Protocol for nutritional intervention in neonatal rats using the “pup-in-a-cup” artificial rearing system

Summary

Early-life nutrition fundamentally influences newborn development and health. Here, we present a protocol for nutritional intervention in neonatal rats using the “pup-in-a-cup” artificial rearing system. We describe steps for rat milk substitute preparation, cheek cannulation and maintenance, and nutritional manipulation during the suckling period. This protocol enables investigation into the role of nutritional factors in newborns by artificially rearing rats away from the mother with experimental diets starting at postnatal day 4 for up to 18 days.

For complete details on the use and execution of this protocol, please refer to Wang et al.,¹ Choudhary et al.,² and Mu et al.^3,4

Graphical abstract

Highlights

• •A protocol for artificial rearing of newborn rats for nutritional manipulation
• •Detailed requirements for rat milk substitutes and housing apparatus
• •Procedures for cheek cannula placement and daily maintenance
• •Adaptable for other dietary investigations

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Early-life nutrition fundamentally influences newborn development and health. Here, we present a protocol for nutritional intervention in neonatal rats using the “pup-in-a-cup” artificial rearing system. We describe steps for rat milk substitute preparation, cheek cannulation and maintenance, and nutritional manipulation during the suckling period. This protocol enables investigation into the role of nutritional factors in newborns by artificially rearing rats away from the mother with experimental diets starting at postnatal day 4 for up to 18 days.

Before you begin

This protocol describes the details of nutritional intervention in neonatal Sprague-Dawley rats using the “pup-in-a-cup” artificial rearing system (Figure 1). If other rodents or strains are used, the details provided below for the Sprague-Dawley rat may need to be modified to meet specific needs. While other early life feeding systems exist (e.g., oral gavage, intragastric cannula system), the combined cheek cannula and pump system described here offers several advantages. Compared to oral gavage, the advantages include: 1) The continuous feeding method ensures consistent intake of nutrients or substances among pups by allowing precise control over feeding rate, volume, and duration; 2) Continuous feeding during nighttime more closely mirrors the natural eating pattern of pups, providing a more relevant model for studying early-life nutrition; 3) Pump systems used in continuous feeding are ideal for long-term studies, sustaining feeding over extended periods. This is crucial for observing developmental changes and studying the effects of prolonged nutrient exposure; 4). Unlike oral gavage, which may risk trauma, continuous feeding is less risky and stressful for pups during long-term feeding. Compared to an intragastric cannula system, although both are continuous feeding systems, the cheek cannula adopted here is less invasive and restricts movement less than the intragastric cannula used in previously described rat studies.^5,6 Other considerations to optimize this artificial rearing system have been taken into account. To minimize the effects of variable litter size on access to maternal milk and weight gain in pups prior to the initiation of artificial rearing, litters are culled to 10–12 pups at birth when possible. The rat milk substitute used in this protocol contains 12 g fat, 5.25 g protein and 7.5 g total carbohydrates, with a calculated energy density of 6359.7 kJ/L (152 kcal /100 mL), which is comparable to that of actual rat milk (6485–8452 kJ/L (155–202 kcal/100 mL)). It is possible to manipulate the ingredients of the milk substitute (i.e., substitutions or additions to that described) to study the effects of other dietary components on pup development. If the energy density of the milk substitute needs to be adjusted to induce a state of overnutrition or malnutrition, the specific adjustments require further experimental verification. On postnatal day (PD) 4–5, a cannula is inserted into the cheek of Sprague Dawley rats to allow for the delivery of customized rat milk substitutes to the pup. A clean surgical area with bright lights and heating pads is required to ensure hygienic standards for cannulation and recovery of body temperature after hypothermic anesthesia. The entire artificial rearing system is placed in a humidity-controlled room maintained at 30°C with a 12-h normal light–dark cycle. For more detailed preparation of the rat milk substitute and ‘pup-in-a-cup’ rearing system setup, please refer to the key resources table and materials and equipment sections for a detailed list of ingredients, materials, and equipment.

Figure 1.

Schematic workflow of overall protocol

Institutional permissions

All animal experiments included in this protocol followed ethical guidelines and received approval from the University of Calgary Animal Care Committee (#AC19-0104) in compliance with the Guidelines of the Canadian Council on Animal Care. Users of this protocol must obtain ethical permission from a local Animal Ethics Committee and undergo training in animal handling and basic surgery through a local training program.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
Evaporated milk	Carnation	https://www.carnationmilk.ca/En/Products/Carnation-Evaporated-Milk
Maize oil	Mazola	https://www.mazola.com/products/mazolareg-corn-oil/
FeSO4·7H2O	Sigma-Aldrich	215422-5G
CuSO4·5H2O	Sigma-Aldrich	C8027-500G
ZnSO4·7H2O	Sigma-Aldrich	221376-100G
L-methionine	Dyets, Inc.	403000
L-tryptophan	Dyets, Inc.	404800
AIN-93VX	Dyets, Inc.	310025
Evaporated milk	Carnation	https://www.carnationmilk.ca/En/Products/Carnation-Evaporated-Milk
Experimental models: Organisms/strains
Rattus norvegicus: Sprague-Dawley 4-day-old rat	In-house breeding	Strain code: 400; RRID:
Software and algorithms
R version 4.2.2	Bell Laboratories	https://mirror.rcg.sfu.ca/mirror/CRAN/
Prism version 10.0.2	GraphPad	https://www.graphpad.com/
Other
DART 12 oz foam cups	Dart	12J12
3M Vetbond tissue adhesive	3M	1469SB
Sand, sea (washed)	Fisher Chemical	S25-3
BD Intramedic PE tubing (PE10)	BD	22-204008
BD Intramedic PE tubing (PE20)	BD	14-170-11A
BD Intramedic PE tubing (PE90)	BD	14-170-11D
L5M-A laboratory mixer	Silverson	L5M-A
Signature rocking platform shaker	VWR	12620-908
Precision general purpose baths	Thermo Scientific	S37365
NE-1200 twelve channel programmable syringe pump	MULTI-PHASER	NE-1200
Weighing scale	Sartorius	BCE622-1S

Materials and equipment

Ingredients found in the rat milk substitute

Reagent	Final concentration	Amount (/100 mL)
Milk base
Evaporated milka	75% (v/v)	75 mL
Fat
Corn oilb	6% (v/v)	6 mL
Mineral solution
FeSO4.7H2O	0.27 g/L	27 mg
CuSO4.5H2O	0.15 g/L	15 mg
ZnSO4. 7H2O	0.16 g /L	16 mg
Amino acid
L-Methionine	1 g/L	0.1 g
L-Tryptophan	0.5 g/L	0.05 g
Vitamin mix
AIN-93VXc	15 g/L	1.5 g
Sterile water	17% (v/v)	17 mL
Energy density	N/A	152 kcal

The rat milk substitute formula was adopted and modified from Smart et al.⁷ For customized applications, rat milk substitutes can be adjusted accordingly and should be aliquoted in sterile containers for 2–3 days use. Storage at 4°C is recommended. Before use, it is essential to warm the milk substitutes to 37°C using a water bath. Avoid freezing the milk substitutes or exposing them > 4 °C °C for extended periods.

^aper 100 g, Carnation evaporated milk provides: 134 kcal; 4.6 g saturated fat, 0.2 g polyunsaturated fat, 2.3 g monounsaturated; 29 mg cholesterol; 106 mg sodium; 303 mg potassium; 7 g protein, and 10 g total carbohydrate (3′ sialyllactose < 20 mg, 6′ fucosyllactose < 2 mg).

^bper 14 g, Mazola corn oil provides: 120 kcal; 2 g saturated fat, 7 g polyunsaturated fat, 4 g monounsaturated fat; and 2 mg vitamin E.

^cper 1 g, vitamin mixture AIN-93VX provides: 6 mg Thiamin HCl, 6 mg Riboflavin, 7 mg Pyridoxine HCl, 180 mg Niacin, 16 mg Calcium, 2 mg Folic Acid, 0.2 g Biotin, 25 g Cyanocobalamin (B12), 4000 IU Vitamin A Palmitate, 75 IU Vitamin E Acetate, 1000 IU Vitamin D3, and 0.75 mg Vitamin K1.

Step-by-step method details

Rat milk substitutes preparation

Timing: 1 h

This step prepares a basal rat milk substitute to meet the nutritional requirements of pups and allows for a wide range of additions of ingredients of interest. The dosage or combination of tested ingredients can be flexibly manipulated to study their effects on pup development.

• 1.Prepare and mix ingredients for the basal milk substitute in autoclaved containers as needed for 2–3 days’ use.

CRITICAL: Mix the evaporated milk and fat thoroughly until no fat droplets are visible and then add other ingredients to prevent fat droplets from affecting the subsequent homogenization of powdered ingredients.

• 2.Introduce any other experimental components/ingredients into the basal milk mixture as required.
• 3.Homogenize ingredients 3 times × 10 min each using immersion dispersers at a speed of 15,500 rpm/s to avoid delamination as shown in Figure 2A.

CRITICAL: Alternative homogenization methods may exist. If an immersion disperser is used, the separation of the milk mixture during storage can only be effectively avoided when the rotation speed is equal to or exceeds 15,500 rpm/s.

Note: Prolonged high-speed homogenization may generate heat. It is recommended to introduce 3–5 minute intervals at 20°C–22°C between each homogenization.

• 4.Store the homogenized milk substitute(s) in 90 mL sterile containers at 4°C until used.

Figure 2.

Rat milk substitute preparation with immersion dispersers

Cheek cannula preparation

Timing: 1 h

This step prepares the cannula that is connected to the milk delivery tubing system and inserted through the cheek to deliver milk to the pup. The cheek cannula can be detached from the PE20 milk delivery tubing for daily flushing and leakage and blockage checks.

• 5.Apply heat to soften the ends of polyethylene PE10, PE20, and PE90 tubing. Shape them into capped ends, as depicted in Figure 3A.

CRITICAL: The capped end of the PE10 tubing should maintain a normal inside diameter to ensure proper milk flow. Additionally, ensure that all ends are shaped as flat as possible to enable a tight fit against the skin.

• 6.Cut the shaped PE10, PE20 and PE90 tubing into lengths of 5, 0.5, and 0.1 cm, respectively.
• 7.Assemble the PE10, PE20 and PE90 tubing as shown in Figures 3A and 3B.

Figure 3.

Cannula preparation

Schematic (A) and side (B) views of cheek cannula.

“Pup-in-a-cup” rearing system setup

Timing: 1 h

This step assembles the key components of the “pup-in-a-cup” artificial rearing system, including housing condition, syringe pump and tubing system, and the water bath and cup system.

• 8.
  Housing condition setup.

  • a.
    Place the entire artificial rearing system in a humidity-controlled room maintained at 30°C with a 12-h normal light–dark cycle.
  • b.
    Avoid excessive noise in the environment throughout the process.
• 9.
  Milk delivery system connection.

  • a.
    Connect rocking platform shaker, syringe pump, 12 mL syringes, and PE20 tubing.
  • b.
    Label the tubing with colored tape and secure it to the stand.
  • c.
    Flush the tubing with autoclaved water and check for leaks.
  • d.
    Set up the pump to pause for 15-min every 2 h of feeding to allow for gastric emptying.
  • e.
    Homogenize and pre-heat the milk to 37°C before use.

    Note: For the duration of the 12 h feeding period, the milk is left at the designated temperature of the room which is set to 30°C.

  • f.
    Repeat homogenization and pre-heating at next feeding period.

    Note: Reserve milk that is not needed until the next feeding period is stored at 4°C.

    Note: The milk is delivered via sterile lines and therefore would not be expected to degrade over the 12 h time frame. The formulation also contains preservatives and anti-caking agents including Sodium Ascorbate and Silicon Dioxide. Separation of the milk (due to high fat content) is of greater concern and is therefore prevented by having the syringe pump on an oscillating rocking platform.

• 10.
  Water bath and cup setup.

  • a.
    Fill the bottom of the Styrofoam cup with one-third full of sand to weigh the cup down in the bath (11 cm diameter × 15 cm deep).
  • b.
    Fill the second Styrofoam cup with bedding to one-third full (11 cm diameter × 15 cm deep).
  • c.
    Add water to the bath and secure the stacked cups in the water bath as shown in Figure 4.

Figure 4.

“Pup-in-a-cup” rearing system setup

Cheek cannulation

Timing: 30 min

This step performs cheek cannulation on pups under hypothermic anesthesia. Proper cheek cannulation procedures ensure the feasibility of long-term artificial rearing and daily maintenance.

• 11.
  Surgery area, materials, and animal preparation.

  • a.
    Pre-warm a heating pad to 37°C.
  • b.
    Clean the surgery area with 70% ethanol and make sure bright light is available.
  • c.
    Cut a piano wire (stainless steel, 0.25 mm diameter) into lengths of 3–5 cm and soak in 70% ethanol for skin insertion.
  • d.
    Prepare 3M Vetbond Tissue Adhesive for skin closure.
  • e.
    Separate PD4‒5 pups from dams, record their initial body weight and label each pup with a unique ID.
• 12.
  Cheek cannula insertion under hypothermic anesthesia.

  • a.
    Apply 2 min of hypothermic anesthesia with crushed ice to the pups prior to surgery. Protect the pup by placing it on a latex glove.
  • b.
    Cover the piano wire with a cap shaped PE10 tubing and pierce it through the inside lining of the cheek as shown in Figures 5A and 5B.

    CRITICAL: When piercing the cheek, avoid blood vessels to avoid bleeding and infection.

  • c.
    Reinforce the PE10 tubing with pre-made PE90 and PE20 shaped caps and use 3M skin adhesive to keep them in place.
  • d.
    Once in place, flush the PE10 tubing with sterile water to check for blockage and leaks.
• 13.
  Post-anesthesia care.

  • a.
    Place the cannulated pup on a pre-warmed heating pad and monitor its breathing and body temperature.
  • b.
    Calculate the initial milk flow rate based on the average body weight of all of the pups that will be fed by the pump.
  • c.
    Transfer the pups to the pre-warmed “pup-in-cup” system and start feeding.

Figure 5.

Pups at postnatal day 4 with cheek cannula in place (left) and schematic illustration (right)

Daily management

Timing: up to 18 days

This step maintains the daily health of the pups and the hygiene of the entire system. Daily adjustments of milk flow provide the flexibility to supply appropriate energy and nutrients for pup development.

Daily management, including changing milk every 12 h, daily cleaning of the tubing system, daily checking of intestinal motility and fecal discharge, mimicking maternal care, and milk flow speed adjustment, are critical to meeting pup nutritional needs and reducing mortality. The procedure for daily manipulation is suggested as follows.

• 14.
  Daily body weight measurement for milk flow adjustment.

  • a.
    Record body weight before morning milk changes.
  • b.
    Monitor intestinal motility by checking the volume of the “milk belt” in the stomach (visible milk in the stomach).
  • c.
    Stimulate the pup’s bowel movements twice daily using a cotton-tip applicator and provide gentle downward pressure on the pup’s abdominal region.
  • d.
    Consistently handle the pups to mimic maternal care.
  • e.
    Calculate the flow speed based on age and the daily average body weight of the pups using the formula: Flow Speed (mL/h) = {0.35 + 0.02 ∗ [Age (days) -4]} ∗ Body Weight (g) / Feeding hours.
• 15.
  Artificial rearing system maintenance.

  • a.
    Refill new syringes with fresh milk pre-warmed with a water bath every 12 h.
  • b.
    Detach PE10 cannula from PE20 tubing and flush both with boiled sterile water (cooled to 50°C–60°C for use) every 12 h.

    CRITICAL: Following the instructions and frequency of this step is essential to avoiding milk spoilage and tubing blockage and leakage.

  • c.
    Monitor the activity status of the pups and adjust activity area for pups as needed.

    Note: When pups become more active, usually around PD15‒17, add an additional cup layer (i.e. 4–5 cm of the top of an empty cup) to increase cup height and also add more bedding to the bottom of the cup, or use other possible means such as using larger-sized water bath and containers, to increase the diameter of the activity area for pups.

• 16.Termination.

Note: This protocol allows rats to be artificially reared on an experimental diet for up to 18 days, after which time euthanasia and sample collection can be performed as needed.

Note: The age of postnatal day 6–13 is a critical stage for structural and functional changes in lung development in newborn rats.^8,9 Rat pups with poor adaptability to increasing flow rate (needed to maintain proper weight gain and growth) are prone to choking on the milk and suffocation when the flow rate increases sharply after 8 days of age. At this stage, the condition of the pups should be closely monitored, and corresponding measures taken if necessary. Refer to Problem 1 for more details.

Expected outcomes

This protocol is applicable for neonatal nutrition intervention starting from postnatal day 4 to day 21 or transition to solid feed. The use of rat milk substitute and the established artificial system herein enables precise control of nutrient input as needed, independent of any maternal factors. The cheek cannula employed is less invasive than the previously described gastric cannula¹⁰ and allows for daily maintenance of hygiene levels. An example growth curve of nine Sprague-Dawley male rats fed rat milk substitute from postnatal day 4 to day 21 is shown in Figure 6A. The Lee index (weight 0 · 33/naso-anal length × 1000), body composition and organ weights of pups measured on 21 days of age are shown in Figures 6B–6D, respectively.

Figure 6.

Growth parameters in Sprague Dawley rats

Growth curve (A) of pups during 18 days of artificial rearing with rat milk substitute and Lee index (B), body composition (C) and organ weights (D) of pups at 21 days of age.

Limitations

The complexity of establishing the pup-in-a-cup model and the limited number of ‘cups’ that can be placed in the water bath at one time limit the scale of the model. In this case, although we affix an additional 4–5 cm of Styrofoam cup to the top to increase cup height and add more bedding to the bottom of the cup, thus increasing the diameter of the activity area for pups, the use of foam cups generally limits the activity space for pups, particularly for pups after PD15‒17.

While early separation of the pups from their mother holds clinical relevance for infants requiring special care (e.g., neonatal intensive care), the lack of typical maternal care, grooming, and other factors in this rodent model may affect brain development and later behavior. For studies that include behavioral outcomes or focus on early development of brain functions, the applicability of this protocol should be carefully evaluated. Procedures to mimic maternal care and grooming, including consistent handling and putting gentle downward pressure on the pup’s abdomen to stimulate bowel movements should be performed regularly.

Delivering milk substitutes to all pups with the same flow speed based on age and daily average body weight would not allow for investigation of factors influencing neonatal feed intake and eating behaviors, which have been shown to be critical to early-life growth and development.^11,12 Additionally, during the daily milk change, we warm up the milk from 4°C to 37°C before use and keep reserve milk at 4°C until the next milk change. Storage at 4°C helps maintain the milk quality and warming prior to administration makes it closer to body temperature, avoiding intestinal distress and disturbances for the pups. However, prolonged 30°C exposure of milk in the tubing system may lead to degradation of temperature-sensitive compounds, which limits the application of our model to the addition of these types of components to milk. In this case, researchers could increase the number of milk changes to shorten the 30°C exposure if necessary and without causing undue stress to the pups.

Troubleshooting

Problem 1

Abdominal bloating may occur with the increase in flow speed as pups age (Figure 7), which is one of the causes of mortality when using this artificial rearing model (Steps 14–15).

Figure 7.

Abdominal bloating in a pup on postnatal day 6

Potential solution

• •Monitor bowel movements and milk intake every day. If abdominal bloating occurs, gently stroke the pup’s abdomen with a cotton-tip applicator immediately and an additional 2 times during the day to encourage the passage of feces. Give a dose of 0·1–0·2 mL deoxycholate (0.5 g/100 mL) via the cannula if the bloating persists for more than 24 h.⁷ Given the interaction of deoxycholate with host and gut microbiome metabolism, careful consideration should be given to the use of deoxycholate-treated pup samples when experimental designs involve blood metabolomics and gut microbiome measurements.¹³ If after 48 h the bloating does not subside, then the pup will be euthanized.

Problem 2

As the duration of artificial rearing and the age of pups increases, the cheek cannula may fall off due to inflammation caused by milk residue or pulling by the pup (Step 15-c).

Potential solution

• •In addition to thorough daily cleaning of residual milk, carefully check and adjust loose cheek cannulas and reinforce them with 3M Vetbond Tissue Adhesive as needed. If the cannula falls off completely, the pup will be euthanized.

Problem 3

The use of the Styrofoam cups may limit activity space for pups, especially when the pups become more active after PD15‒17 (Step 15).

Potential solution

• •When pups become active (usually around PD15‒16), cut the upper end (4–5 cm) of an empty foam cup and attach this part to the outside top of each cup to extend cup height and also add more bedding to the bottom to enlarge the diameter of the pup’s activity area (approximately 0.5–1 cm). If needed, cover the top of the cups with mesh to prevent pups from accidentally jumping out of the cups. Given the space limitation of the cups used in this protocol, this model is best suited to the early life period (P4-P12) when the pups are less active.

Problem 4

Although both the milk and pup’s cheek cannulas are flushed every 12 h to avoid milk blockage/leakage, occasionally blockages and leakages are still observed at the connection between PE10 cheek cannula and PE20 milk delivery tubing (Step 15).

Potential solution

• •When piercing the skin with a piano wire to install the cheek cannula, avoid making the insertion wound too large. Further, using 3M Vetbond Tissue Adhesive to reinforce the insertion effectively avoids all cannula leakage. Routine flushing of both the milk tubing and the cheek cannula (every 12 h) prevents the occurrence of blockage caused by the milk. If persistent cannula leakage occurs and is not effectively stopped, it will cause malnutrition in pups and leaking milk is also the main cause of inflammation in the cheeks. In this case, it is recommended that the pup should be euthanized after consultation with the veterinarian.
• •Leakage may also occur at the connection between the PE10 cheek cannula and the PE20 delivery tubing during subsequent feedings. Because 50°C–60°C hot sterile water is used every day to clean the tubing system, it may cause the tubing to loosen over time. This problem can be combatted by cutting ∼2 cm off the end of the tubing to allow for a snug fit between the PE10 cheek cannula and PE20 tubing connection. If the leaking issue persists, it is recommended to replace the PE20 feeding tube.

Problem 5

The main energy source of neonatal rats is liquid milk which does not produce large amounts of fecal matter. When it is necessary to collect solid fecal samples (e.g., for potential gut microbiota analysis), it may be difficult to collect sufficient fecal samples from the pups (Step 15).

Potential solution

• •We recommend that feces should be collected no earlier than PD7 and should be collected at the same time as weighing and defecation stimulation in the morning to avoid additional stress. When a large amount of feces is required, it is recommended to collect samples for three consecutive days and pool them before use to ensure that the collection amount is sufficient for most pups.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Raylene A. Reimer reimer@ucalgary.ca.

Technical contact

For questions about cheek cannulation and rearing system setup contact Dr. Weilan Wang (weilan.wang@bcn.edu), for all other questions contact Dr. Raylene Reimer (reimer@ucalgary.ca).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate or analyze new datasets or code.