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).

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&#39;s laboratory. Details on the reagents and the equipment used are listed in the Table of Materials.
1. Pregnancy diagnosis
<ol>
	<li>On the 12th to 14th 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.
	<ol>
		<li>Hold the neck skin behind the rabbit&#39;s ears with one hand, let the rabbit&#39;s buttocks shrink forward, close to the abdominal cavity of the rabbit&#39;s buttocks, and then use the other hand to open the palm and gently palpate the back end of the abdomen.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>If conditions permit, use ultrasound on the 15th 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.</li>
	<li>During the period of the 16th-18th 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 16th-18th days due to the decrease of luteal hormone.</li>
</ol>
2. Antenatal care
<ol>
	<li>Allow pregnant rabbits to eat freely during pregnancy and add fresh green feed once a week.</li>
	<li>On the morning or evening of the 25th day after embryo transfer, transfer the pregnant rabbits to the delivery cage.
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>Provide adequate feed and drinking water.</li>
		<li>Gently move the pregnant rabbits to the delivery cage.</li>
		<li>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 28th 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.</li>
	</ol>
	</li>
</ol>
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 0.5th 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 0.5th day of pregnancy.
<ol>
	<li>Prenatal monitoring 
	NOTE: The gestation period of rabbits is 28-32 days. If a rabbit gives birth before the 30th 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 30th day.
	<ol>
		<li>On the morning of the 30th day of pregnancy in rabbits, perform abdominal palpation to roughly determine the number, size, and activity of the fetus.
		<ol>
			<li>Hold the neck skin behind the rabbit&#39;s ears with one hand, let the rabbit&#39;s buttocks shrink forward, close to the abdominal cavity of the rabbit&#39;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.</li>
		</ol>
		</li>
		<li>If it is found that the number of fetuses conceived by pregnant rabbits is only 1-2, on the 30th day of pregnancy, inject 0.02 mg of cloprostenol intramuscularly at 5 p.m. to induce the rabbits&#39; delivery the next day. 
		NOTE: Pregnant rabbits have only 1-2 fetuses, which are prone to dystocia or delivery failure when mature.</li>
	</ol>
	</li>
	<li>Hasten parturition 
	NOTE: If the pregnant rabbit does not give birth on the morning of the 31st day of pregnancy, it can consider whether it needs to hasten parturition according to the following conditions.&#160;If there are more than 2 fetuses, no further action will be taken.
	<ol>
		<li>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 31st day of pregnancy, inject 10 units of oxytocin into the muscles to induce labor.&#160;</li>
		<li>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.</li>
		<li>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 32nd 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.</li>
	</ol>
	</li>
</ol>
4. Midwifery
<ol>
	<li>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 &#215; 30 cm &#215; 15 cm in length &#215; width &#215; height.
	<ol>
		<li>Add sterile absorbent padding. Corncob granules are recommended.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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 &#176;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 &#176;C. Female rabbits usually have 6-8 nipples. If there are too many babies, some babies can be fed by other mother rabbits.</li>
</ol>
5. Postpartum care
<ol>
	<li>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.
	<ol>
		<li>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.</li>
		<li>Gently massage each nipple and gently squeeze out the first drop of milk.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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).</li>
		<li>As the hair of the baby rabbit grows, remove some down cotton from the incubator every day.</li>
		<li>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 8th day after birth.</li>
		<li>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.</li>
		<li>After the young rabbit opens its eyes (about 2 weeks old), move it to a nursing cage with a towel.&#160;Add some green feed and daily diet. 
		NOTE: The suitable temperature for baby rabbits (2-3 weeks old) is 23-30 &#176;C.</li>
		<li>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&#39;s nipple.</li>
	</ol>
	</li>
	<li>Postpartum care of the mother rabbit
	<ol>
		<li>During lactation, allow mother rabbits to drink and eat freely and feed them with appropriate green feed to ensure adequate nutrition.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
6. Ablactation
<ol>
	<li>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 &#176;C. The suitable temperature for young rabbits (3-6 months old) and adult rabbits is also 15-25 &#176;C.</li>
	<li>Consider whether to vaccinate conventional vaccines according to the experimental requirements after weaning.</li>
</ol>

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The authors have nothing to disclose.

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 h...

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 models1,2. 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 growth3,4. 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 therapy7,8,9, stem cell therapy10,11,12,13, brain science research14, and other scientific fields1,15,16,17,18, 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&#39;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.

<table><tbody><tr><td>Artificial down cotton</td><td>Yangzhou Anguang Textile Co., Ltd, China</td><td>5490-Jinyu</td><td></td></tr><tr><td>Cloprostenol Sodium Injection</td><td>Shanghai Quanyu Biotechnology (Zhumadian) Animal Pharmaceutical Co., Ltd, China</td><td>163232207</td><td></td></tr><tr><td>Corncob granules</td><td>Guangdong Provincial Medical Laboratory Animal Center, China</td><td>YUMIXIN</td><td></td></tr><tr><td>Electronic platform scale</td><td>Yongkang Runjin weighing instrument Co., Ltd, China</td><td>rj-09</td><td></td></tr><tr><td>Gentamicin</td><td>Shanxi Jinfukang Biological Pharmaceutical Co., Ltd, China</td><td>041531504</td><td></td></tr><tr><td>Glucose injection</td><td>Henan Kelun Pharmaceutical Co., Ltd, China</td><td>H41022251</td><td></td></tr><tr><td>Incubator</td><td>Foshan Chancheng Hualong Plastic Factory, China</td><td>AAA-2</td><td>For newborn rabbits</td></tr><tr><td>Incubator box&amp;nbsp;</td><td>Hebei mabao wire mesh products Co., Ltd., China</td><td>https://qr.1688.com/s/AJ1K7O3p</td><td>As a nest for the mother rabbits</td></tr><tr><td>Inslin Syringe</td><td>Becton,Dickinson and Company, USA</td><td>Ultra-Fine,328421</td><td></td></tr><tr><td>Oxytocin injection</td><td>Guangzhou Baiyunshan Mingxing Pharmaceutical Co., Ltd</td><td>H44025245</td><td></td></tr><tr><td>Penicillin injection</td><td>Guangdong Kangtaiyuan Animal Husbandry Co., Ltd, China</td><td>300012430</td><td></td></tr><tr><td>Rabbit delivery cage</td><td>Suzhou Suhang Technology Equipment Co., Ltd., Suzhou, China</td><td>RB42-8G</td><td></td></tr><tr><td>Towel</td><td>Zhejiang Jieliya Co., Ltd, China</td><td>W3290</td><td></td></tr></tbody></table>

pregnancy and nursing management for embryo-transferred and genetically modified 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.

This article describes a pregnancy and feeding management procedure for embryo-transferred and genetically modified rabbits. At the end of 2022, the author&#39;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 brea...

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.

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

With the advancement of scientific research, the demand for gene-edited rabbit models is increasing. However, there are limited pregnancy and feeding ...

pregnancy-nursing-management-for-embryo-transferred-genetically

Research

JoVE Journal

Biology

179 Views.  Guangzhou Medical University. 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.

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

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Medicine

Production of Apolipoprotein C-III Knockout Rabbits using Zinc Finger Nucleases

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins (HDL). It has been recognized that high levels of plasma ApoCIII constitutea risk factor for cardiovascular diseases (CVD). Elevated plasma ApoCIII level often correlates with insulin resistance, obesity, and hypertriglyceridemia. Invaluable knowledge on the roles of ApoCIIIin lipid metabolisms and CVD has been obtained from transgenic mouse models including ApoCIII knockout (KO) mice; however, it is noted that the metabolism of lipoprotein in mice is different from that of humans in many aspects. It is not known until now whether elevated plasma ApoCIII is directly atherogenic. We worked to develop ApoCIII KO rabbits in the present study based on the hypothesis that rabbits can serve as a reasonablemodelfor studying human lipid metabolism and atherosclerosis. Zinc finger nuclease (ZFN) sets targeting rabbit ApoCIIIgene were subjected to in vitro validation prior to embryo microinjection. The mRNA was injected to the cytoplasm of 35 rabbit pronuclear stage embryos, and evaluated the mutation rates at the blastocyst state. Of sixteen blastocysts that were assayed, a satisfactory 50% mutation rate (8/16) at the targeting site was achieved, supporting the use of Set 1 for in vivo experiments. Next, we microinjected 145 embryos with Set 1 mRNA, and transferred these embryos to 7 recipient rabbits. After 30 days gestation, 21 kits were born, out of which five were confirmed as ApoCIII KO rabbits after PCR sequencing assays. The KO animal rate (#KO kits/total born) was 23.8%. The overall production efficiency is 3.4% (5 kits/145 embryos transferred). The present work demonstrated that ZFN is a highly efficient method to produce KO rabbits. These ApoCIII KO rabbits are novel resources to study the roles of ApoCIII in lipid metabolisms.

Recent development in gene targeting tools makes production of knockout (KO) rabbits possible. In the present work, we generated five Apolipoprotein (Apo) C-III KO rabbits using Zinc Finger Nucleases (ZFN). This work demonstrated that ZFN is a highly efficient method to produce KO rabbits.

Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins ...

Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal and postnatal consequences. To ensure the innocuousness of ART applications, studies in animal models are necessary. In addition, as a last step, embryo development studies require evaluation of their capacity to develop full-term healthy offspring. Here, embryo transfer to the uterus is indispensable to perform any ARTs-related experiment.
The rabbit has been used as a model organism to study mammalian reproduction for over a century. In addition to its phylogenetic proximity to the human species and its small size and low maintenance cost, it has important reproductive characteristics such as induced ovulation, a chronology of early embryonic development similar to humans and a short gestation that allow us to study the consequences of ART application easily. Moreover, ARTs (such as intracytoplasmic sperm injection, embryo culture, or cryopreservation) are applied with suitable efficiency in this species.
Using the laparoscopic embryo transfer technique and the cryopreservation protocol presented in this article, we describe 1) how to transfer embryos through an easy, minimally invasive technique and 2) an effective protocol for long-term storage of rabbit embryos to provide time-flexible logistical capacities and the ability to transport the sample. The outcomes obtained after transferring rabbit embryos at different developmental stages indicate that morula is the ideal stage for rabbit embryo recovery and transfer. Thus, an oviductal embryo transfer is required, justifying the surgical procedure. Furthermore, rabbit morulae are successfully vitrified and laparoscopically transferred, proving the effectiveness of the described techniques.

Assisted reproductive techniques (ARTs) are in continuous evaluation to improve outcomes and reduce the associated risks. This manuscript describes a minimally invasive embryo transfer procedure with an efficient cryopreservation protocol that allows the use of rabbits as an ideal animal model of human reproduction.

Assisted reproductive techniques (ARTs), such as in vitro embryo culture or embryo cryopreservation, affect natural development patterns with perinatal ...

Developmental Biology

Intraductal Delivery to the Rabbit Mammary Gland

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and adverse effects1,2,3,4,5,6,7. However, several challenges remain before these treatments can be applied more widely. The development and validation of intraductal therapeutics in an appropriate animal model facilitate the development of intraductal therapeutic strategies for patients. While the mouse mammary gland has been widely used as a model system of mammary development and tumorigenesis, the anatomy is distinct from the human gland. A larger animal model, such as the rabbit, may serve as a better model for mammary gland structure and intraductal therapeutic development. In contrast to mice, in which ten ductal trees are spatially distributed along the body axis, each terminating in a separate teat, the rabbit mammary gland more closely resembles the human gland, with multiple overlapping ductal systems that exit through separate openings in one teat. Here, we present minimally invasive methods for the delivery of reagents directly into the rabbit mammary duct and for visualization of the delivery itself with high-resolution ultrasound imaging.

Here, we describe a technique for the localized delivery of reagents to the rabbit mammary gland via an intraductal injection. In addition, we describe a protocol for visualization and the confirmation of delivery by high-resolution ultrasound imaging of contrast agents.

Localized intraductal treatments for breast cancer offer potential advantages, including efficient delivery to the tumor and reduced systemic toxicity and ...

Bioengineering

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits (Oryctolagus cuniculus)

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of this research model are the ease of breeding and sample collection, relatively short gestation period, and large litter sizes. Commonly assessed routes of immunization include subcutaneous, intramuscular, intranasal, and intradermal. Nonterminal sample collection for the chronological detection of the immunologic responses to these immunizations include the collection of blood, from both dams and kits, and milk from the lactating does. In this article, we will demonstrate techniques our lab has utilized in studies of maternal immunization in New Zealand White rabbits (Oryctolagus cuniculus), including intranasal immunization and milk collection.

Intranasal Immunization and Milk Collection in Studies of Maternal Immunization in New Zealand White Rabbits Oryctolagus cuniculus

This article describes and demonstrates the administration of intranasal vaccines and the collection of milk from lactating rabbits (Oryctolagus cuniculus) as a means to assess mucosal immunity in a translationally appropriate model of maternal immunization.

Due to similarities in placentation and antibody transfer with humans, rabbits are an excellent model of maternal immunization. Additional advantages of ...

Immunology and Infection

Simplifying Transcutaneous Intratracheal Drug Delivery in the Newborn Preterm Rabbit

Intratracheal (IT) drug delivery allows the direct delivery of pharmaceutical substances to the lung, maximizing potential pulmonary benefit and minimizing systemic drug exposure. The transcutaneous technique is simple and allows for the IT delivery of substances to the lung of prematurely born rabbits shortly after birth. Newborn pups are anesthetized with inhaled Isoflurane before being placed in a supine position with the neck extended. The larynx is identified and stabilized before transcutaneous placement of a 26-gauge (G) catheter into the trachea. Following catheterization of the trachea, a 30 G blunt needle attached to a Hamilton syringe is introduced into the IT catheter and is used for delivering a precise volume into the trachea during spontaneous respiration. After the IT injection is completed, the needle and catheter are withdrawn, and the pup is allowed to recover from anesthesia. Transcutaneous IT injection delivers a large proportion of the injected substance to the lung, with the majority remaining in the lung 3 hours after the intervention. The injections are well tolerated from the day of birth and can be repeated for multiple consecutive days without influencing survival. This technique can be used to investigate the effect of pharmaceutical agents on lung development and in the prevention of neonatal lung injury in preterm rabbits.

Transcutaneous intratracheal injection allows for effective intrapulmonary drug delivery during spontaneous respiration. Single and multiple injections are well tolerated with no effect on survival. The technique is simple to perform and can examine the effect of substances on lung development and the prevention of lung injury in newborn rabbits.

Intratracheal (IT) drug delivery allows the direct delivery of pharmaceutical substances to the lung, maximizing potential pulmonary benefit and ...

Implantation of Electroencephalogram and Electrocardiogram Telemetry Devices in Neonatal Rabbit Kits

Pathogenic variants in ion channel genes are associated with a high rate of sudden unexpected death in epilepsy (SUDEP). Mechanisms of SUDEP are poorly understood but may involve autonomic dysfunction and cardiac arrhythmias in addition to seizures. Some ion-channel genes are expressed in both the brain and the heart, potentially increasing the risk of SUDEP in patients with ion-channelopathies associated with epilepsy and cardiac arrhythmias. Transgenic rabbits expressing epilepsy variants provide a whole organism to study the complex physiology of SUDEP. Importantly, rabbits more closely replicate human cardiac physiology than do mouse models. However, rabbit models have additional health and anesthesia considerations when undergoing invasive monitoring procedures. We have developed a novel method to surgically implant a telemetry device for long-term simultaneous electroencephalogram (EEG) and electrocardiogram (ECG) monitoring in neonatal rabbit kits. Here, we demonstrate surgical methods to implant a telemetry device in P14 (weight range 175-250 g) kits with detailed attention to surgical approach, appropriate anesthesia and monitoring, and postoperative care, resulting in a low complication rate. This method allows for continuous monitoring of neural and cardiac electrophysiology during critical points in the development of cardiac arrhythmias, seizures, and potential SUDEP in rabbit models of genetic or acquired epilepsies.

Mechanisms of sudden unexpected death in epilepsy (SUDEP) are poorly understood and challenging to translate from current models. Transgenic rabbits may offer insights into these mechanisms. We describe a method for long-term, continuous electroencephalography and electrocardiography recordings in transgenic rabbit kits to evaluate serious events that may lead to death.

Pathogenic variants in ion channel genes are associated with a high rate of sudden unexpected death in epilepsy (SUDEP). Mechanisms of SUDEP are poorly ...

Modeling Metastatic Endometrial Cancer  Model: A Technique to Generate Endometrial Cancer with Lymph Node Metastasis in Rabbit Models

Source: Lauren Philp et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/t/59340">An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells.</a> J. Vis. Exp. (2019)
This video demonstrates the generation of a rabbit model of endometrial cancer by injecting a highly metastatic VX2 cell suspension into the sutured uterine horns of the rabbit. This leads to generation of primary endometrial cancer with retroperitoneal lymph node metastases.

Source: Lauren Philp et al. An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 ...

JoVE Encyclopedia of Experiments

Cancer Research

Gynecologic Cancer

Animal model studies

Embryo Transfer Into Rabbit: A Procedure for Laparoscopy-Guided Transfer of Embryos Into Oviduct of Ovulating Recipient Female Rabbit Model

Source: Garcia-Dominguez, X. et al., <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/58055">Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model.</a> J. Vis. Exp. (2019)
This video describes the technique of transferring embryos into the oviduct of a recipient female rabbit via Laparoscopy. It allows embryo remodeling in rabbits during its migration through the oviduct, eventually helping in successful implantation. This technique can also be used for stored embryos with good efficiency.

Source: Garcia-Dominguez, X. et al., Minimally Invasive Embryo Transfer and Embryo Vitrification at the Optimal Embryo Stage in Rabbit Model. J. Vis. Exp. ...