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. Mouse Milking Protocol
The methods described in this protocol were approved by IACUC.
1. Separation of the Dam from Offspring
<ol>
	<li>Select the dam to be milked. Choose a dam with a litter of 4 or more pups that is 8-12 days post parturition to provide maximal milk collection&#8212;although collection is possible at any time point post parturition up to 21 days.</li>
	<li>Separate the dam from her litter at least 2 hr before milking. 
	Note: Should one desire to milk more than one dam in a given time period, it is acceptable to house the separated dams in the same cage with adequate food and water supply.</li>
</ol>
2. Administration of Oxytocin, Anesthesia and Eye Lubricant
<ol>
	<li>Administer 2 IU/kg of oxytocin intraperitoneally (IP). Oxytocin is a hormone that acts on the mammary glands of lactating females to stimulate the release of milk. Oxytocin can be acquired from a veterinarian.</li>
	<li>Administer an anesthetic mixture containing 80-100 mg/kg ketamine in combination with 5-10 mg/kg xylazine IP. For example, a 10 ml stock anesthetic mixture could be prepared in advance for mice weighing approximately 30 g and would include 0.1 ml xylazine, 8.9 ml H2O, and 1.0 ml ketamine. 0.25 ml total anesthetic will sedate a mouse of this size for approximately 20-30 min, which should provide adequate time for milk collection. 
	Notes: To determine that the animal has been successfully anesthetized, gently place her on her stomach on a solid surface; she should not be moving her feet or attempting to rise to her feet if she is fully anesthetized. In the case of a mouse not being fully anesthetized with the original dosage, the researcher may administer another &#188; or &#189; of the original dose of the anesthetic mixture depending on the size of the mouse and/or level of sedation that the mouse has already achieved. However, this could increase the chance of an adverse reaction to anesthesia, and also may result in the mouse being more deeply anesthetized and/or anesthetized for a longer period of time. 
	Note: If milking more than one dam, it is necessary to stagger oxytocin and anesthetic injections due to the short half-life of oxytocin and sedation times for ketamine and xylazine.</li>
	<li>Apply a small amount of eye lubricant to the corner of each eye and spread to prevent the mouse&#8217;s eyes from drying out while under anesthesia.</li>
</ol>
3. Milk Collection
The milk collection is most easily performed with two researchers: one researcher to hold the anesthetized mouse while manually expressing the milk (referred to as R1), and one researcher to collect the milk ( R2). This method can also be performed with one person, if the mouse is secured on a flat surface to avoid harm.
<ol>
	<li>R2 should be equipped with sterile alcohol prep pads, a P-200 pipetman, a clean pipette tip for each mouse, and a container to hold the milk, such as a 1.5 ml Eppendorf tube or a 1.2 ml cryovial (see Table of Materials).</li>
	<li>As the oxytocin begins to take effect, milk letdown will become visible in the mouse&#8217;s mammary area (see Figure 1A). R1: Unwrap a sterile alcohol prep pad and gently wipe the mammary area of the mouse to clean the area prior to milking. Manually express milk from the teat by using the thumb and forefinger to gently massage and squeeze the mammary tissue in an upward motion until a visible bead of milk begins to form at the base of the teat.</li>
	<li>Press the P-200 pipetman plunger to its first stop to release air out of the pipette tip and prepare for milk collection.</li>
	<li>Position the pipette tip at the top of the drop of milk, and gently pull the milk into the pipette tip by slowly releasing pressure on the plunger. Take care not to put the tip too close to the teat or the skin (see Figure 1B). It is not necessary to completely release the plunger as this will pull the milk all the way to the top of the pipette tip and will make it difficult to expel the often viscous milk into the holding container.</li>
	<li>Expel the milk into the container by using the thumb to press the plunger past the first stop (see Figure 1C). To facilitate expulsion of small volumes, press the pipette tip against the side of the tube so that no liquid is lost.</li>
	<li>Continue to manually express milk from each teat, moving in either a clockwise or counterclockwise direction so as not to skip any teats. It is both acceptable and useful to come back to and continually express milk from the same teat should it produce more milk than another.</li>
</ol>
4. Anesthetic Reversal in the Dam
<ol>
	<li>In most cases, it is common for the dam to begin to begin to wake up during the milking process. If this is not the case, place the dam in a cage indirectly underneath a heat source, such as a heated circulating blanket, until she begins to wake up. 
	Note: As the dam begins to wake up, keep close observation of her activity until she is able to walk about her cage on her own.</li>
	<li>Once the dam is moving about the cage without struggle, place her back into her original cage with her litter. No ill effects have been noted with respect to the pups&#8217; ability to feed after milk collection.</li>
</ol>
2. Muntjac Milking Protocol
The methods described in this protocol were approved by IACUC.
1. Separation of the Doe from Offspring
<ol>
	<li>Select the doe to be milked. Reeves&#8217; muntjac deer generally give birth to one fawn2. Milk the doe the day after birthing to allow the fawn opportunity to gain access to essential nutrients in colostrum8. 
	Note: The fawn usually hides and does not reappear during the procedure; separation of the mother-offspring pair is not necessary.</li>
</ol>
2. Administration of Oxytocin, Anesthesia and Eye Lubricant
<ol>
	<li>Administer 10 IU/kg of Oxytocin intramuscularly (IM). Oxytocin is a hormone that acts on the mammary glands of lactating females to stimulate the let-down of milk. Oxytocin can be acquired from a veterinarian.</li>
	<li>Administer the BAM anesthetic IM11 containing butorphanol 0.45 mg/kg, azaperone 0.25 mg/kg, medetomadine 0.07 mg/kg. One dose of the BAM anesthetic mixture will sedate a muntjac for approximately 20-30 min, which provides ample time for complete milk collection. Another option for anesthetic is midazolam at 1-2 mg/kg, which will sedate a muntjac for approximately 10-15 min. While this provides adequate time for milk collection, this anesthetic is metabolized more quickly than BAM and the animal may attempt to escape at the end of the shorter time period. 
	Note: Minimal response to pain, minor reflexes upon touching the ears, followed by the ability of R1 to hold the MJ without struggle indicates that the animal has been successfully anesthetized. 
	Note: In the case of a muntjac not being fully anesthetized with the original dosage, the researcher may administer another &#188; or &#189; of the original dose of the anesthetic mixture depending on the size of the animal and/or level of sedation that has already been achieved. However, this could increase the chance of an adverse reaction to anesthesia, and also may result in the muntjac being more deeply anesthetized and/or anesthetized for a longer period of time.</li>
	<li>Apply a small amount of lubricant to the corner of each eye and spread to prevent the muntjac&#8217;s eyes from drying out while under anesthesia.</li>
</ol>
3. Milk Collection
The milk collection is most easily performed with two researchers; one researcher to hold the anesthetized muntjac in the lap while manually expressing the milk (Referred to as R1), and a second researcher to keep the muntjac&#8217;s head in a comfortable and safe position and to collect the milk (R2).
<ol>
	<li>Depending upon the anesthetic used, milking should be performed either 1) in their pens to provide a safe place for them should they wake up during the procedure (midazolam-induced) or 2) in a bedding-free anteroom to maintain cleanliness (BAM-induced).</li>
	<li>R1 and R2 should be equipped with ethanol-soaked gauze and several 15 ml conical polypropylene tubes to hold the milk.</li>
	<li>As the oxytocin begins to take effect, milk letdown will become visible in the muntjac&#8217;s mammary area. R1: Sanitize gloved hands and fingers, as well as the entire udder and each individual teat of the animal with the ethanol-soaked gauze. R2: Hold the muntjac&#8217;s head in a safe and comfortable position to allow ease of breathing and help prevent rumen regurgitation. Hold the collection tube close to the teat being milked without touching it to prevent potential contamination.</li>
	<li>Express milk from one teat by using the thumb and forefinger to gently squeeze the mammary tissue in an upward motion until the teat is engorged with milk, and gently roll the milk out of the teat and into the tube that R2 is holding (see Figure 2).</li>
	<li>Note: The milk comes out in a stream, so it is important for R2 to have the collection tube positioned so that little to no milk will be wasted.</li>
	<li>The muntjac&#8217;s udder is divided into quadrants. R1: Continue to express milk from each quadrant until the milk no longer flows easily. If milk cannot be expressed from one teat, express from all other teats before returning to the first. It is both acceptable and useful to come back to and continually express milk from the same teat should it produce more milk than another.</li>
</ol>
4. Anesthetic Reversal in the Doe
It is common for midazolam-induced doe to begin to wake up during the milking process. In most cases, the doe will attempt to escape before all the milk has been expressed. Minor restraint is used in an effort to increase the milk yield; however the safety and comfort of both the researchers and the doe are equally important.
<ol>
	<li>Administer atipamezole at 2.5 mg/mg medetomadine subcutaneously for rapid reversal of BAM anesthetized doe. 
	Note: With both types of anesthetic, it is necessary to maintain close observation of the doe until she is able to walk on her own without stumbling or falling, and is able to eat and drink without fear of aspiration or choking. No ill effects have been noted with respect to the fawn&#8217;s ability to nurse after milk collection.</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
There are several factors to take into consideration when collecting milk from a mouse, including 1) the amount of time set aside for collection, 2) the dose of oxytocin administered, 3) how many pups the dam is currently nursing and 4) the amount of time that has passed since parturition at the time of collection. Using previous studies as guidelines, we set out to optimize conditions for milk yield.
Previous research has directly correlated ...

Animal models provide insight into disease pathology that cannot be gained by in vitro analysis. To provide the most efficacious results, it is important to use an animal model that is closely related to the disease and species of interest. For example, the Reeves&#8217; muntjac (Muntiacus reevesi), a small Asian deer1,2, and transgenic mice expressing the cervid prion protein (CerTgPrP)3 are useful animal models for cervid species. Both species are polyestrous, allowing year-round breeding, and therefore a consistent source for pregnancy-related tissues and fluids to study specific mechanisms in cervid biology. Studies of milk have a vast array of applications that are more simply (and inexpensively) accomplished in animal models than in humans. Researchers could investigate milk and colostrum as a potential source of 1) infectious disease transmission, 2) immunoglobulins transferred from mother to offspring in the development of passive immunity4 and 3) lactoferrin, a protein found in human breast milk involved in passive immunity that researchers are currently attempting to commercially produce5.
Collecting a substantial amount of milk from small animals can prove to be a difficult task. Rogers proposed an approach to collect milk from rats6, which was subsequently used in mice. DePeters and Hovey proposed two methods for milk collection, one using a manually-generated vacuum produced by a rubber pipette bulb attached to a Pasteur pipette, and a second requiring the construction of a milking unit, which is then attached to a vacuum source (such as a faucet) to harvest mouse milk7. Here we propose a simple, low cost method for collecting milk from both mice and Reeves&#8217; muntjac deer, which requires only readily available laboratory equipment and basic technical skills. Our method yields sufficient volumes of milk for various applications.

<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
From our experiments, we have determined that it is possible to collect approximately 100-400 &#956;l of milk from one laboratory mouse, dependent on several variables. These variables include 1) the amount of time set aside for collection, 2) the dose of oxytocin administered, 3) how many pups the dam is currently nursing and 4) the amount of time post parturition. Our studies have shown that the highest yield is obtained when the dams are separated from their offspring...

Watch this Scientific Journal Video about Milk Collection Methods for Mice and Reeves' Muntjac Deer at JoVE.com

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.

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 ...

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

Research

JoVE Journal

Biology

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.

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

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.

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.

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.

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.

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

Repeated Blood Collection for Blood Tests in Adult Zebrafish

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from zebrafish has not been established. The previous methods for blood collection from zebrafish are lethal, such as lateral incision, decapitation and tail ablation. Thus we have developed a novel &#8220;repeated&#8221; blood collection method, and present here a detailed protocol outlining this procedure. This method is minimally invasive and results in a very low mortality rate (2.3%) for zebrafish, thus enabling repeated blood sampling from the same individual. The maximum volume of blood sampling is dependent on body weight of the fish. The volume for repeated blood sampling at intervals should be&#160;&#8804;0.4% of body weight every week or &#8804;1% every 2 weeks, which were evaluated by measurements of blood hemoglobin. Additionally, hemoglobin, fasting blood glucose, plasma triacylglycerol (TG) and total cholesterol levels in male and female adult zebrafish were measured. We also applied this method to investigate the dysregulation of glucose metabolism in diet-induced obesity. This blood collection method will allow many applications, including glucose and lipid metabolism and hematological studies, which will increase the use of zebrafish as a human disease model organism.

Repeated blood sampling is necessary and important in animal research. We developed a novel, non-lethal and reliable method for repeated blood collection from adult zebrafish, and applied this method to the study of blood biochemistry, including glucose metabolism.

Repeated blood collection is one of the most common techniques performed on laboratory animals. However, a non-lethal protocol for blood collection from ...

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.

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.

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.

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.

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 ...