Hand-Rearing Method for Infant Marmosets

Summary

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.

Abstract

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.

Introduction

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 risks^1,2,3,4,5,6. 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 processing^{7,8,9,10,11,12,13,14}, vocal communication^{15,16,17,18,19}, models of spinal cord injury^20,21,22,23, Parkinson's disease^{24,25,26,27,28}, and age-related diseases²⁹. Compared with other NHPs, the common marmoset has a relatively high reproduction rate, which is potentially useful for transgenic modification^30,31,32. This primate is also widely used in pharmacology, angiography, and pathogen and immune studies^{33,34,35,36,37,38,39}. 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 settings^40,41,42 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.

Protocol

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

1. Set the colony room with a 12 h:12 h day/night cycle, the temperature at 26-28 °C, and the relative humidity at 45%-55%.
2. Pair male and female marmosets at 2-6 years old, and hold them in cages (850 mm x 800 mm x 800 mm) with sufficient space and fresh air with a 24 h ventilation system.
3. Provide resting boards, swings, perches, and hammocks in the cages.
4. Feed the pair of marmosets with fresh water and 30-40 g of food twice a day, including cereal, eggs, sweet potatoes, honey, fruit, vegetables, and mealworms.
   ​NOTE: Veterinarians and experimenters must inspect the animal facility at least once per day to ensure that any sick individuals are diagnosed and treated immediately.

2. Preparation before the birth of the marmoset infants

1. Care for the pregnant marmosets
   NOTE: The time of conception was diagnosed by palpation (typically 10-20 days after the start of the embryonic period), and we also referred to the reproductive history of the animal subjects.
   1. Provide a larger space and minimal human disturbance for the breeding pairs.
   2. Feed the breeding pairs with additional food such as mealworms, eggs, yogurt, and dried fruit to guarantee nutrition for the females.
   3. Take care of and frequently check the pregnant marmosets to prepare for their parturition.
      NOTE: The gestation period of the marmoset is estimated to be 148 days ± 4.3 days⁴⁵.
2. Prepare the following items: an animal incubator (855 mm [W] x 470 mm [L] x 440 mm [H]), disposable diaper pads (M/L sizes), baby wipes, plush toys (Figure 1A), toy rollers (Figure 1B), climbing frames (Figure 1B), blankets (10 cm х 10 cm, Figure 1C), and an electronic scale (precision of 0.2 g, Figure 1F).
   ​NOTE: To avoid animal hanging, the plush toys should not have loop structures.
3. Food and feeding appliance preparation
   1. Prepare the following items: baby formula (suitable for 0-12 months of age), baby rice paste (suitable for 0-6 months of age), electric water kettle, beaker (100 mL), heating pad, plastic weighing dish (80 mm x 80 mm x 22 mm, Figure 1D), sterile centrifuge tubes (50 mL), disposable sterile syringes (1-5 mL), intravenous injectors (for custom-made feeding nipples) (Figure 1E), and swabs (80-100 cm).
      NOTE: To make a nipple for feeding, a cut is made 1 cm from the end of an intravenous injector attached to a syringe (Figure 1E).
4. Recording form preparation
   1. Prepare a form, usually several pages long, for each infant marmoset to record basic information such as the name, birth date, birth weight, parents, other basic information of interest such as the head circumference and tail length, breeding information such as the breeding date and time, the amount (mL) of the food intake, the defecation state (yes/no, hard/loose), and the incubator temperature and humidity.
      ​NOTE: Usually, the body weight is measured and recorded twice a day, once before the first meal and once before the last meal.

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

1. Clean and sterilize the room ahead of the due date.
   1. Spray hypochlorous acid or 75% ethyl alcohol on the floor and table, leave for 30 s, and then wipe the table, and mop the floor.
2. Set the temperature of the incubator at 35 °C and the humidity at 40%. Usually, to simulate the basic temperature requirement of infant marmosets, as Table 1 shows, before postnatal day 14 (P14), keep the incubator temperature at 35 °C, and from P15 on, lower the temperature by 0.5 °C every 3 days. Keep the humidity at 40%-45% inside the incubator, which is close to the colony humidity and keeps the fur of the infants dry.
3. Tile a disposable diaper pad to cover the chassis of the incubator.
4. To minimize the stress of the infants, put a couple of blankets and plush toys in the incubator ahead of introducing the infant marmosets, which tend to mimic the adult marmosets.
   NOTE: The blankets and plush toys are put in the home cage of the breeding pairs for 1 day before use.
5. Put the infant marmosets into the incubator and place them on the plush toys once they are separated from their parents in the home cage.
   NOTE: To avoid social isolation and in accord with animal welfare, generally, two infants are chosen together for hand-rearing.
   1. Wear sterilized personal protective equipment (PPE, Figure 1G) before feeding.
   2. Warm a couple of blankets to 35 °C.
   3. Gently hold the infant marmoset with warm blankets, and obtain the weight of the animal as the birth weight.
   4. Transfer the infant marmoset into the incubator with warm blankets.
   5. Take records, as mentioned in step 2.4.
6. Blend food ingredients and feed the infant marmoset.
   1. Dissolve 5 g of baby formula in 30 mL of 50 °C boiled water in a 50 mL sterile centrifuge tube.
      NOTE: Infant marmosets at different postnatal ages need different food recipes. Table 2 includes the different dosages of baby formula, rice paste, and water at postnatal ages from P1 to P60. The dosage is usually enough for 1 day. Blend the ingredients before the first meal, and store the rest of the food in a 4 °C fridge. Heat the food to 30-35 °C for each meal.
   2. Take 1 mL of food with a 1 mL syringe, and lid the syringe with a custom-made feeding nipple.
      NOTE: Select a syringe of the proper size, referring to Table 2.
   3. Keep the food temperature at 30-35 °C with the heating pad.
   4. Warm the hands before feeding, and gently hold the infant marmoset with a warm blanket in one hand in the incubator.
   5. Put the feeding nipple into the mouth of the infant marmoset while the head of the infant marmoset is softly held with the thumb and index finger of the holding hand of the caregiver, and slowly push the food out of the syringe at a constant velocity.
      NOTE: Never push the food faster than the infant marmoset swallows. Pushing fast can cause choking, with food coming out from the nose through the throat. This can cause diseases such as pneumonia, which can even lead to death. If the food overflows, the infant marmoset will struggle. Whenever this occurs, stop feeding and wipe the food off the animal's face carefully. Continue to feed after the marmoset starts behaving normally.
7. After the infant marmoset consumes a proper amount of food, wipe its anus with a swab with warm water, which both cleans the anus and promotes defecation.
8. Observe the animal for a few minutes, and check the locomotion and defecation of the animal.
9. Record the feeding time, the amount (mL) of the food intake, the defecation state (yes/no, hard/loose), and the incubator temperature and humidity.
   NOTE: Wrap the infant marmoset with warm blankets during weighing to avoid the infant getting cold or injured.
10. Keep the chassis of the incubator clean by picking up solid feces or changing to a new disposable diaper pad.
11. Before P50, feed the infant marmoset following steps 3.3-3.7, and use the dosage of food ingredients and the feeding time and frequency shown in Table 2.
12. From P50 on, the infant marmoset is usually ready for voluntary eating.
    1. Use plastic weighing dishes instead of syringes. Prepare the food by mixing food ingredients directly in the dish; refer to Table 2 for the amounts.
    2. Put the food dish in the incubator, and fix the bottom in case it is turned over. Observe for a few minutes to make sure the infant marmosets eat the food. For the first few times, guide the animal to eat voluntarily by luring the animal to the food dish and guiding its mouth to touch the food several times.
       ​NOTE: Never let the animal's nose touch the food. Usually, the animal learns to voluntarily eat in 1 day.

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.

1. Move the infant marmosets from the animal incubator to small cages (45 cm x 45 cm x 40 cm), which are similar to a birdcage. Hang a water bottle (50 mL) on each small cage.
2. Transfer the small cages with the infant marmosets to the colony, and position them close to the family cage.
3. Feed the infant marmosets separately with a plastic weighing dish for 1 week, mixing the food according to the daily recipes prepared for the whole colony.
4. Make a record of the body weight and defecation status once per day.

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.

1. Put the infant marmosets back into the family cage in the morning.
2. Observe the animals for at least 15 min to make sure there is no biting, fighting, or chasing between the family members and the new entrants.
   NOTE: If biting, fighting, or chasing occurs, separate the infant from the others as soon as possible; and try to return the infant to its family one more time on another day. If the failure occurs again, select another family to foster it. The family groups that have rich parenting experience are the first choice for fostering. Put a plush toy in a new cage to accompany the infant monkey if no family accepts it.
3. Stop feeding the infant marmosets separately, and start feeding them using the diet for the colony.
4. Pay close attention to the activity of the infant marmosets for 1 week after they return to the family cage.
5. Measure the body weights of the infant marmosets every 2 days, and make a record. If they lose weight, feed them extra nutritional food by syringe in the family cage.
6. Provide daily care for the infant marmosets, as for the marmosets in the colony.

Results

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...

Discussion

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 ...

Materials

Name	Company	Catalog Number	Comments
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