Here, we describe a hand-rearing method for raising infant marmosets in an animal incubator. This method greatly increases the survival rate of marmoset infants, which provides the opportunity to study the development of marmoset infants with similar genetic backgrounds raised in different postnatal environments.
The common marmoset (Callithrix jacchus) is a small and highly social New World monkey with high reproduction rates, which has been proven to be a compelling non-human primate model for biomedical and neuroscience research. Some females give birth to triplets; however, the parents cannot raise all of them. To save these infants, we have developed a hand-rearing method for raising newborn marmosets. In this protocol, we describe the formula of the food, the time for feeding, the configuration of the temperature and humidity, as well as the adaptation of the hand-reared infants to the colony environment. This hand-rearing method significantly increases the survival rate of marmoset infants (without hand-rearing: 45%; with hand-rearing: 86%) and provides the opportunity to study the development of marmoset infants with similar genetic backgrounds raised in different postnatal environments. As the method is practical and easy to use, we anticipate that it could also be applied to other labs working with common marmosets.
The common marmoset (Callithrix jacchus) is a small and arboreal New World monkey originating from South and Central America. The use of marmosets in biomedical research has grown rapidly over the past decades due to several key advantages of marmosets compared with other non-human primates (NHPs), including their smaller body size, easier handling and breeding in captivity, shorter gestation time, earlier sexual maturation, and lower zoonotic risks1,2,3,4,5,6. The common marmoset has a similar brain structure and brain function to humans and displays a rich repertoire of vocalizations and highly social behavior with rich emotions. It is a compelling NHP model for different types of neuroscience studies, such as studies on sensory processing7,8,9,10,11,12,13,14, vocal communication15,16,17,18,19, models of spinal cord injury20,21,22,23, Parkinson's disease24,25,26,27,28, and age-related diseases29. Compared with other NHPs, the common marmoset has a relatively high reproduction rate, which is potentially useful for transgenic modification30,31,32. This primate is also widely used in pharmacology, angiography, and pathogen and immune studies33,34,35,36,37,38,39. However, the supply of marmosets remains very limited, especially in China, and cannot meet the rapidly growing needs of scientific research.
In marmoset colonies, the adult animals are fed once or twice per day, and a few institutions alter the diet for juvenile marmosets40. Generally, infant marmosets usually grasp firmly onto the body of the father or elder siblings for daily care and are handed to the mother several times per day for milk. Some female marmosets give birth to triplets, and in this case, one or two infants cannot survive due to a lack of milk; moreover, some parents do not take care of their infants because they lack nursing experience or for other unknown reasons. This is a big loss for many laboratories. A few studies have reported methods of nutrition management for adult marmosets in captive settings40,41,42 utilizing foods and formulas with different macronutrient compositions, vitamins, and minerals, as well as different feeding protocols for enrichment (mashed, gelled, purified, or canned)2,41. One previous study reported a collaborative rearing method for marmoset triplets43, in which caregivers take one infant per day, hand-feed it throughout the day, and exchange it for another of the triplets on the next day. Although this method allows the infants to have parental care, it requires an experienced caregiver to grab the infant from the body of the parents every day and is labor-intensive. So far, no study has reported a detailed, step-by-step hand-rearing method for newborn marmosets.
The goal of the current study is to provide a hand-rearing method for those interested in marmoset development but with limited resources. In contrast to the previous collaborative rearing method43, the current method is an alternative that causes less disturbance to the infant's family and is easy to learn. Based on the basic rules of breastfeeding and 5 years of practice, this paper describes a hand-rearing method for raising infant marmosets that includes the preparation of the food, a timetable for feeding, the configuration of the temperature and humidity of the animal incubator, as well as the adaptation of the infant animals to the colony environment.
All the experimental procedures were approved by the Animal Use and Care Committee of Zhejiang University and followed the National Institutes of Health (NIH) guidelines.
1. Housing and husbandry44
2. Preparation before the birth of the marmoset infants
Figure 1: Photos of the items in the incubator and the feeding tools and accessories. (A) Plush toys; (B) toy roller and climbing frames; (C) blanket; (D) plastic weighing dish; (E) intravenous injector and syringe with a custom-made feeding nipple; (F) electronic scale; (G) caregiver with personal protective equipment. Please click here to view a larger version of this figure.
3. Hand-rearing procedure
4. Acclimation before the return of the infant marmosets to the colony
NOTE: Usually, the hand-rearing is finished when the infant marmosets learn to eat by themselves. There are a few adaptation procedures to be carried out before they are returned to the home cage in the marmoset colony.
5. Infant marmosets returning to the family cage
NOTE: After living in the small cage for 7-10 days, the infant marmosets usually adapt to the colony environment well and exhibit no more anxiety.
Body weight is a key index of animal body development and is used as an indicator of the health status of the marmosets in this protocol. In this work, the body weights of the hand-reared animals increased gradually with age (Figure 2A, n = 16), similar to the weights of newborn infants in a previous study46. To minimize the disturbance to the breeding families in the colony, we did not weigh the infant marmosets in the colony every day. We obtained the weights of the parent-raised animals 1 month after birth and later, and these were compared with the weights of the hand-reared infants at the same ages. There were no significant differences in the body weights between the parent-raised infants and the hand-reared infants (P30, hand-rearing group, 52.25 g ± 2.10 g, n = 18; colony group, 57.34 g ± 2.77 g, n = 7; t = −1.3417, P = 0.1928; P60, hand-rearing group, 91.76 g ± 3.44 g, colony group, 93.06 g ± 4.68 g; t= −0.2019, P = 0.8424; Student's t-test) (Figure 2B).
Figure 2: Body weights of the hand-reared infant marmosets and the parent-raised ones in the colony. (A) The body weight of each hand-reared infant. The gray lines indicate individual body weights, and the orange curve indicates the population average. (B) Comparison of the body weights between the hand-reared infants (orange) and the parent-raised infants (green) at P30 and P60. The gray circles represent individuals. Data shown as Mean ± SEM. ns indicates no significant difference. Please click here to view a larger version of this figure.
Table 1. Configuration of the temperature in the animal incubator. Please click here to download this Table.
Table 2: Feeding recipes. The columns under "food ingredients" indicate the milk recipe for 1 day. For example, at postnatal days 1-14, we mix 5 g of baby milk powder and 30 mL of water before the first meal and take 0.5-1.5 mL of this milk each time for feeding. The dosages of "baby milk powder" and "rice paste" are for the powder forms. Please click here to download this Table.
The common marmoset is a very useful NHP model for biomedical and neuroscience research. However, marmoset resources are too limited to meet the rapidly growing needs. In this work, we have developed a hand-rearing method that not only increases the survival rate of marmoset infants but also provides an opportunity to study their postnatal development. This hand-rearing method is practical and easy to learn and is, therefore, easily applicable to other labs working with common marmosets.
Some congenital defects usually develop in the first 2 weeks of life. Until now, we have identified several cases of newborn marmosets with innate defects on the eyelid, the lung, the intestine, and the brain, and they rarely survive until P14. Therefore, P1-14 is a critical period for screening out healthy newborn marmosets that can be raised by the hand-rearing method. In addition, if infant marmosets are dropped from the body of their parents due to negligence or lack of milk, they cannot survive. Thus, the hand-rearing method helps to save their lives. In this work, without hand-rearing, the survival rate was 45% (n = 13/29), and it increased to 86% (n = 25/29) after we applied the hand-rearing method.
There are several critical points to note. The caregivers must be patient during feeding; pushing the syringe too fast is not good, as it does not allow the infant marmosets to swallow, which may lead to choking or ileus and, thus, impair the respiratory system or the digestive system of the animals. The caregivers must be gentle and quiet during feeding to minimize the stress to the infant animals, because stress could significantly affect the gut microbiome and is associated with chronic diarrhea in marmosets47,48. During the hand-rearing, all the people near the infant marmosets have to behave gently throughout. Sometimes, opening the door of the incubator in a hurry makes sudden loud sounds, which can frighten the infants and lead to a stress reaction.
The body weights of the animals must be monitored every day, because body weight is a key indicator of an animal's health status. Underfeeding, overfeeding, malnutrition, or dyspepsia can be detected quickly by a change in body weight. It has been previously reported that marmosets with low body weights are more prone to concurrent bone and gastrointestinal diseases49. Generally, the body weights of the infant marmosets remained constant in the P1-5 stage and gradually increased by 1-4 g per day when using the hand-rearing method. From P5 onward, if no increase or a gradual loss of body weight over 2 days is observed, the caregivers must be alert and check the food ingredients, as well as the body temperature and excretion status of the animals. It is useful to add probiotics in this case.
It is not necessary to feed the animals overnight. We tried to feed the infant marmosets every 2 h to 4 h around the clock and found that it did not promote the growth of the infant marmosets (data not shown). Lacking adequate physical exercise or overfeeding could induce metabolic syndromes, such as obesity, which has been linked to dyslipidemia, altered glucose metabolism, and insulin resistance in marmosets50,51,52. The infants must be kept warm during feeding and weighing, especially in the first few days after birth. We found that one infant had diarrhea after it caught a cold; diarrhea rapidly weakens the animal's vitality and reduces the body weight.
The grip strength and body temperature are the main indicators of the health status of infant marmosets, and these must be monitored carefully. Whenever an infant marmoset is being fed, it grips the blanket or the feeder's finger. The grip strength is usually weakened when the animal has diarrhea. Once this happens, one should add pea-sized probiotics to the food and feed the affected infant for 3 days until the diarrhea is gone. If the diarrhea lasts over 3 days, one should add montmorillonite powder instead of probiotics. In contrast, if the infant is constipated, one should gently wipe its anus with a swab dipped in warm water to promote defecation, and lactulose should be used if necessary. In addition, the body temperature of the infant marmoset is usually higher than that of humans. If the animal's temperature is lower than normal, which is often associated with a weakened grip strength, the animal's behavior must be monitored closely and the feeding adjusted accordingly.
When the infant marmosets start to climb, they must be provided with climbing frames and toy rollers in the incubator under the supervision of the breeder. The animals are allowed to practice climbing before and after each meal from P25 onward. At P50, the infant marmosets can climb and jump well, and this is a prerequisite for voluntary eating. Infant marmosets like to grind their teeth by biting blankets, plush toys, or disposable diaper pads, but these may form small loops that threaten their lives due to self-strangulation. Hence, torn pieces and thread should be cleaned promptly to avoid injuries or deaths.
Sometimes, the feeding procedure can be modified slightly due to differences in appetite, vitality, and environmental conditions. If an infant marmoset is strong enough, it will probably start to practice climbing before P25, learn voluntary eating before P50, and even go back to the family before P60. Meanwhile, we lower the temperature by 0.5 °C in the incubator every day or every 2 days so that the infant marmoset can adjust to the same temperature as that in the colony before it eventually goes back to the cage. If an infant marmoset has a poor appetite for a certain period, less food must be provided per meal but at a higher frequency per day, and the incubator must be kept warmer than the colony for a longer time. In the current study, 88.9% (n = 16/18) of infants were successfully accepted by the birth parents, 11.1% (n = 2/18) were fostered by other families, and none were rejected.
Studying juvenile marmosets with different family structures reveals differences in their social and mental development; indeed, social interactions between parents and children and between peers during development potentially sculpt social brain function53,54,55. Therefore, whether one or two animals are selected for hand-rearing depends on the aims of the experiment. Our lab usually hand-rears two infants, if possible, to both meet animal welfare demands and help more infants survive. The collaborative rearing method for marmoset triplets43 is also a good choice. Hand-reared marmosets with limited parental interactions may not be suitable for experiments for the study of social behavior.
In conclusion, the hand-rearing method in the current study provides instructions on how to feed infant marmosets from P1 to P60. With this protocol, it takes ~15 min for an experienced breeder to manually feed one infant marmoset. The successful operation depends on multiple factors, including the proper temperature of the food and the environment, the feeding time and technique, and the excretion of the animals after meals. This method is feasible and easy to learn. It has been continually optimized with 5 years of practice. This method may be applied to other laboratories working with common marmosets.
The authors have nothing to disclose.
The authors would like to thank Mingxuan Li for his editing of the grammar and polishing of the early version of this manuscript. This work was supported by the Zhejiang Province Natural Science Foundation of China (LD22H090003); the Natural Science Foundation of China (32170991 and 32071097), STI2030-Major Projects 2021ZD0204100 (2021ZD0204101) and 2022ZD0205000 (2022ZD0205003); and the MOE Frontier Science Center for Brain Science & Brain-Machine Integration, Zhejiang University.
animal incubator | RCOM, Korea | MX – BL600N, 855 mm (W) x 470 mm (L) x 440 mm (H) | |
baby milk powder | Meadjohnson, America | suitable for 0-12 months of age, executive standard – GB25596 | |
baby rice paste | HEINZ, China | suitable for 0-6 months of age, executive standard – GB10769 | |
baby wipes | babycare, China | soft | |
beaker | ShuNiu, China | 100 mL | |
blankets | Grace, China | 10 cm × 10 cm, soft | |
climbing frame | WowWee, China | firm and no small circular structures | |
disposable diaper pads | Hi Health Pet, China | either M or L size | |
disposable sterile syringe | Cofoe, China | 1 mL, 2.5 mL, 3 mL, 5 mL, 10 mL | |
electronic scale | YouSheng, China | measuring range from 0 to 6,000 g with precision of 0.2 g | |
intravenous injector | HD, China | 0.55 mm x 20 mm needle | |
kettle | FGA, China | warm-keeping kettle 1,500 mL | |
lactulose | BELCOL, China | to solve constipation | |
plastic weighing dish | SKSLAB, China | 80 mm x 80 mm x 22 mm, used as a bowl | |
plush toy | Lebiyou, China | soft | |
probiotic powder | G-Pet, China | to regulate gastrointestinal environment | |
sterile centrifuge tube | NEST, China | 50 mL | |
swab | OYEAH, China | 80 – 100 mm | |
toy roller | WowWee, China | firm and no small circular structures |