Milk collection from animal models facilitates various research avenues: understanding passive immunity, identifying pathogens responsible for vertical transmission and, through the use of transgenic mice, even commercial production of proteins found in human breast milk. Here we illustrate a simple method for milk collection in mice and Reeves’ muntjac deer.
Animal models are commonly used throughout research laboratories to accomplish what would normally be considered impractical in a pathogen’s native host. Milk collection from animals allows scientists the opportunity to study many aspects of reproduction including vertical transmission, passive immunity, mammary gland biology, and lactation. Obtaining adequate volumes of milk for these studies is a challenging task, especially from small animal models. Here we illustrate an inexpensive and facile method for milk collection in mice and Reeves’ muntjac deer that does not require specialized equipment or extensive training. This particular method requires two researchers: one to express the milk and to stabilize the animal, and one to collect the milk in an appropriate container from either a Muntjac or mouse model. The mouse model also requires the use of a P-200 pipetman and corresponding pipette tips. While this method is low cost and relatively easy to perform, researchers should be advised that anesthetizing the animal is required for optimal milk collection.
Animal models provide insight into disease pathology that cannot be gained by in vitro analysis. To provide the most efficacious results, it is important to use an animal model that is closely related to the disease and species of interest. For example, the Reeves’ muntjac (Muntiacus reevesi), a small Asian deer1,2, and transgenic mice expressing the cervid prion protein (CerTgPrP)3 are useful animal models for cervid species. Both species are polyestrous, allowing year-round breeding, and therefore a consistent source for pregnancy-related tissues and fluids to study specific mechanisms in cervid biology. Studies of milk have a vast array of applications that are more simply (and inexpensively) accomplished in animal models than in humans. Researchers could investigate milk and colostrum as a potential source of 1) infectious disease transmission, 2) immunoglobulins transferred from mother to offspring in the development of passive immunity4 and 3) lactoferrin, a protein found in human breast milk involved in passive immunity that researchers are currently attempting to commercially produce5.
Collecting a substantial amount of milk from small animals can prove to be a difficult task. Rogers proposed an approach to collect milk from rats6, which was subsequently used in mice. DePeters and Hovey proposed two methods for milk collection, one using a manually-generated vacuum produced by a rubber pipette bulb attached to a Pasteur pipette, and a second requiring the construction of a milking unit, which is then attached to a vacuum source (such as a faucet) to harvest mouse milk7. Here we propose a simple, low cost method for collecting milk from both mice and Reeves’ muntjac deer, which requires only readily available laboratory equipment and basic technical skills. Our method yields sufficient volumes of milk for various applications.
1. Mouse Milking Protocol
The methods described in this protocol were approved by IACUC.
1. Separation of the Dam from Offspring
2. Administration of Oxytocin, Anesthesia and Eye Lubricant
3. Milk Collection
The milk collection is most easily performed with two researchers: one researcher to hold the anesthetized mouse while manually expressing the milk (referred to as R1), and one researcher to collect the milk ( R2). This method can also be performed with one person, if the mouse is secured on a flat surface to avoid harm.
4. Anesthetic Reversal in the Dam
2. Muntjac Milking Protocol
The methods described in this protocol were approved by IACUC.
1. Separation of the Doe from Offspring
2. Administration of Oxytocin, Anesthesia and Eye Lubricant
3. Milk Collection
The milk collection is most easily performed with two researchers; one researcher to hold the anesthetized muntjac in the lap while manually expressing the milk (Referred to as R1), and a second researcher to keep the muntjac’s head in a comfortable and safe position and to collect the milk (R2).
4. Anesthetic Reversal in the Doe
It is common for midazolam-induced doe to begin to wake up during the milking process. In most cases, the doe will attempt to escape before all the milk has been expressed. Minor restraint is used in an effort to increase the milk yield; however the safety and comfort of both the researchers and the doe are equally important.
Mouse
From our experiments, we have determined that it is possible to collect approximately 100-400 μl of milk from one laboratory mouse, dependent on several variables. These variables include 1) the amount of time set aside for collection, 2) the dose of oxytocin administered, 3) how many pups the dam is currently nursing and 4) the amount of time post parturition. Our studies have shown that the highest yield is obtained when the dams are separated from their offspring for at least 2 hr, and at least 45 min to an hour is set aside per mouse for milking. Maximum milk volumes were harvested 8-12 days post parturition using 2 IU/kg of oxytocin. Our results are mostly in accordance with other studies where litter size was positively correlated with milk yield9 (see Figure 3).
Muntjac
Although the amount of milk harvested from the muntjac was variable from day to day, we were able to collect 5 to 30 ml milk/dam/session, depending on the anesthesia used and/or time post parturition. Muntjac deer are polyestrous; milk can be collected at various time points throughout the year2 (see Table 1). Midazolam-induced doe remained under anesthesia for as little as 10 min, allowing for smaller volume collections (typically 5-30 ml), whereas BAM-induced doe sustained a longer period of anesthetic effect (20-30 min), allowing complete milk expression (15-130 ml). Milk collections were discontinued when milk was no longer being produced in sufficient amounts by the doe, and the corresponding fawn was being nourished on a diet of mostly hay forage.
Figure 1. (A) The dam’s teats become engorged with milk upon oxytocin injection. (B) Collection of milk from an anesthetized dam using a P-200 pipetman. (C) Ejection of milk into a 1.2 ml cryovial.
Figure 2. Muntjac milk is collected into a 15 ml conical tube.
Figure 3. Approximate milk yield collected from mice based on litter size, and age of pups.
Table 1. Milk collection start and finish dates for lactating Reeves’ muntjac dams.
Mouse
There are several factors to take into consideration when collecting milk from a mouse, including 1) the amount of time set aside for collection, 2) the dose of oxytocin administered, 3) how many pups the dam is currently nursing and 4) the amount of time that has passed since parturition at the time of collection. Using previous studies as guidelines, we set out to optimize conditions for milk yield.
Previous research has directly correlated milk yield with litter size8. Unfortunately, litter size is a variable that cannot be controlled. However, the time of milking post parturition can be controlled. According to The Laboratory Mouse maximum milk secretion is at 8-10 days post parturition9. Our experience is in congruence with this statement.
It has been shown that oxytocin plays an irreplaceable role in milk production post parturition10. Oxytocin doses ranging from 0.1 IU/kg9 to 4 IU/kg6 have been reported for collection of milk from rodents6,7,9. We began our study by administering the lowest dose suggested, 0.1 IU/kg9. Using this dosage, the amount of milk produced by the mouse was too small to measure or collect. Upon increasing the oxytocin dose to 2 IU/kg6,7, milk yield was considerably higher at approximately 120 μl. Because we wanted to use the lowest dosage of oxytocin to achieve optimal milk collection, we did not administer doses higher than 2 IU/kg.
Milk yield is also dependent upon the amount of time set aside for collection. We have determined that a minimum of 3 hr is necessary for optimal collection—2 hr for the pups to be separated from the dam and 1 hr for material preparation and collection. Collecting milk from several dams in one session will decrease the overall time necessary to harvest a specific volume of milk, i.e., 2 hr to separate pups, 30 min to setup materials and 20-30 min to collect milk from each dam. Each dam must be monitored until they are awake before being placed back with their pups. We have noted that the more time set aside for milking leads to higher milk yields, because the mouse can be milked until she regains consciousness.
Instances do arise with animal models that will affect milk yield. One common issue is the presence of blood or exudate in the milk of older dams that have been bred repeatedly and have large litters, or in dams that are nursing their first litter. Should this occur, we suggest discontinuing milk collection from that particular teat. It is advisable to switch pipet tips at this time, and discard any exudate or blood.
Researchers carrying out this method should also be aware that, as is often the case when using animal models, results vary (Figure 3). If a less than optimal amount of milk is collected in one session, it is preferable to repeat the procedure at a later time (e.g. 24-48 hr) rather than alter the experimental conditions to force milk release.
Muntjac
The volume of milk collected from Reeves’ muntjac dams was dependent upon the anesthetic used. Our initial anesthetic of choice was midazolam. Midazolam provided sufficient sedation to allow restraint for short periods of time—i.e., if R1 had a calm disposition and could keep the muntjac calm, R2 could take over the milking to increase the yield before the muntjac would awaken—the safety and comfort of both animal and handler were of utmost importance and we abandoned the milking effort if we felt either was compromised. We discovered that muntjac doe quickly become tolerant to midazolam and that higher doses would be necessary after repeated exposure, making milk collection twice weekly increasingly difficult.
The shortage of midazolam, coupled with a building tolerance, led us to research an alternative anesthetic. A mixture of Butorphanol, Azaperone and Medetomadine (BAM) is effectively used for the immobilization of white tail deer11. In our hands, the BAM cocktail provided sufficient anesthesia to allow collection of acceptable volumes of milk in a safe manner, with less drug tolerance after repeated use, and with better control in reversing the effects of the anesthetic.
Some muntjac dams suffered from sensitive udders and/or were first-time mothers. These muntjac developed bruising on the teats during the milking process, and the milk flow slowed or stopped completely, prompting us to discontinue the ongoing session. Should this occur, one should use a more gentle milking technique in an attempt to prevent discomfort or bruising in the teats.
Milk production appeared to wax and wane on several occasions. Low milk harvests may be caused by 1) the fawns having nursed just prior to collections or 2) a decline in milk production due to the natural process of fawn weaning.
While it can be a trying process to obtain a sizeable volume of milk from small animal models, our work illustrates that it is indeed a possibility for both mouse and muntjac models; neither requiring extensive skill nor deep pockets, simply perseverance.
The authors have nothing to disclose.
These experiments were funded by the NIH grant #RAI093634A. We express immense gratitude to Jenny Powers, DVM, PhD, for her assistance in muntjac milking.
Materials-Mouse | |||
Name of Material/Reagent | Vendor | Catalog Number | Comments |
Ketamine (KetaVed 100 mg/mL) (dose: 80-100 mg/kg) | MWI/Vedco | 011075 | |
Xylazine (TranquiVed 100 mg/ml) (dose:5-10 mg/kg) | MWI/Vedco | NADA 139236 | |
Eye lubricant | MWI/Apexa | 701013 | |
Oxytocin (2 IU/kg) | Vet One | 501013 | |
Materials- Muntjac | |||
Name of Material/Reagent | Vendor | Catalog Number | Comments |
Midazolam (1-2 mg/kg) | Amerisource Bergen Drug Corp | 924326 | optional: researcher can either use Midazolam on its own or BAM combination as an anesthetic |
Butorphanol tartrate (Torbugesic) (0.45 mg/kg) | Fort Dodge Animal Health | NADA 135-780 | |
Azaperone tartrate (0.25 mg/kg) | ZooPharm | ||
Medetomadine HCl (0.07 mg/kg) | ZooPharm | ||
Antipamezol hydrochloride (Antisedan) (dose: 0.25 mg/kg) | Orion Corporation | NADA 141-033 | |
Oxytocin (10 IU/kg) | Vet One | 501013 | |
Equipment- Mouse | |||
Name of Equipment | Vendor | Catalog Number | Comments |
P-200 Pipetman | Gilson | F123601 | |
200 μL pipette tips | Light Labs | B-2004-SH | |
1 ml syringes | MWI | 005427 | |
27 gauge needles | MWI | 002031 | |
Equipment- Muntjac | |||
Name of Equipment | Vendor | Catalog Number | Comments |
15 ml conical tubes | VWR | 21008-918 | |
22 gauge needles | MWI | 873 | |
3 ml luer lock syringes | MWI | 001377 |