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><tbody><tr><td colspan="4">Materials&amp;#8212;Mouse</td></tr><tr><td>Ketamine (KetaVed 100 mg/ml) (dose: 80-100 mg/kg)</td><td>MWI/Vedco</td><td>011075</td><td></td></tr><tr><td>Xylazine (TranquiVed 100 mg/ml) (dose: 5-10 mg/kg)</td><td>MWI/Vedco</td><td>NADA 139236</td><td></td></tr><tr><td>Eye lubricant</td><td>MWI/Apexa</td><td>701013</td><td></td></tr><tr><td>Oxytocin (2 IU/kg)</td><td>Vet One</td><td>501013</td><td></td></tr><tr><td colspan="4">Materials&amp;#8212;Muntjac</td></tr><tr><td>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><td>Butorphanol tartrate (Torbugesic) (0.45 mg/kg)</td><td>Fort Dodge Animal Health</td><td>NADA 135-780</td><td></td></tr><tr><td>Azaperone tartrate (0.25 mg/kg)</td><td>ZooPharm</td><td></td><td></td></tr><tr><td>Medetomadine HCl (0.07 mg/kg)</td><td>ZooPharm</td><td></td><td></td></tr><tr><td>Antipamezol hydrochloride (Antisedan) (dose: 0.25 mg/kg)</td><td>Orion Corporation</td><td>NADA 141-033</td><td></td></tr><tr><td>Oxytocin (10 IU/kg)</td><td>Vet One</td><td>501013</td><td></td></tr><tr><td colspan="4">Equipment&amp;#8212;Mouse</td></tr><tr><td>P-200 Pipetman</td><td>Gilson</td><td>F123601</td><td></td></tr><tr><td>200 &amp;mu;l pipette tips</td><td>Light Labs</td><td>B-2004-SH</td><td></td></tr><tr><td>1 ml syringes</td><td>MWI</td><td>005427</td><td></td></tr><tr><td>27 G needles</td><td>MWI</td><td>002031</td><td></td></tr><tr><td colspan="4">Equipment&amp;#8212;Muntjac</td></tr><tr><td>15 ml conical tubes</td><td>VWR</td><td>21008-918</td><td></td></tr><tr><td>22 G needles</td><td>MWI</td><td>873</td><td></td></tr><tr><td>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' 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.

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

19.8K Views.  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

Harvesting Sperm and Artificial Insemination of Mice

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals. Peromyscus species are used in a wide range of research including toxicology, epidemiology, ecology, behavioral, and genetic studies. Here they provide a useful model for demonstrations of artificial insemination. 

Methods similar to those displayed here have previously been used in several deer mouse studies, yet no detailed protocol has been published. Here we demonstrate the basic method of artificial insemination. This method entails extracting the testes from the rodent, then isolating the sperm from the epididymis and vas deferens. The mature sperm, now in a milk mixture, are placed in the female&#x2019;s reproductive tract at the time of ovulation. Fertilization is counted as day 0 for timing of embryo development. Embryos can then be retrieved at the desired time-point and manipulated.

Artificial insemination can be used in a variety of rodent species where exact embryo timing is crucial or hard to obtain. This technique is vital for species or strains (including most Peromyscus) which may not mate immediately and/or where mating is hard to assess. In addition, artificial insemination provides exact timing for embryo development either in mapping developmental progress and/or transgenic work. Reduced numbers of animals can be used since fertilization is guaranteed. This method has been vital to furthering the Peromyscus system, and will hopefully benefit others as well.

This protocol demonstrates methods for extracting sperm from the testes of males and then inseminating female mice. This procedure is useful when precise time is needed in developmental studies as well as transgenic work.

Rodents of the genus Peromyscus (deer mice) are the most prevalent native North American mammals.  Peromyscus species are used in a wide range of research ...

Protocol for Assessing the Relative Effects of Environment and Genetics on Antler and Body Growth for a Long-lived Cervid

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which promotes growth of large weaponry and body size. Populations of the same species display each phenotype depending on environmental conditions. Although antler and body size of male white-tailed deer (Odocoileus virginianus) varies by physiographic region in Mississippi, USA and is strongly correlated with regional variation in nutritional quality, the effects of population-level genetics from native stocks and previous re-stocking efforts cannot be disregarded. This protocol describes how we designed a controlled study, where other factors that influence phenotype, such as age and nutrition, are controlled. We brought wild-caught pregnant females and six-month-old fawns from three distinct physiographic regions in Mississippi, USA to the Mississippi State University Rusty Dawkins Memorial Deer Unit. Deer from the same region were bred to produce a second generation of offspring, allowing us to assess generational responses and maternal effects. All deer ate the same high-quality (20% crude protein deer pellet) diet ad libitum. We uniquely marked each neonate and recorded body mass, hind foot, and total body length. Each subsequent fall, we sedated individuals via remote injection and sampled the same morphometrics plus antlers of adults. We found that all morphometrics increased in size from first to second generation, with full compensation of antler size (regional variation no longer present) and partial compensation of body mass (some evidence of regional variation) evident in the second generation. Second generation males that originated from our poorest quality soil region displayed about a 40% increase in antler size and about a 25% increase in body mass when compared to their wild harvested counterparts. Our results suggest phenotypic variation of wild male white-tailed deer in Mississippi are more related to differences in nutritional quality than population-level genetics.

Phenotypic differences among cervid populations may be related to population-level genetics or nutrition; discerning which is difficult in the wild. This protocol describes how we designed a controlled study where nutritional variation was eliminated. We found that phenotypic variation of male white-tailed deer was more limited by nutrition than genetics.

Cervid phenotype can be placed into one of two categories: efficiency, which promotes survival over extravagant morphometric growth, and luxury, which ...

Environment

Modified Blood Collection from Tail Veins of Non-anesthetized Mice with a Vacuum Blood Collection System and Eyeglass Magnifier

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. The tail veins of mice are small, and it is sometimes difficult to obtain the required blood volume by conventional puncture methods. This study investigates the superiority of repeated blood sample collection from tail veins of mice through use of a vacuum blood collection system and eyeglass magnifier (experimental group) compared to conventional blood sampling methods (conventional group), performed by beginners and experts, respectively. With the help of an eyeglass magnifier, a butterfly needle tip is inserted into the tail vein of each mouse in the experimental group. When the vein is penetrated successfully, a blood sample is collected in the vacuum collection tube by insertion of the rubber end of a butterfly needle into the vacuum blood collection tube. The plunger is then used to collect blood without the help of the eyeglass magnifier in the conventional group. Success rates of blood sample collection by the beginners and experts were shown to be 70% vs. 100% (p &#60; 0.01) in the experimental group and 35% vs. 85% (p &#60; 0.01) in the conventional group. For both beginners and experts, puncture times required for obtaining required blood sample were significantly lower in the experimental group compared to the conventional group (2.40 &#177; 0.75 vs. 2.90 &#177; 0.31, p &#60; 0.05; 1.15 &#177; 0.37 vs. 1.55 &#177; 0.76, p &#60; 0.05). In conclusion, the presented blood sampling technique is feasible and easy to practice and enables frequent sampling of adequate blood volumes from non-anesthetized mice.&#160;

This study reports blood sampling from tail vein in mice using a vacuum extraction tube system with eyeglass magnifier. Our method is easy to practice and could be used for repeat blood sampling in mice.

Blood sample collection is the basis of experimental animal research. It is of importance to obtain adequate blood samples for various research purposes. ...

Behavior

Transabdominal Ultrasound for Pregnancy Diagnosis in Reeves' Muntjac Deer

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of prion disease transmission and pathogenesis. Vertical transmission is an area of interest among researchers studying infectious diseases, including prion disease, and these investigations require efficient methods for evaluating the effects of maternal infection on reproductive performance. Ultrasonographic examination is a well-established tool for diagnosing pregnancy and assessing fetal health in many animal species1-7, including several species of farmed cervids8-19, however this technique has not been described in Reeves' muntjac deer. Here we describe the application of transabdominal ultrasound to detect pregnancy in muntjac does and to evaluate fetal growth and development throughout the gestational period. Using this procedure, pregnant animals were identified as early as 35 days following doe-buck pairing and this was an effective means to safely monitor the pregnancy at regular intervals. Future goals of this work will include establishing normal fetal measurement references for estimation of gestational age, determining sensitivity and specificity of the technique for diagnosing pregnancy at various stages of gestation, and identifying variations in fetal growth and development under different experimental conditions.

Transabdominal ultrasound is described as an effective, noninvasive means for assessing reproductive status in Reeves' muntjac deer. These methods can be used to achieve early pregnancy diagnosis and to evaluate fetal viability. Future applications of this technique include estimation of gestational age and effects of maternal disease on fetal development.

Reeves' muntjac deer (Muntiacus reevesi) are a small cervid species native to southeast Asia, and are currently being investigated as a potential model of ...

Medicine

Reducing Research Animal Stress with Micropipette-Guided Drug Administration

Use of Micropipette-Guided Drug Administration as an Alternative Method to Oral Gavage in Rodent Models

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the stomach of research animals. Unfortunately, this method comes with difficulties for both the operator and the research animal. Studies have shown that OG may lead to complications, including esophagitis, perforation of the esophagus, and inadvertent tracheal drug administration. In addition, OG is associated with increased plasma and fecal corticosterone levels (due to stress), altered blood pressure, and increased heart rate, which could negatively influence or bias study results. A previously developed alternative method termed micropipette-guided drug administration (MDA) incentivizes the animal to consume treatments readily in a minimally invasive manner. Herein, we present examples of the use of the MDA technique with treatments reconstituted in different vehicles and demonstrate effective delivery of the varied treatments to multiple different mouse strains. We further demonstrate that MDA is a technique that decreases the timing and invasiveness of drug administration and does not affect the gut microbiome composition as assessed by quantitative analysis of core gut microbial species. Overall, MDA may offer a less stressful and effective alternative to OG.

Here, we describe micropipette-guided drug administration (MDA) as an alternative method to oral gavage that incentivizes the research animal to ingest treatments readily with minimal stress and discomfort.

Oral gavage (OG) with the use of a cannula attached to a syringe is one of the most common methods used to deliver precise dosing of compounds to the ...

Cancer Research

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus)

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of this research model are the ease of breeding and sample collection, relatively short gestation period, and large litter sizes. Commonly assessed routes of immunization include subcutaneous, intramuscular, intranasal, and intradermal. Nonterminal sample collection for the chronological detection of the immunologic responses to these immunizations include the collection of blood, from both dams and kits, and milk from the lactating does. In this article, we will demonstrate techniques our lab has utilized in studies of maternal immunization in New Zealand White rabbits (Oryctolagus cuniculus), including intranasal immunization and milk collection.

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits Oryctolagus cuniculus

This article describes and demonstrates the administration of intranasal vaccines and the collection of milk from lactating rabbits (Oryctolagus cuniculus) as a means to assess mucosal immunity in a translationally appropriate model of maternal immunization.

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of ...

Immunology and Infection

Blood Collection Through Subclavian Vein Puncture in Mice

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in research work. Tail blood collection is a popular method when a small amount of blood sample is needed. Orbital artery could be considered if a large amount of blood is needed but this blood collection method has ethical issues. Formerly, we demonstrated the feasibility and safety of blood sample collection through subclavian vein puncture in rats, and here we investigate whether this method could be used in mice. We report that this method is safe and practical for blood collection in mice. Blood collection through the subclavian vein puncture in mice can be a convenient method in daily research works.

Here, we present a protocol to take blood samples from the subclavian vein of mice.

The mouse is the foremost mammalian model for studying human disease and human health. However, blood sample collection from mice is challenging in ...

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 ...

Optimized Single&mdash;Person Method for Milk Collection in Lactation Studies of Mice

An Efficient Single-Person Technique for Milk Sampling from Laboratory Mice

Rodent models have been widely used in biology since the early 20th century to study basic physiology and biochemical mechanisms. In lactation and neonatal development research, there is a need to sample milk to determine the composition and how milk may be affected by experimental manipulations, thereby impacting newborn development. Collecting biofluids in sufficient amounts from small rodents can be difficult. Several studies have demonstrated different techniques to obtain milk from mice. However, these techniques often require at least two trained personnel, which may be challenging in some cases.&#160;
Here, we demonstrate a modified milk sampling technique based on a method published in 2009. With this affordable and easy-to-assemble method, one trained person can routinely obtain 0.1&#8211;0.5 mL of milk from a dam under anesthesia in less than 10 min. Using this method, we collected sufficient milk on post-delivery day 2 (PD2) and post-delivery day 10 (PD10). We measured milk macronutrient components and compared them with the existing literature to validate our collection method.&#160;
We recorded that on PD2, milk protein averaged at 70.1 &#177; 5.2 g/L, milk fat was 174.4 &#177; 7.1 g/L, and milk lactose was 12.3 &#177; 0.6 g/L. On PD10, while milk lactose and protein remained at similar concentrations as on PD2, milk fat was significantly higher (224.4 &#177; 17.1 g/L). We also observed that the relative abundance of individual milk proteins varied between PD2 and PD10. More specifically, &#945;S1-casein and whey acidic protein were higher, while &#946;-casein was lower at PD10 compared with PD2. Overall, we demonstrate an efficient single-person technique for milk sampling from mice using a device that can easily be assembled with commonly used laboratory equipment.

An Efficient Single&mdash;Person Technique for Milk Sampling from Laboratory Mice

This article describes how to assemble a device that can be used to collect milk from laboratory mice. This device is affordable, portable, and allows a collection of 0.1&#8211;0.5 mL of milk by one trained person. A detailed milking protocol is also included.&#160;