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 appea...

<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. C., Laitinen, K., Salminen, S., Isolauri, E., Mira, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+milk+microbiome+changes+over+lactation+and+is+shaped+by+maternal+weight+and+mode+of+delivery.">The human milk microbiome changes over lactation and is shaped by maternal weight and mode of delivery.</a> Am. J. Clin. Nutr. 96 (3), 544-551 (2012).</li><li>Hallam, M. C., Barile, D., Meyrand, M., German, J. B., Reimer, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+high-protein+or+high-prebiotic-fiber+diets+affect+maternal+milk+composition+and+gut+microbiota+in+rat+dams+and+their+offspring.">Maternal high-protein or high-prebiotic-fiber diets affect maternal milk composition and gut microbiota in rat dams and their offspring.</a> Obesity. 22 (11), 2344-2351 (2014).</li><li>Munch, E. 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=Transcriptome+Profiling+of+microRNA+by+Next-Gen+Deep+Sequencing+Reveals+Known+and+Novel+miRNA+Species+in+the+Lipid+Fraction+of+Human+Breast+Milk.">Transcriptome Profiling of microRNA by Next-Gen Deep Sequencing Reveals Known and Novel miRNA Species in the Lipid Fraction of Human Breast Milk.</a> PLoS ONE. 8 (2), e50564 (2013).</li><li>Li, M., Sloboda, D. M., Vickers, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+obesity+and+developmental+programming+of+metabolic+disorders+in+offspring:+Evidence+from+animal+models.">Maternal obesity and developmental programming of metabolic disorders in offspring: Evidence from animal models.</a> Exp Diabetes Res. 2011, (2011).</li><li>Ellis, P. J. I., 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=Thrifty+metabolic+programming+in+rats+is+induced+by+both+maternal+undernutrition+and+postnatal+leptin+treatment,+but+masked+in+the+presence+of+both:+implications+for+models+of+developmental+programming.">Thrifty metabolic programming in rats is induced by both maternal undernutrition and postnatal leptin treatment, but masked in the presence of both: implications for models of developmental programming.</a> BMC Genomics. 15, 49 (2014).</li><li>Gomez-Gallago, C., 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=A+method+to+collect+high+volumes+of+milk+from+mice+(Mus+musculus).">A method to collect high volumes of milk from mice (Mus musculus).</a> An. Vet. Murcia. 29, 55-61 (2013).</li><li>Wang, C. D., Chu, P. S., Mellen, B. G., Shenai, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Creamatocrit+and+the+nutrient+composition+of+human+milk.">Creamatocrit and the nutrient composition of human milk.</a> J. Perinatol. 19 (5), 343-346 (1999).</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>Godbole, V. Y., Grundleger, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+of+rat+milk+from+day+5+to+20+of+lactation+and+milk+intake+of+lean+and+preobese+zucker+pups.">Composition of rat milk from day 5 to 20 of lactation and milk intake of lean and preobese zucker pups.</a> J. 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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat+and+energy+contents+of+expressed+human+breast+milk+in+prolonged+lactation.">Fat and energy contents of expressed human breast milk in prolonged lactation.</a> Pediatrics. 116 (3), e432-e435 (2005).</li></ol>

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

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12.2K vistas.  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.