The overall goal of this procedure is to describe how to manually collect milk from a lactating rat for laboratory analysis of milk composition. This method can be used to help answer key questions in the field of maternal and child health, such as how maternal diet can influence milk composition. The main advantage of this technique is that it can be performed by a single investigator without any specialized vacuum or suction equipment.
No.To begin, separate the dam from her offspring for a minimum of five minutes prior to milking. Next, collect all of the materials that are required for the milking procedure as shown here. Then place an absorbent bench pad onto a heated surgical table, or use a separate heat pad to prevent heat loss from the dam during the milking procedure.
Next, check the anesthetic system in order to ensure that the system has sufficient oxygen and iso fluorine prior to the procedure. Then attach the mask that will be used for the initial anesthesia induction to the machine and set the maintenance mask nearby. Open the oxygen tank and set the flow to one liter per minute.
Then turn on the flow of isof fluorine and set its flow to 5%Once anesthetized, switch the dam to the maintenance mask. Confirm a sufficient level of anesthetization by checking for the lack of a pedal reflex. Then place the dam in a supine position on the absorbent bench pad.
Reduce the flow of isof fluorine down to two to 3%Continually monitor the dam throughout the procedure and adjust the flow of isof fluorine as necessary to avoid pain and respiratory depression. Next, use a sterile alcohol wipe or cleansing pad to disinfect a vial of oxytocin using a one milliliter syringe equipped with a 25 gauge needle, draw up 0.1 milliliters of oxytocin. Then insert the needle into the lower right quadrant of the dam's abdomen with the needle pointing towards the head at an angle of 15 to 30 degrees, about 0.5 centimeters deep.
Pull back on the plunger to ensure negative pressure prior to injection. If no fluid is aspirated, inject the oxytocin and discard the needle and syringe immediately into a biohazard container. Wait approximately five to 15 minutes for the oxytocin to stimulate milk.
Let down during this time, choose the tets from which milk will be collected. Use trimmers to gently remove the fur around the selected tets as fur may cause difficulty in sample collection. Then clean the tets with lukewarm water.
Prepare at least two sites as more than one site may be required for milk collection. Once the milk has let down, gently squeeze the base of the Tet to manually expel milk. For collection, collect the milk droplets into a 50 microliter capillary tube.
Once full, dispense the milk from the capillary tube into a sterile micro centrifuge tube by touching the end of the tube that was used to draw milk from the teat to the side of the micro centrifuge tube. Then use an 18 gauge needle attached to a one milliliter syringe to blow out any milk that was not drawn into the tube. Repeat the milk collection and transfer it into the micro centrifuge tube until sufficient milk has been collected.
For the chosen analysis, try to limit the total time that the dam is under anesthetic to less than one hour. When the milking is complete, turn off the flow of isof fluorine and oxygen and remove the mask from the dam. Continue to monitor the dam until it has regained sufficient consciousness to maintain sternal recumbent.
If no further analyses are required, breeze the milk at negative 80 degrees. To prepare for CCR measurement, load 15 to 20 microliters of milk into a micro hematocrit tube directly from the Tet. Then seal the end of the tube with a clay sealant.
Place the capillary tube into the hematocrit spinner with the seal dent pointing towards the outside and ensure that the centrifuge is balanced. Then spin the sample at 13, 700 times G for two minutes. Once the sample finishes spinning, remove the tube from the centrifuge.
Observe the sample separation into a cream layer and a clear layer. Measure and record the total length of fluid in the tube and the length of the fat or cream layer using a ruler or calipers. Use this information to calculate the CCR as described in the accompanying text protocol in order to determine how diet during pregnancy and lactation affects milk composition.
Milk samples were collected at weaning from Worcester dams who are fed either a high protein, a high prebiotic fiber, or a controlled diet. The milk collected from dams on the high prebiotic fiber diet had the numerically highest C hematocrit, bad concentration and energy value. However, these differences were not statistically significant when it comes to protein.
However, milk collected from the dams on the high protein diet had the highest protein concentration. Though again, this difference was not statistically significant. Once mastered, this technique can be performed in less than an hour from the initial point of anesthetization of the dam if done correctly.
When attempting this procedure, it's important to remember to continually monitor the dam to ensure that depression of respiration does not incur while under anesthesia following this procedure. Other methods like mass spectrometry can be used to answer additional questions like the determination of the milk's oligosaccharide composition. After watching this video, you should have a good understanding of how to perform the manual collection of milk from a lactating rat.
