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

Summary

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.

Abstract

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.

Introduction

Milk is the sole source of nutrition for newborn mammals, providing energy and nutrients for infant growth and development^1,2. 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 microRNA^1,2. 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, highligh....

Protocol

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

1. 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 separation^1,6,13, 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 ....

Results

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 .......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Equipment - Milking
1 ml syringes	BD-Canada	309602
25 G needles	BD-Canada	305122
18 G needles	BD-Canada	305196
50 μl 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