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 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 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.
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
2. Set-up and Preparation
3. Anesthetize the Dam Using Isoflurane
4. Oxytocin Injection
5. Preparation of Milking Sites
6. Milk Collection
7. Creamatocrit Measurement
8. Protein Concentration Determination
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 fat concentration and energy value according to: Fat concentration (g/L) = (creamatocrit (%)-0.59)/0.146 (Figure 1B); Energy value (kcal/L) = 290 + (66.8*creamatocrit (%) (Figure 1C)19. Milk protein concentration was determined using the Bio-Rad DC protein assay (Figure 2). There were no differences in creamatocrit (p = 0.674), fat concentration (p = 0.674), energy value (p = 0.674), or protein concentration (p = 0.127) based on maternal diet (one-way ANOVA).
Figure 1. Milk creamatocrit, fat concentration, and energy value. Milk samples were collected at weaning from Wistar dams on Control (n = 5), High Protein (n = 5), or High Prebiotic Fibre (n = 4) diets throughout pregnancy and lactation. Creamatocrit measurements (A) were used to calculate milk fat concentration (B) and energy value (C). Please click here to view a larger version of this figure.
Figure 2. Total milk protein concentration. Milk samples were collected at weaning from Wistar dams on Control (n = 5), High Protein (n = 5), or High Prebiotic Fibre (n = 4) diets throughout pregnancy and lactation. Total protein concentration was determined using the Bio-Rad DC protein assay. Please click here to view a larger version of this figure.
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 method requires no specialized vacuum equipment and can be performed by a single person.
While this protocol is intended for use at a single time point during lactation, others have performed serial milk collections throughout their studies12,15. However, as serial milking may affect milk composition17, it is advised that researchers determine the frequency and number of milk collections that best suit their study, based on the outcome of interest. Additionally, while others have used injectable anesthetics during milking procedures in the rat14,17, this protocol involves anesthetization of the rat using isoflurane. Isoflurane is an inhalable anesthetic, and its advantages include rapid induction and recovery rates20 and the ability to easily adjust the time the animal is anesthetized. However, in mice, isoflurane may result in decreased milk yield in comparison to injectable anesthetics10, although this does not seem to have been tested in rats. Furthermore, protocols involving vacuum suction may not require anesthetic, and may result in higher milk yield depending on the efficiency of the suction apparatus12. However, if proper suction does not occur, milk yield will decrease.
It is possible for the researcher to encounter difficulty in manual expression of milk, which can prolong time under anesthesia or decrease the milk yield. If difficulty in milking is encountered, some possibilities are: 1) sufficient time has not passed for the oxytocin to take effect; 2) improper or inexperienced milking technique; 3) physical differences in the nipple; 4) incorrect intraperitoneal injection of the oxytocin. The researcher may therefore wait a few more minutes prior to attempting to milk again; if this does not result in increased milk yield, the researcher may choose to move to a different milking site. Using a vacuum, each set of teats (upper thoracic, lower thoracic, upper abdominal, and lower abdominal) was equally successful in expelling milk when the milker is proficient, and approximately 0.5 ml can be obtained from each teat12. Therefore, if one milking site is proving difficult, moving to another site may help. The authors are not aware of differences in milk composition between milking sites. Finally, a second dose of oxytocin may be administered to further stimulate milk letdown. Diluting the oxytocin to a standard dose volume/kg body weight can also be attempted, for example 1 ml/kg body weight12, however be aware of maximum intraperitoneal injection volumes.
It is also possible that other factors, including but not limited to, rodent strain, health status, parity, litter number, level of stress, diet, time of day, and food intake may affect the outcome of the milk collection. While the discussion of all these variables is beyond the scope of this protocol, it is encouraged that the investigators consider the multitude of factors that may influence the efficiency of milk collection in their experiments.
Finally, a simple method of measuring the proportion of cream in the milk, the creamatocrit, which is an accurate estimation of the amount of lipid in the samples11, is described herein. The creamatocrit can then be used to estimate the fat concentration and energy value of the milk. Though there were no differences in creamatocrit, fat concentration, energy value, or protein concentrations between the maternal dietary groups in the results presented above, others have found differences in creamatocrit values based on maternal characteristics such as gestational age21 and duration of lactation22. Differences in oligosaccharide content in maternal milk has also been demonstrated with maternal dietary manipulation6.
In summary, milk contains a plethora of bioactive compounds whose specific roles in early life development of offspring, and their subsequent effect on disease susceptibility throughout life, remain to be clarified. The protocol reported here allows milk to be collected from lactating rats by a single individual, requires no specialized vacuum apparatus, and provides sufficient milk for subsequent analysis. Mastering this technique will provide the researcher with the opportunity to contribute significant insight into the processes involved in early-life development and establishment of health.
The authors have nothing to disclose.
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's Hospital Research Institute Training Award in Genetics, Child Development, and Health.
Equipment – Milking | |||
1 ml syringes | BD-Canada | 309602 | |
25 G needles | BD-Canada | 305122 | |
18 G needles | BD-Canada | 305196 | |
50 ul Microdispenser Capillary Tubes | Fisher Scientific | 21-169D | |
Oxytocin (20 USP Units/ml) | Bimeda-MTC | 1OXY015 | |
PPC Vet Isoflurane Inhalation Anesthetic, 250 ml | Fresenius Kabi | M60302 | Used on the order of a veterinarian |
Sterile Alcohol Prep Pad | Dukal | 853 | |
Absorbent Bench Underpad | VWR | 82020-845 | |
Maxi-Therm Hyper/Hypothermia Blanket | Cincinnati Sub-Zero | 274 | |
Rodent Anesthesia Machine with Vaporizer | Benson Medical Industries Inc. | Subject to individual laboratory needs | |
Animal Masks | Benson Medical Industries Inc. | 50100/50102 | |
Microcentrifuge Tubes | Axygen | MCT-060-C | |
ChroMini Professional Trimmer | Wahl | – | |
Equipment – Creamatocrit | |||
StatSpin SafeCrit Plastic Microhematocrit Tubes (Untreated) | Fisher Scientific | 22-274-914 | |
Critoseal Capillary Tube Sealant Tray | VWR | 470161-478 | |
StatSpin CritSpin Microhematocrit Centrifuge | Beckman Coulter, Inc | X00-004999-001 |