This study focuses on improving milk sampling techniques in lactation research, particularly in mice. We present a method that overcomes challenges such as sample loss, limited volume, and labor-intensive processes, offering a streamlined solution for rodent milk collection and a valuable tool for researchers in the field. One of the biggest challenges with conducting research in mice is their small size, especially when it comes to sampling biofluids such as milk.
It is difficult to get sufficient volume without pooling samples or having two people manually collect it, which makes the process less practical in many cases. Our protocol simplifies milk collection by allowing one person to collect sufficient volume in under 10 minutes using readily available lab tools. Unlike other methods, it minimizes sample loss with continuous suction, pulling milk directly into collection tubes, saving time, and preserving volume for analysis.
To begin, attach one end of the 5/32 inch inner diameter PVC tubing to the vacuum pump, and secure the attachment with tape. Using a barbed straight connector, connect the other end of the 5/32 inch inner diameter PVC tubing to the 1/8 inch inner diameter PVC tubing. Next, use a connector to attach the other end of the 1/8 inch inner diameter PVC tubing to an 18 gauge hypodermic needle.
Using scissors, cut the end of a P200 pipette tip, approximately 0.2 centimeters, to create a larger opening. Insert the modified P200 pipette tip through the opening of the septum stopper so that the tip points upward, and attach the septum stopper to a sterile collecting tube. Using the 18 gauge hypodermic needle, puncture the septum stopper.
Now, turn on the vacuum pump to check the suction pressure by placing a gloved finger over the pipette tip opening On the milking day, separate the dam from her pups to allow milk accumulation in the glands. Keep the pups warm in a nest. After anesthetizing the dam, squeeze a foot of the dam firmly to assess the animal's perception of sensation and pain.
Then, apply veterinary ophthalmic ointment to the eyes of the dam to prevent dryness while under anesthesia. Place the anesthetized dam on her back on a solid working surface. Gently clean the mammary area of the dam with a sterile alcohol prep wipe.
Administer one to two units of oxytocin intraperitoneally between the fourth and fifth pair of teats on the side of the peritoneal cavity that has not received any injection. Gently scruff the neck area of the dam with the non-dominant hand. Then, squeeze the mammary tissue from the base to the tip of the teat until a bead of milk is visible.
Turn on the vacuum device by pressing the power button. To start milking, gently apply the inverted P200 pipette tip to a teat and use a gentle pulling motion to draw out milk. Milk all teats until a sufficient volume of milk is collected.
Afterward, return the anesthetized dam to the cage and ensure she is kept warm during the anesthetic recovery period. 0.1 to 0.5 milliliters of milk can be comfortably collected from each dam in under 10 minutes. Milk fat concentration increased significantly from 174.4 grams per liter on post-delivery day two to 224.4 grams per liter on post-delivery day 10.
Lactose and total protein concentrations did not show significant differences between post-delivery day two and post-delivery day 10. The relative abundance of alpha-S1-casein and whey acidic protein was higher in post-delivery day 10 milk compared to post-delivery day two milk, while beta-casein was more abundant in post-delivery day two milk than in post-delivery day 10 milk. Protein band intensities on the electrophoresis gel visually confirmed differences in relative abundances of individual milk proteins.
