesario escalonar la oxitocina y las inyecciones anestésicas debido a la corta vida media de la oxitocina y los tiempos de sedación para la ketamina y xilazina. </li><li> Aplique una pequeña cantidad de lubricante ocular a la esquina de cada ojo y se extendió a prevenir los ojos del ratón se seque mientras está bajo anestesia. </li></ol> 3. Recogida de Leche La recogida de la leche se realiza más fácilmente con dos investigadores: un investigador para sostener el ratón anestesiado al tiempo que expresa manualmente la leche (denominado R1), y un investigador para recoger la leche (R2). Este método también se puede realizar con una sola persona, si el ratón está fijado sobre una superficie plana para evitar daño. <ol><li> R2 debe estar equipado con almohadillas con alcohol estériles, un pip P-200Etman, una punta de pipeta limpia para cada ratón, y un recipiente para contener la leche, tal como un tubo Eppendorf de 1,5 ml o 1,2 ml criovial (véase la Tabla de Materiales). </li><li> Como la oxitocina empieza a hacer efecto, bajada de la leche se hará visible en la zona mamaria del ratón (véase la Figura 1 A). R1: Separar una almohadilla con alcohol estéril y frote suavemente la zona mamaria del ratón para limpiar la zona antes del ordeño. Expresar manualmente la leche de la teta utilizando el pulgar y el índice para masajear suavemente y apretar el tejido mamario en un movimiento hacia arriba hasta que una gota visible de la leche empieza a formar en la base de la tetina. </li><li> Pulse el Pipetman émbolo P-200 a su primera parada para liberar el aire de la punta de la pipeta y prepararse para la recogida de leche. </li><li> Coloque la punta de la pipeta en la parte superior de la gota de leche, y tire suavemente de la leche en la punta de la pipeta al liberar lentamente la presión sobre el émbolo. Tenga cuidado de no poner la punta demasiado cerca del pezón o la piel (verFigura 1B). No es necesario para liberar completamente el émbolo como este se tire de la leche todo el camino hasta la parte superior de la punta de pipeta y hará que sea difícil para expulsar la leche a menudo viscoso en el recipiente de retención. </li><li> Expulsar la leche en el contenedor utilizando el pulgar para presionar el émbolo más allá de la primera parada (véase la Figura 1C). Para facilitar la expulsión de pequeños volúmenes, presione la punta de la pipeta contra el lado del tubo, de manera que no se pierda líquido. </li><li> Continuar expresar manualmente la leche de cada pezón, moviéndose en sentido horario o en sentido contrario para que no se salte ninguna pezones. Es aceptable y útil para volver y expresar continuamente la leche de la misma teta debería producir más leche que el otro. </li></ol> 4. Reversión anestésico en la presa <ol><li> En la mayoría de los casos, es común que la presa para empezar a comenzar a despertar durante el proceso de ordeño. Si este no es el caso, coloque la presa en una cage indirectamente por debajo de una fuente de calor, como una frazada que circula calienta, hasta que comienza a despertar. Nota: Como la presa comienza a despertar, mantener una estrecha observación de su actividad hasta que ella es capaz de caminar sobre su jaula por su cuenta. </li><li> Una vez que la represa se está moviendo alrededor de la jaula sin lucha, colocarla de nuevo en su jaula original con su camada. No hay efectos nocivos se han observado con respecto a la capacidad de las crías a alimentarse después de la recogida de leche. </li></ol> 2. Protocolo de ordeño Muntjac Los métodos descritos en este protocolo fue aprobado por el IACUC. 1. Separación de los Pérez de Offspring <ol><li> Seleccione la gama de ser ordeñadas. Ciervos muntjac Reeves general dan a luz a un cervatillo 2. Ordeñar la cierva el día después del parto para permitir la oportunidad cervatillo para ganar acceso a los nutrientes esenciales en el calostro 8. Nota: El cervatillo suele ocultar y no vuelve a aparecer durante el procedure; la separación del par madre-descendencia no es necesario. </li></ol> 2. La administración de oxitocina, Anestesia y lubricantes para ojos <ol><li> Administrar 10 UI / kg de oxitocina por vía intramuscular (IM). La oxitocina es una hormona que actúa sobre las glándulas mamarias de las hembras lactantes para estimular la bajada de la leche. La oxitocina puede ser adquirido de un veterinario. </li><li> Administrar el anestésico BAM IM 11 que contiene butorfanol 0,45 mg / kg, azaperona 0,25 mg / kg, medetomadine 0,07 mg / kg. Una dosis de la mezcla anestésica BAM sedar un muntjac por aproximadamente 20 a 30 min, que proporciona el tiempo suficiente para la recolección de la leche completa. Otra opción para anestesia es de midazolam en 1-2 mg / kg, que sedar un muntjac de aproximadamente 10-15 minutos. Si bien esto proporciona un tiempo adecuado para la recogida de leche, este anestésico se metaboliza más rápidamente que BAM y el animal pueden intentar escapar al final del período de tiempo más corto. Nota: respuesta mínima al dolor, Reflejos menores al tocar los oídos, seguidos por la capacidad de R1 para mantener el MJ sin lucha indica que el animal ha sido anestesiado con éxito. Nota: En el caso de un muntjac no está totalmente anestesiado con la dosis original, el investigador puede administrar otra de ¼ o ½ de la dosis original de la mezcla anestésica dependiendo del tamaño del animal y / o el nivel de sedación que ya ha sido alcanzado. Sin embargo, esto podría aumentar la posibilidad de una reacción adversa a la anestesia, y también puede resultar en la muntjac ser más profundamente anestesiados y / o anestesiado durante un período de tiempo más largo. </li><li> Aplicar una pequeña cantidad de lubricante a la esquina de cada ojo y propagación para evitar que los ojos del muntjac de la desecación, mientras que bajo anestesia. </li></ol> 3. Recogida de Leche La recolección de la leche se realiza más fácilmente con dos investigadores; uno de los investigadores para mantener el muntjac anestesiado enla vuelta al tiempo que expresa manualmente la leche (Conocida como R1), y un segundo investigador para mantener la cabeza del muntjac en una posición cómoda y segura y para recoger la leche (R2). <ol><li> Dependiendo de la anestesia utilizada, el ordeño debe llevarse a cabo ya sea 1) en sus corrales para proporcionar un lugar seguro para ellos en caso de que despierta durante el procedimiento (midazolam-inducida) 2) en una antesala-ropa de cama de forma gratuita o de mantener la limpieza (BAM- inducida). </li><li> R1 y R2 deben estar equipados con una gasa empapada en etanol y varios tubos de 15 ml de polipropileno cónicos para mantener la leche. </li><li> Como la oxitocina empieza a hacer efecto, bajada de la leche se hará visible en la zona mamaria del muntjac. R1: Desinfectar las manos y los dedos enguantados, así como toda la ubre y cada pezón individual del animal con la gasa empapada en etanol. R2: Sostenga la cabeza del muntjac en una posición segura y cómoda para permitir la facilidad de la respiración y ayudar a prevenir la regurgitación rumen. Mantenga los cl tubo de recogida<html> OSE al pezón ordeñada sin tocarlo para evitar la contaminación potencial. </li><li> Extraiga la leche de un pezón con los dedos pulgar e índice para apretar suavemente el tejido mamario en un movimiento hacia arriba hasta que el pezón se hincha con la leche, y rodar suavemente la leche del pezón y en el tubo que R2 está llevando a cabo (véase la Figura 2 ). </li><li> Nota: La leche sale en un arroyo, por lo que es importante para el R2 para que el tubo de extracción colocado de manera que poco o nada de leche será en vano. </li><li> La ubre de la muntjac se divide en cuadrantes. R1: Continuar para expresar la leche de cada cuadrante hasta que la leche ya no fluye fácilmente. Si la leche no se puede expresar de una teta, expresa de todas las otras tetinas antes de regresar a la primera. Es aceptable y útil para volver y expresar continuamente la leche de la misma teta debería producir más leche que el otro. </li></ol> 4. Inversión de anestésico en el DOE t &quot;&gt; Es común que doe inducida midazolam-para empezar a despertar durante el proceso de ordeño. En la mayoría de los casos, la cierva intentará escapar antes de que toda la leche se ha expresado. moderación Minor se utiliza en un esfuerzo por aumentar la leche rendimiento; sin embargo, la seguridad y el confort tanto de los investigadores y la hembra son igualmente importantes. <ol><li> Administrar atipamezol a 2,5 mg / mg medetomadine subcutánea para la rápida reversión de BAM anestesiado doe. Nota: Con ambos tipos de anestesia, es necesario mantener una estrecha observación de la hembra hasta que ella es capaz de caminar por sí misma sin tropezar o caer, y es capaz de comer y beber sin temor a la aspiración o atragantamiento. No hay efectos nocivos se han observado con respecto a la capacidad del cervatillo para amamantar después de la recogida de leche. </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.

Ratón Hay varios factores a tener en cuenta en la recogida de leche de un ratón, incluyendo 1) la cantidad de tiempo dedicado a la recolección, 2) la dosis de oxitocina administrada, 3) el número de crías de la presa es actualmente de enfermería y 4) la cantidad de tiempo que ha pasado desde el parto en el momento de la recolección. A partir de estudios anteriores como guía, nos dispusimos a optimizar las condiciones para la producción de leche. <p class="jove_c...

Los modelos animales proporcionan información sobre la patología de la enfermedad que no puede ser adquirida mediante el análisis in vitro. Para proporcionar los resultados más eficaces, es importante utilizar un modelo animal que está estrechamente relacionado con la enfermedad y de las especies de interés. Por ejemplo, el muntjac Reeves (Muntiacus reevesi), un pequeño ciervo de Asia 1,2, y los ratones transgénicos que expresan la proteína priónica cérvido (CerTgPrP) 3 son los modelos animales útiles para las especies de cérvidos. Ambas especies son poliestro, lo que permite la cría durante todo el año, y por lo tanto una fuente constante de tejidos y fluidos relacionadas con el embarazo para estudiar mecanismos específicos en la biología cérvido. Los estudios de la leche tienen una amplia gama de aplicaciones que son más simplemente (y barata) realizados en modelos animales que en los seres humanos. Los investigadores podrían investigar la leche y el calostro como fuente potencial de 1) la transmisión de enfermedades infecciosas, 2) las inmunoglobulinas transferidos de madre a hijo en el desarrollo de ciertasllo de la inmunidad pasiva 4 y 3) la lactoferrina, una proteína presente en la leche materna humana implicada en la inmunidad pasiva que los investigadores están tratando actualmente para producir comercialmente 5. La recolección de una cantidad sustancial de la leche de animales pequeños puede llegar a ser una tarea difícil. Rogers propone un enfoque para recoger la leche de ratas 6, que se utilizó posteriormente en ratones. De Peters y Hovey proponen dos métodos para la recogida de leche, uno utilizando un vacío generado manualmente-producida por un bulbo de pipeta de goma en una pipeta Pasteur, y una segunda que requiere la construcción de una unidad de ordeño, que luego se une a una fuente de vacío (tales como un grifo) para cosechar la leche del ratón 7. Aquí proponemos un método simple y económica para la recogida de leche de ambos ratones y ciervos muntjac Reeves, que requiere equipos de laboratorio sólo disponibles y habilidades técnicas básicas. Nuestro método proporciona un volumen suficiente de leche para variosaplicaciones.

<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.

Ratón Desde nuestros experimentos, hemos determinado que es posible recoger aproximadamente 100-400 l de leche de un ratón de laboratorio, dependen de diversas variables. Estas variables incluyen: 1) la cantidad de tiempo dedicado a la recolección, 2) la dosis de oxitocina administrada, 3) el número de crías de la presa es actualmente de enfermería y 4) el importe de la post parto vez. Nuestros estudios han demostrado que se obtiene el máximo rendimiento cuando las pr...

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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 recolección de leche Ratones y 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 ...

milk-collection-methods-for-mice-and-reeves-muntjac-deer

Research

JoVE Journal

Biology

Milk Collection Methods for Mice and Reeves' Muntjac Deer

19.6K vistas.  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. 

Video: 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 ascósporas de producción y de recolección de experimentos de microarrays.

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

Investigación

Biología

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.

El agua de mar de muestreo y recolección

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.

La necropsia de diagnóstico y selecta colección de tejidos y muestras en ratas y ratones

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 de sincronización y de observación

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.

Los métodos para la piel Heridas y ensayos para Respuestas de heridas en<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.

Leche Colección de la rata utilizando tubos capilares y Estimación del contenido de grasa de la leche por 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 ...

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.

Los métodos para el aislamiento, cultivo y caracterización funcional de los miocitos del nodo sinusal ratones 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.

Determinación de ácidos siálicos en hígado y muestras de leche de tipo salvaje y<em&gt; CMAH</em&gt; Ratones knock-out.

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.

Gamete Collection y Fertilización 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.

Colección de tejidos de murciélagos para análisis de émica y cultivo celular primario

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