Pregnancy and Nursing Management for Embryo-Transferred and Genetically Modified Rabbits

This protocol describes a pregnancy and feeding management technique for embryo-transferred and genetically modified rabbits, aimed at reducing newborn rabbit mortality and enhancing the preparation efficiency of gene-edited rabbits.

With the advancement of scientific research, the demand for gene-edited rabbit models is increasing. However, there are limited pregnancy and feeding management systems for gene-edited rabbits, leading to low survival rates among gene-edited rabbits prepared by many inexperienced researchers. Therefore, proper guidance is essential. This article summarizes the pregnancy and feeding practices for genetically modified rabbits developed in the author's laboratory and outlines a set of fundamental processes. These include pregnancy diagnosis, antenatal care, midwifery, assisted breastfeeding, weaning, and other procedures, along with the rescue and care of weak newborn rabbits. Compared to the traditional natural childbirth and nurturing methods used in rabbit farms, this approach involves more refined management, requiring additional time and effort but significantly increasing the survival rate of suckling rabbits. The methods described in this article are suitable for most laboratory breeding scenarios involving gene-edited or embryo-transferred rabbits and provide a straightforward and effective reference for other researchers.

Rabbits are a classic animal model for biomedical research and are increasingly becoming the preferred translational model to bridge the gap between rodent models and large animal models^1,2. Compared with large animals, rabbits have moderate body size, small feeding space, low feeding cost, and convenient blood collection, which are conducive to repeated collection of research data and surgical operations. Rabbits are characterized by good reproductive ability, a short gestation period (28-32 days for New Zealand white rabbits), a large number of births, and fast growth^3,4. Compared with rodents, rabbits are closer to humans in phylogenesis⁵. It is a cost-effective and practical experimental substitute for pigs and non-human primates⁶. Compared with other large and medium-sized gene editing animals (pigs, cattle, sheep, cats, dogs, monkeys), the preparation cost of gene editing rabbits is low, and the cycle is short. With the development of gene therapy^7,8,9, stem cell therapy^10,11,12,13, brain science research¹⁴, and other scientific fields^{1,15,16,17,18}, the demand for nonrodent species in gene editing is increasing.

Gene editing rabbits were once considered to be one of the middle-sized gene editing animals that could realize industrial applications⁶. However, until today, gene-editing rabbits have not been able to achieve large-scale production. One of the main reasons is that the production and breeding of gene-editing rabbits are challenging, and the difficulty is much greater than that of gene-editing mice or rats. There are not many laboratories that can efficiently cultivate gene-editing rabbits. It is often difficult for beginners to successfully prepare gene editing rabbits, and they often encounter such unpleasant problems as low pregnancy rate¹⁹, abortion²⁰, dystocia²¹, refusal of lactation by female rabbits²², and death of suckling rabbits²³. However, there are few systematic data or literature guidelines on the pregnancy and feeding of gene-editing rabbits or embryo-transferred rabbits. Most of them are based on the experience of ordinary rabbit farms.

Therefore, this article summarizes the mature experience of pregnancy and nursing management of genetically modified rabbits from author's laboratory, and introduces a set of basic processes of embryo-transferred rabbits and genetically modified rabbits, including pregnancy diagnosis, antenatal care, midwifery, artificial assisted feeding, ablactation, as well as the rescue and nursing of weak baby rabbits. This process is specially established for breeding gene-editing rabbits or embryo-transferred rabbits. Compared with the traditional natural childbirth and nurturing method of rabbit farms, the pregnancy and nursing management model takes more time and energy, but it can greatly reduce the mortality of baby rabbits, and the effort is worth it.

All experimental protocols were approved by the Ethics Committee for Animal Experiments of the Guangdong Medical Laboratory Animal Center. The ethical review number for the MKRN3 gene-modified rabbit breeding involved in this study is B202210-6. The study adheres to procedures in compliance with ethical standards outlined in the Helsinki Declaration of 1975 (revised 1983). The New Zealand rabbits used in this study were obtained from the Guangdong Medical Laboratory Animal Center in China. The pregnancy and feeding process of genetically modified rabbits described in this article is a summary of the daily animal breeding practices in the author's laboratory. Details on the reagents and the equipment used are listed in the Table of Materials.

1. Pregnancy diagnosis

1. On the 12^th to 14^th day after embryo transfer (choose one of the time points), confirm pregnancy through direct palpation.
   NOTE: This step can be skipped. If palpation is too rough and forceful, it can lead to miscarriage in pregnant rabbits.
   1. Hold the neck skin behind the rabbit's ears with one hand, let the rabbit's buttocks shrink forward, close to the abdominal cavity of the rabbit's buttocks, and then use the other hand to open the palm and gently palpate the back end of the abdomen.
   2. Press the abdomen to a certain degree. If a continuous string of smooth and elastic flesh balls, similar in size to fava beans, is palpated, it is considered pregnancy²⁴.
2. If conditions permit, use ultrasound on the 15^th day after embryo transfer to determine if the rabbit is pregnant. The pregnancy diagnosis of rabbits is based on the presence of small, strong echogenic points (gestational sacs) in the liquid dark zone.
3. During the period of the 16^th-18^th days after embryo transfer, observe the rabbit twice a day in the morning and evening, for approximately 15 min each time. If the rabbit is observed to exhibit behaviors such as restlessness, rapid breathing, plucking hair to build nests, etc., during labor or estrus, it indicates that the female rabbit is pseudopregnant and has not truly conceived.
   NOTE: If embryo transfer fails, female rabbits will have pseudopregnancy similar to parturient or oestrus on the 16^th-18^th days due to the decrease of luteal hormone.

2. Antenatal care

1. Allow pregnant rabbits to eat freely during pregnancy and add fresh green feed once a week.
2. On the morning or evening of the 25^th day after embryo transfer, transfer the pregnant rabbits to the delivery cage.
   1. Disinfect the delivery cage, animal incubator, paper scraps, or shavings in advance.
      NOTE: Choose a rabbit delivery cage that meets international criteria in size. Ensure that the delivery cage has sufficient space for the mother rabbit to move. Choose an incubator that can resist mother rabbit nibble. Choose thick paper scraps with good water absorption. According to the requirements and suggestions of local animal facility management, select appropriate disinfection methods.
   2. Fix the incubator on one side of the delivery cage, and put towels, shavings, or paper scraps into the animal incubator for the pregnant rabbits to make a nest.
   3. Provide adequate feed and drinking water.
   4. Gently move the pregnant rabbits to the delivery cage.
   5. Pay attention to whether the pregnant rabbits have made a nest in the incubator every day. If not, add some paper scraps and towels outside the incubator on the morning of the 28^th day of pregnancy.
      NOTE: If the pregnant rabbits do not nest in the incubator, it will likely not give birth in the incubator. Put some towels and paper scraps outside the incubator to prevent newborn rabbits from losing temperature and dying outside the box.

3. Parturition

NOTE: Day 0 refers to shortly after fertilization and prokaryotic formation. Rabbits are ovulation-stimulating animals that ovulate 10-12 h after mating. The single-cell stage embryos obtained on the day after mating are considered to be on the 0.5^th day. If single-cell stage embryos are cultured in vitro for only 2-3 h and then transferred to surrogate rabbits, it is also considered to be the 0.5^th day of pregnancy.

1. Prenatal monitoring
   NOTE: The gestation period of rabbits is 28-32 days. If a rabbit gives birth before the 30^th day of pregnancy, the size of the fetus is usually relatively small, and there are rare cases of difficult labor. No excessive intervention is needed before the 30^th day.
   1. On the morning of the 30^th day of pregnancy in rabbits, perform abdominal palpation to roughly determine the number, size, and activity of the fetus.
      1. Hold the neck skin behind the rabbit's ears with one hand, let the rabbit's buttocks shrink forward, close to the abdominal cavity of the rabbit's buttocks, and then use the other hand to open the palm and gently palpate the back end of the abdomen. The force must be light.
         NOTE: Pressing into the abdomen to a certain extent will palpate several egg-sized ellipsoids, which can feel the movement of the fetus.
   2. If it is found that the number of fetuses conceived by pregnant rabbits is only 1-2, on the 30^th day of pregnancy, inject 0.02 mg of cloprostenol intramuscularly at 5 p.m. to induce the rabbits' delivery the next day.
      NOTE: Pregnant rabbits have only 1-2 fetuses, which are prone to dystocia or delivery failure when mature.
2. Hasten parturition
   NOTE: If the pregnant rabbit does not give birth on the morning of the 31^st day of pregnancy, it can consider whether it needs to hasten parturition according to the following conditions. If there are more than 2 fetuses, no further action will be taken.
   1. If the pregnant rabbit is only pregnant with 1-2 fetuses and has been injected with cloprostenol the day before, on the morning of the 31^st day of pregnancy, inject 10 units of oxytocin into the muscles to induce labor. 
   2. If the pregnant rabbit has signs of delivery (such as restlessness, rapid breathing, ear vascular dilation, plucking and making a nest, planning the ground, etc.), inject 10 units of oxytocin into the muscles to induce labor.
   3. If the pregnant rabbit has no signs of delivery and fetal movement is normal, continue to observe. If there is no delivery in the morning of the 32^nd day of pregnancy, inject 10 units of oxytocin into the muscles to induce labor.
      NOTE: Usually, the use of oxytocin can solve difficult labor. Generally, 5-10 min after oxytocin injection, the pregnant rabbits will start delivery. Oxytocin is effective for about half an hour and can be administered again after half an hour. It is not recommended to induce labor with oxytocin more than 3 times. If oxytocin is ineffective, have a veterinarian perform a cesarean section. Otherwise, the fetus will die in the uterus, soften, and be discharged from the vagina.

4. Midwifery

1. Arrange and prepare the incubators (Figure 1).
   NOTE: Prepare in advance before the rabbit gives birth. The size of the incubator used in this study is 45 cm × 30 cm × 15 cm in length × width × height.
   1. Add sterile absorbent padding. Corncob granules are recommended.
   2. Spread a towel as a mat covered with artificial down cotton.
      NOTE: The material of artificial down cotton is baby-grade polyester fiber, which is non-toxic. Artificial down cotton is very suitable for newborn rabbits keeping warm. However, it cannot be used to make a nest for the mother rabbit, or it may be eaten into the stomach.
2. Quickly wipe the mucus on the newborn rabbits with a towel. Ensure they breathe smoothly, and then put them into the incubator.
   NOTE: If the breathing of newborn rabbits stops, immediately press the chest for auxiliary breathing (25-35 times/min) to reduce the chest diameter by 25%-30% until spontaneous breathing is restored.
3. After the delivery of pregnant rabbits, count the number of fetuses born. Stick information cards on the incubator: modified genes, number of newborn rabbits, date of birth, etc.
   NOTE: The suitable environmental temperature for newborn rabbits is 30-32 °C. Placing an appropriate amount of rabbit hair or artificial down cotton inside the incubator can achieve a good insulation effect. Rabbit house should be maintained at 16-25 °C. Female rabbits usually have 6-8 nipples. If there are too many babies, some babies can be fed by other mother rabbits.

5. Postpartum care

1. Baby rabbit care (Figure 2)
   NOTE: After giving birth, let the mother rabbit rest for about 1 h before breastfeeding. Rabbits usually breastfeed 1-3 times a day.
   1. Clean all nipples of the mother rabbit with alcohol cotton before the first feeding. Except for breastfeeding, keep mother rabbits and baby rabbits separately at other times.
   2. Gently massage each nipple and gently squeeze out the first drop of milk.
   3. Feeding of 1-2-day-old baby rabbits: Each time, place the mother rabbit on the mat in a lateral position or a prone position and place the sucking rabbits one by one to their nipples to suck milk (Figure 2A). 1-2 day-old rabbits need to be fed 2-3 times a day.
      NOTE: Premature rabbits or low birth weight baby rabbits are prone to hypoglycemia. Feed 1 mL of 5% glucose and consume breast milk as soon as possible. If the baby rabbit is too weak to drink, then directly inject 1 mL of 5% glucose into the abdominal cavity.
   4. On the second day after birth, use a marker pen to number the back of each baby rabbit. Trim a small amount of tail tissue for genotyping.
   5. Feeding baby rabbits over 2 days old: When feeding, place the mother rabbit in an incubator and let it breastfeed on its own (Figure 2B). 2-9-day-old rabbits need to be fed 1-2 times a day, and over 9-day-old rabbits need to be fed once a day until weaning. Make sure the baby rabbits are full (Figure 2C).
      NOTE: If the mother rabbit is unwilling to breastfeed, it can be fed some fresh vegetables to make it more willing to cooperate. If necessary, mother rabbits can be breastfed after being placed in a stable position. Gently squeeze out the first drop of milk from each nipple of the mother rabbit to alleviate breast swelling and pain, which is beneficial for the baby rabbits to suckle on the milk. If the above methods are ineffective, other lactating mother rabbits should be used for breastfeeding.
   6. During the first 4 days after birth, clean the perineum of the baby rabbit twice a day with wet cotton balls to stimulate the excretion of meconium and urine (Figure 2D).
   7. As the hair of the baby rabbit grows, remove some down cotton from the incubator every day.
   8. Change the towel once a day until the baby rabbit grows to the point where its entire body is covered in hair.
      NOTE: Generally, the towel is no longer needed on the 8^th day after birth.
   9. If baby rabbits (1-2 weeks old) experience diarrhea, immediately feed 3 drops of animal-grade gentamicin (concentration is 0.04 g/mL) twice a day for 5 consecutive days.
      NOTE: Diarrhea in baby rabbits is usually caused by sucking milk from mother rabbits with mastitis, or by the high temperature and humidity of the environment. The mortality rate of baby rabbits with diarrhea is very high.
   10. After the young rabbit opens its eyes (about 2 weeks old), move it to a nursing cage with a towel. Add some green feed and daily diet.
       NOTE: The suitable temperature for baby rabbits (2-3 weeks old) is 23-30 °C.
   11. Separate the mother rabbit from the baby rabbits and only open the baffle once a day for breastfeeding.
       NOTE: This can prevent the baby rabbits from biting the mother rabbit's nipple.
2. Postpartum care of the mother rabbit
   1. During lactation, allow mother rabbits to drink and eat freely and feed them with appropriate green feed to ensure adequate nutrition.
   2. If the mother rabbit suffers from mastitis, stop breastfeeding immediately and administer appropriate veterinary antibiotics for treatment (cephalosporins are recommended). Feed the young rabbits to other lactating mother rabbits with similar delivery times.

6. Ablactation

1. Ensure that most young rabbits are weaned at 35 days of age. Weak rabbits are weaned at 40 days of age.
   NOTE: The suitable temperature for young rabbits (3 weeks to 3 months old) is 20-25 °C. The suitable temperature for young rabbits (3-6 months old) and adult rabbits is also 15-25 °C.
2. Consider whether to vaccinate conventional vaccines according to the experimental requirements after weaning.

This article describes a pregnancy and feeding management procedure for embryo-transferred and genetically modified rabbits. At the end of 2022, the author's laboratory initiated the development of a preparation platform for gene-modified rabbits. During this period, several common issues were encountered, including abortion, failure to deliver at term, dystocia, failure to pluck hair for nest preparation before delivery, the unwillingness of mother rabbits to breastfeed or incidents of cannibalism, insufficient breast milk due to large litter sizes, and baby rabbits failing to drink milk. These challenges significantly reduced the breeding efficiency of gene-modified rabbits.

By summarizing experiences and learning from these issues, a pregnancy and nursing management protocol for embryo-transferred and genetically modified rabbits was developed (Figure 3). This protocol has substantially improved the survival rate of newborn rabbits and reduced mortality during lactation (Table 1). For most cases, except for specific genetically modified disease models with severe postnatal genetic disease phenotypes leading to higher mortality rates, this approach has proven effective and acceptable.

Currently, the author's laboratory has transitioned from traditional natural childbirth and nurturing methods to the newly established pregnancy and nursing management model. To date, this method has facilitated the successful breeding of more than ten new varieties of genetically modified rabbits, resulting in the production of numerous offspring. Although this approach increases the workload, it is highly effective. Figure 4 illustrates a real-life example of breeding MKRN3 gene-modified rabbit offspring, showcasing the process from birth to weaning.

Figure 1: Arrangement of baby rabbit incubators. Steps involved in preparing an incubator for baby rabbits: (1) Preparing the incubator; (2) Padding; (3) Spreading a towel; (4) Adding artificial down cotton; (5) Placing the baby rabbit inside; (6) Attaching the information card. Please click here to view a larger version of this figure.

Figure 2: Assisted breastfeeding for baby rabbits. (A) Feeding of 1-2-day-old baby rabbits. (B) Feeding of baby rabbits over 2 days old. (C) Comparison of rabbit pups' abdomens before and after feeding, showing noticeable bulging and visible white breast milk in the stomach (indicated by the red circle). (D) Excretion of meconium from the gastrointestinal tract within 3 days of birth (indicated by the red arrow). The perineum requires regular cleaning with a wet cotton ball (indicated by the yellow arrow). Please click here to view a larger version of this figure.

Figure 3: Pregnancy and nursing management process for embryo-transferred and genetically modified rabbits. Steps in the management process: Pregnancy diagnosis; Antenatal care; Midwifery; Postpartum care; Ablactation. Please click here to view a larger version of this figure.

Figure 4: Growth and development of genetically modified rabbits from birth to ablactation. (A) Images of baby rabbits from 1-8 days old. (B) Fifteen-day-old baby rabbits, with the mother rabbit on the left and the baby rabbits on the right. (C) Thirty-five-day-old weaned young rabbits. (D) Growth curve of baby rabbits within the first three weeks after birth. Baby rabbits were from the same litter: ID1, ID2, and ID3 represent mkrn3 gene-modified rabbits, while ID4, ID5, and ID6 represent wild-type rabbits. Please click here to view a larger version of this figure.

Table 1: Survival of rabbit pups under different breeding management modes. A comparison of rabbit pup survival rates across various breeding management strategies.

The main steps in the pregnancy and nursing management of embryo-transferred and genetically modified rabbits include midwifery and early assisted breastfeeding. Difficult childbirth is a common issue in rabbits¹⁹. Prolonged delivery times often result in fetal ischemia, hypoxia, and death²⁵. The appropriate use of oxytocin can effectively shorten delivery times and reduce instances of fetal dystocia²⁶. Many primiparous mother rabbits fail to pluck hair to prepare nests, increasing the risk of temperature loss and mortality in newborn rabbits^27,28. Typically, after an intramuscular injection of oxytocin, rabbits begin delivery within 5-10 min, which facilitates artificial delivery and improves the survival rate of newborns. Poor maternal behavior or inexperience often hinders the proper growth of rabbit pups. Assisted nursing management ensures that newborn rabbits receive adequate breastfeeding and are housed in comfortable incubators, significantly improving their survival²⁹.

In this study, a simple incubator was utilized, employing artificial down cotton and towels for warmth. The optimal environmental temperature for newborn rabbits is 30-32 °C³⁰. If available, temperature and humidity control equipment can further enhance outcomes. In the late stages of pregnancy, female rabbits may exhibit symptoms such as accelerated breathing, respiratory rales, ear vasodilation, and reluctance to eat or move³¹. These symptoms resemble human pregnancy-induced hypertension and require immediate attention. In severe cases, maternal and fetal death may occur. Under such circumstances, pregnant rabbits should be housed in more spacious cages with paper scraps to encourage nest-making, promoted to exercise, and provided with fresh green feed. These measures generally alleviate adverse symptoms in pregnant rabbits. However, in critical cases, an intramuscular injection of 0.02 mg of cloprostenol is necessary to induce early pregnancy termination³².

This study establishes a pregnancy and nursing management protocol for embryo-transferred and genetically modified rabbits, significantly improving the breeding efficiency of genetically modified rabbits. Compared to traditional rabbit farm breeding methods, this approach requires more manpower and material resources and involves a more meticulous process. However, for standard laboratory settings, this method meets experimental needs and offers practical insights and skills.

When breeding rabbit models for specific genetic diseases, such as muscular atrophy rabbits or hairless rabbits, the weak congenital constitution of these models often results in their inability to compete with wild-type rabbits in the same litter for breast milk, leading to high mortality rates shortly after birth^33,34. Assisted nursing management effectively mitigates these challenges, preventing the premature death of rabbit pups. Additionally, this protocol allows researchers to observe and determine the genotype of newborn rabbits promptly after birth, reducing concerns about female rabbits biting or eating their offspring. Overall, this protocol has promising applications in the generation of embryo-transferred and genetically modified rabbits.

The authors have nothing to disclose.

This research was supported by the National Natural Science Foundation of China (Grant No. 82101937), the Guangdong Medical Science and Technology Research Fund Project, China (Grant No. B2024069), and the Guangzhou Science and Technology Plan Project, China (Grant No. SL2023A04J02229, Assignment No. 2024A04J4923).