This works was supported through grants from the Natural Sciences and Engineering Research Council of Canada (RGPIN 238382-2011) and Canadian Institutes of Health Research (MOP115076). Heather Paul was supported by a Natural Sciences and Engineering Research Council of Canada Postgraduate Scholarship and an Alberta Innovates Health Solutions scholarship. Megan Hallam was supported by a Natural Sciences and Engineering Research Council Postgraduate Scholarship, a Frederick Banting and Charles Best Canada Graduate Scholarship, and an Alberta Children&#39;s Hospital Research Institute Training Award in Genetics, Child Development, and Health.

This protocol was approved by the University of Calgary Animal Care Committee and conformed to the Guide for the Care and Use of Laboratory Animals.
1. Separate Dam from Offspring
<ol>
	<li>Separate the dam from her offspring for a minimum of 5 min prior to milking12. 
	NOTE: The dam can be milked up to 5-6 hr after separation1,6,13, however periods of separation longer than 4 hr may alter milk composition14. While separation time does not appear to impact milk collection volume12, it is advised that a consistent separation time be maintained throughout the study. Milk composition may change throughout lactation15, therefore attempts should be made to keep the day of milk collection consistent. Maximum milk production is suggested to occur on lactation day 1412.</li>
	<li>Using a warming chamber, ensure the pups are able to maintain proper body temperature without the presence of their mother for the duration of the milking procedure. 
	NOTE: In the study presented below, milking was performed at weaning, when the dams were approximately 22 weeks old and the offspring 21 days old, therefore no warming chamber was used.</li>
</ol>
2. Set-up and Preparation
<ol>
	<li>Collect all materials required for the milking procedure. 
	NOTE: All materials can be found in the materials and equipment table.</li>
	<li>Place a heating pad on the bench where milking will take place and cover the pad with an absorbent bench under-pad.</li>
	<li>Set up the anesthetic system. Ensure that the system has sufficient oxygen and isoflurane prior to beginning. Attach the anesthetic mask that will be used for initial anesthesia induction to the machine. Place the mask that will be used for anesthesia maintenance nearby if different than the initial anesthetic mask.</li>
	<li>Attach a 25 G needle to a 1 ml syringe for oxytocin injection using aseptic technique.</li>
	<li>Turn on the heating pad so that maternal body temperature is maintained during the milking procedure.&#160;NOTE: Monitor the temperature of the heating pad to ensure the pad does not become too hot and cause burns. Alternatively, use a heat source, such as a heated surgical table, that can be set to a specific temperature.&#160;</li>
</ol>
3. Anesthetize the Dam Using Isoflurane
<ol>
	<li>Open the oxygen tank and turn the flow to 1 L (1,000 cc) per min. Turn on the flow of isoflurane and set to 5%. CAUTION: Avoid direct inhalation of anesthetic and prevent accumulation of anesthetic vapors.</li>
	<li>Anesthetize the dam.</li>
	<li>Switch over to the maintenance mask if required, placing the dam supine on the absorbent bench pad. Confirm anesthetization by lack of pedal reflex.</li>
	<li>Reduce the flow of isoflurane to 2-3% for maintenance of anesthesia. Continually monitor dam throughout the procedure to ensure depression of respiration does not occur.&#160;NOTE: Once under anesthesia, the dam&#8217;s eyes should be protected using a sterile eye lubricant to prevent the eyes from drying out or becoming scratched.&#160;</li>
</ol>
4. Oxytocin Injection
<ol>
	<li>Ensure the oxytocin (20 USP Units/ml) has not passed its expiry date. Disinfect the vial of oxytocin with a sterile alcohol wipe/alcohol cleansing pad.</li>
	<li>Using aseptic technique, draw up 2 IU (0.1 ml) of oxytocin into the syringe. Use a new needle and syringe for each dam that will be milked. 
	NOTE: Oxytocin doses generally range from single injections of 1 to 5 IU1,6,12,16. Alternatively, a dose of 4 IU/kg body weight can be used12. A single dose of 2 IU can be repeated once if difficulty milking is encountered.</li>
	<li>Inject the oxytocin intraperitoneally. Insert the needle into the lower right quadrant of the abdomen with the needle pointing towards the head, at an angle of 15-30&#176;, about 0.5 cm deep.</li>
	<li>Pull back on the plunger to ensure negative pressure prior to injection. If any fluid (blood, urine, intestinal contents, etc.) is aspirated into the syringe, remove the needle and attempt the injection with a new needle and syringe. If no fluid is aspirated inject the oxytocin and discard the needle and syringe immediately into a biohazard container.</li>
	<li>Wait approximately 5-15 min for the oxytocin to stimulate milk letdown.</li>
</ol>
5. Preparation of Milking Sites
<ol>
	<li>Choose the sites/teats from which milk will be collected. Milk can be collected from any teat12.</li>
	<li>Gently remove the fur around the teats to be milked with the trimmers, as fur may cause difficulty in sample collection due to wicking of the milk. Be gentle - the skin around the teats is extremely sensitive and may be dry and as such is susceptible to scratches and tears.</li>
	<li>Sterilization of the teat is not necessary, but optionally, clean the teat with lukewarm water after the fur is removed. Prepare at least two sites as more than one site may be required for milk collection. 
	NOTE: If the milk analysis includes microbial profiling, the teat area may require sterilization with iodine5.</li>
</ol>
6. Milk Collection
<ol>
	<li>Gently squeeze the base of the teat, manually expelling the milk for collection. 
	NOTE: If the milk analysis includes microbial profiling, the first few drops of milk should be discarded.</li>
	<li>Collect the milk droplets into a capillary tube, filling the capillary tube. Capillary tubes that accommodate larger volumes (e.g., 50 &#181;l) ease the process. 
	NOTE: If difficulty in collecting milk is encountered, a second dose of oxytocin can be administered. It is recommended the dose not exceed 4 IU total.</li>
	<li>Dispense the milk from the capillary tube into a sterile microcentrifuge tube by touching the end of the tube that was used to draw milk from the teat to the side of the microcentrifuge tube - observe the milk being drawn out of the capillary tube via capillary action. 
	NOTE: &#39;Blow out&#39; the milk that is not drawn into the tube using an 18 G needle attached to a 1 ml syringe.</li>
	<li>Continually monitor the dam for signs of pain or respiratory depression and adjust the flow of isoflurane accordingly.</li>
	<li>Continue to collect milk as described in this section until sufficient milk has been collected for the chosen milk analysis. For the creamatocrit and protein concentration determination described below, collect 0.25 ml of milk. 
	NOTE: Use a different milking site if milk letdown slows or the chosen site does not expel milk sufficiently. A maximum of approximately 2.5 ml of milk per animal can be collected17, or up to 0.5 ml per teat. The authors recommend that total time under anesthetic be limited to approximately 45-60 min, or 45 min of milking.</li>
	<li>Collect milk into a microhematocrit tube for creamatocrit measurement from fresh milk samples, and seal the end of the tube with clay sealant. Label the microhematocrit tube with the dam ID and store the milk sample upright.</li>
	<li>When milking is complete, turn off the flow of isoflurane and oxygen. Remove the mask from the dam and continue to monitor dam until awake.&#160;It is recommended that if not fully conscious, the dam should be placed on an absorbent bench pad during the recovery period, rather than directly on the cage bedding, to prevent the bedding from being aspirated or scratching the dam&#8217;s eyes during recovery.&#160; 
	NOTE: Do not leave the dam unattended until it has regained sufficient consciousness to maintain sternal recumbancy.</li>
	<li>If no further analyses are required, freeze milk at -80 &#176;C. Others have suggested that milk can be stored for up to 3 hr at 4 &#176;C or 5 months at -20 &#176;C18.</li>
</ol>
7. Creamatocrit Measurement
<ol>
	<li>Estimate the fat content in the milk by calculating the milk creamatocrit (the percentage of cream in the milk sample)19. 
	NOTE: Measurements with human milk have demonstrated that either fresh or frozen milk can be used for a creamatocrit measurement, however fresh milk is more highly correlated (r = 0.92 versus r = 0.90) with lipid concentration11. The use of fresh or frozen milk for creamatocrit measurements should be kept consistent across the study, as thawed milk is associated with a small decrease in creamatocrit values11.</li>
	<li>For fresh milk, collect a sample of milk from the teat into a microhematocrit tube; fill at least &#190; full (approximately 15-20 &#181;l). Alternatively, draw fresh milk from the collected sample into the capillary tube after mixing well. Seal the end with clay sealant.</li>
	<li>Place the capillary tube into the hematocrit spinner, with the sealed end pointing towards the outside, ensuring the centrifuge is balanced.</li>
	<li>Begin the hematocrit spin (120 sec at 13,700 x g). 
	NOTE: Spin time or speed may change depending on the model of centrifuge used.</li>
	<li>Remove the tube from the centrifuge after the spin is complete and perform the measurements for calculating the creamatocrit. Observe the sample&#39;s separation into a cream layer and a clear layer.</li>
	<li>Measure and record the total length of fluid in the tube and the length of the fat (cream) layer using calipers or a ruler. 
	NOTE: The creamatocrit is expressed as the percentage of the cream layer within the the milk sample19, calculated as (length of cream layer/total length of milk column) x 100 (Figure 1A). Calculate fat concentration and energy values from the creamatocrit measurement as follows: Fat concentration (g/L) = (creamatocrit (%)-0.59)/0.146 (Figure 1B)19; Energy value (kcal/L) = 290 + (66.8*creamatocrit (%) (Figure 1C)19.</li>
</ol>
8. Protein Concentration Determination
<ol>
	<li>Using Bovine Serum Albumin as a protein standard, determine total milk protein concentration using a standard protein assay, such as a Lowry protein assay6. 
	NOTE: Dilution of the milk may be necessary for the milk protein measurements to fall within the standard curve of the assay.</li>
</ol>

<ol>
	<li>Izumi, H., Kosaka, N., Shimizu, T., Sekine, K., Ochiya, T., Takase, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Time-dependent+expression+profiles+of+microRNAs+and+mRNAs+in+rat+milk+whey.">Time-dependent expression profiles of microRNAs and mRNAs in rat milk whey.</a> PLoS ONE. 9 (2), e0088843 (2014).</li><li>Hsieh, C. C., Hern&#225;ndez-Ledesma, B., Fern&#225;ndez-Tom&#233;, S., Weinborn, V., Barile, D., de Moura Bell, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milk+Proteins,+Peptides,+and+Oligosaccharides:+Effects+against+the+21st+Century+Disorders.">Milk Proteins, Peptides, and Oligosaccharides: Effects against the 21st Century Disorders.</a> BioMed Res. Int. , (2015).</li><li>Rogier, E. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+mother:+Long-term+impact+of+antibodies+in+breast+milk+on+the+gut+microbiota+and+intestinal+immune+system+of+breastfed+offspring.">Lessons from mother: Long-term impact of antibodies in breast milk on the gut microbiota and intestinal immune system of breastfed offspring.</a> Gut Microbes. 5 (5), 663-668 (2014).</li><li>Keen, C. L., L&#246;nnerdal, B., Clegg, M., Hurley, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+composition+of+rat+milk:+trace+elements,+minerals,+protein,+carbohydrate+and+fat.">Developmental changes in composition of rat milk: trace elements, minerals, protein, carbohydrate and fat.</a> J. of Nutr. 111 (2), 226-236 (1981).</li><li>Cabrera-Rubio, R., Collado, M. 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Nutr. 111 (3), 480-487 (1981).</li><li>Del Prado, M., Delgado, G., Villalpando, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+lipid+intake+during+pregnancy+and+lactation+alters+milk+composition+and+production+and+litter+growth+in+rats.">Maternal lipid intake during pregnancy and lactation alters milk composition and production and litter growth in rats.</a> J. Nutr. 127 (3), 458-462 (1997).</li><li>Nicholas, K. R., Hartmann, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Milk+secretion+in+the+rat:+progressive+changes+in+milk+composition+during+lactation+and+weaning+and+the+effect+of+diet.">Milk secretion in the rat: progressive changes in milk composition during lactation and weaning and the effect of diet.</a> Comp. Biochem. Physiol. A. Comp. Physiol. 98 (3-4), 533-542 (1991).</li><li>Azara, C. R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+intake+during+lactation+alters+milk+nutrient+composition+and+growth+and+mineral+status+of+rat+pups.">Ethanol intake during lactation alters milk nutrient composition and growth and mineral status of rat pups.</a> Biol. Res. 41 (3), 317-330 (2008).</li><li>Keen, C. L., L&#246;nnerdal, B., Sloan, M. V., Hurley, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+milking+procedure+on+rat+milk+composition.">Effects of milking procedure on rat milk composition.</a> Physiol. Behav. 24 (3), 613-615 (1980).</li><li>Romeu-Nadal, M., Castellote, A. I., L&#242;pez-Sabater, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+cold+storage+on+vitamins+C+and+E+and+fatty+acids+in+human+milk.">Effect of cold storage on vitamins C and E and fatty acids in human milk.</a> Food Chem. 106 (1), 65-70 (2008).</li><li>Lucas, A., Gibbs, J. A., Lyster, R. L., Baum, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creamatocrit:+simple+clinical+technique+for+estimating+fat+concentration+and+energy+value+of+human+milk.">Creamatocrit: simple clinical technique for estimating fat concentration and energy value of human milk.</a> Br. Med. J. 1 (6119), 1018-1020 (1978).</li><li>Furtado, K., Andrade, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+beneficial+and+adverse+effects+of+inhalable+and+injectable+anaesthetics+in+animal+models:+a+mini-review.">Comparison of the beneficial and adverse effects of inhalable and injectable anaesthetics in animal models: a mini-review.</a> OA Anaesthetics. 1 (2), 20 (2013).</li><li>Hausman Kedem, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+advanced+maternal+age+upon+human+milk+fat+content.">The effect of advanced maternal age upon human milk fat content.</a> Breastfeed. Med. 8 (1), 116-119 (2013).</li><li>Mandel, D., Lubetzky, R., Dollberg, S., Barak, S., Mimouni, F. 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The authors have no conflicts of interest to disclose. All animal experiments were conducted in accordance with CCAC approved protocols.

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

Milk is the sole source of nutrition for newborn mammals, providing energy and nutrients for infant growth and development1,2. While milk mainly consists of cells, lipids, and protein1, it also contains a plethora of bioactive compounds that modulate early life development of offspring including enzymes, carbohydrates, hormones, antibodies, growth factors, cytokines, exosomes, microvesicles, and small RNAs such as microRNA1,2. The fundamental role of maternal milk in the establishment of offspring immune and intestinal health3, coupled with evidence that breastfed infants are less susceptible to disease2, highlights the importance of identifying the milk constituents associated with disease processes in early life and the molecular mechanisms involved in their actions. The developing rat is a popular model for investigating the effect of various nutritional, physiological, and chemical interventions on early-life development4. The analysis of rat milk may therefore provide novel insight into maternal and offspring health.
Current scientific advances now provide increasing opportunities for in-depth investigations of the effects of specific milk constituents on health and disease. For example, sequencing of milk bacterial profiles has elucidated their role in early intestinal colonization of the infant gut5, mass spectrometry analysis of milk oligosaccharides have provided insight into the alteration of milk oligosaccharide profiles via maternal diet6, and deep sequencing of microRNA secreted in the fat globules of breast milk highlights possible roles in gene transcription, metabolism, and immune function7.
Rat models represent one of the most popular model organisms used in maternal studies8,9. One advantage is their short gestation and lactation periods, lasting only approximately 21 days each; therefore the total time from the start of pregnancy to lactation represents a short period of time in which valuable data can be generated. The larger size of rats compared to mice, in the context of milk collection, may provide a significant advantage with respect to volume of milk and ease of milk collection; milk production in the mouse, for example, seems to be dependent on total body weight with heavier mice producing more milk10.
Here, a general description for the manual collection of milk from lactating rats is provided. This protocol requires minimal equipment, is non-invasive, inexpensive, and can be used to collect adequate volumes of milk for further downstream analyses. In brief, the dam is anesthetized with isoflurane, milk letdown is stimulated by oxytocin, and milk is collected into capillary tubes via manual expression of the milk. Finally, as two major components of milk are fat and proteins, a brief description of estimating milk fat content using creamatocrit measurements11 and quantification of total protein concentration using a standard protein assay is presented.

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

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

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

Milk was collected as described at weaning from Wistar dams (approximately 22 weeks old, weighing 350 to 400 g) that consumed a control (AIN-93G, n = 5), high protein (40% casein wt/wt, n = 5), or high prebiotic fibre (21.6% wt/wt, 1:1 ratio of oligofructose and inulin, n = 4) diet throughout pregnancy and lactation. The oxytocin dose was 2 IU. Milk was collected using capillary tubes, and one tube was spun using a hematocrit spinner to determine creamatocrit (Figure 1A), which was then used to estimate ...

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

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

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

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

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

Research

JoVE Journal

Biology

12.2K Views.  University of Calgary. Milk is a primary source of nutrition for the neonate. Analysis of milk components may provide insight into maternal factors that affect offspring health. This protocol describes a manual method of collecting milk samples from the lactating rat, which can then be used for further downstream analysis.

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

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

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

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

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

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

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

Isolation of Rat Portal Fibroblasts by In situ Liver Perfusion

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

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

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

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

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

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

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

Milk Collection Methods for Mice and Reeves' Muntjac Deer

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

Milk Collection 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.

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

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

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

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

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

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

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

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 ...

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. The malfunction of autophagy is associated with aging and multiple human diseases. The autophagy machinery is highly conserved-from yeast to humans.&#160;The larval fat body of Drosophila melanogaster, an analog for vertebrate liver and adipose tissue, provides a unique model for monitoring autophagy in vivo. Autophagy can be easily induced by nutrient starvation in the larval fat body. Most autophagy-related genes are conserved in Drosophila. Many transgenic fly strains expressing tagged autophagy markers have been developed, which facilitates the monitoring of different steps in the autophagy process. The clonal analysis enables a close comparison of autophagy markers in cells with different genotypes in the same piece of tissue. The current protocol details procedures for (1) generating somatic clones in the larval fat body, (2) inducing autophagy via amino acid starvation, and (3) dissecting the larval fat body, aiming to create a model for analyzing differences in autophagy using an autophagosome marker (GFP-Atg8a) and clonal analysis.

Analyzing Starvation-Induced Autophagy in the Drosophila melanogaster Larval Fat Body

The present protocol describes the induction of autophagy in the Drosophila melanogaster larval fat body via nutrient depletion and analyzes changes in autophagy using transgenic fly strains.

Autophagy is a cellular self-digestion process. It delivers cargo to the lysosomes for degradation in response to various stresses, including starvation. ...

Quantification of Skeletal Muscle Fatty Infiltration via Decellularization

Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic disorders, and dystrophies. Clinically in human populations, fatty infiltration is assessed using noninvasive methods, including computed tomography (CT), magnetic resonance imaging (MRI), and ultrasound (US). Although some studies have used CT or MRI to quantify fatty infiltration in mouse muscle, costs and insufficient spatial resolution remain challenging. Other small animal methods utilize histology to visualize individual adipocytes; however, this methodology suffers from sampling bias in heterogeneous pathology. This protocol describes the methodology to qualitatively view and quantitatively measure fatty infiltration comprehensively throughout intact mouse muscle and at the level of individual adipocytes using decellularization. The protocol is not limited to specific muscles or specific species and can be extended to human biopsy. Additionally, gross qualitative and quantitative assessments can be made with standard laboratory equipment for little cost, making this procedure more accessible across research laboratories.

Author Spotlight: Decellularization-Based Quantification of Skeletal Muscle Fatty Infiltration  

The present study describes decellularization-based methodologies for visualizing and quantifying intramuscular adipose tissue (IMAT) deposition through intact muscle volume, as well as quantifying metrics of individual adipocytes that comprise IMAT.

Fatty infiltration is the accumulation of adipocytes between myofibers in skeletal muscle and is a prominent feature of many myopathies, metabolic ...

Estimation of Total Lipid Volume from Decellularized Muscles by Lipid Extraction

Begin by transferring the decellularized skeletal muscles stained with ORO to 200 microliters of isopropanol in the individual wells of a 96 well plate. Agitate the solution by tapping the plate. And mechanically crush the muscle with a pipette tip under a microscope until no red spheres can be seen in the decellularized muscle suspension.Again, mix the cell suspension in each well by pipetting up and down before transferring 75 microliters to two clean wells of the plate. Cover the plate and read the absorbance of the duplicate 75 microliter wells using a spectrophotometer or a plate reader.