Domestic cats serve as laboratory models for human medical research, such as traumatic spinal cord injury [1] and polycystic kidney disease [2]. Assisted reproductive technology (ART) in these animals has made progress, including in terms of artificial insemination, in vitro fertilization, and somatic cell nuclear transfer [3]. By combining these techniques with embryo transfer (ET), offspring could be delivered successfully. However, success is limited by the poor embryo culture system. Successful in vitro production of feline embryos until the morula or blastocyst stage is the key step for successful ET. Several studies have shown that KSOM, PZM [4], or HTM [5] could support the development of feline embryos to the blastocyst stage, but they did not give the best results. In most studies, embryos were transferred into the oviduct or uterus horn by surgery. However, surgical ET not only limits the application of cat ART in veterinary practice, but also increases risk for wild felines during ET [6] because of the immaturity of anesthesia technology. Furthermore, due to animal ethics, surgical ET is not commonly acceptable in commercial pet cloning.

The unique structure of the cat reproductive tract causes difficulties during insertion of the ET catheter through the cervix and into the uterus; the cervix of the female cat cannot be directly visualized due to a narrow and nondistensible cranial vagina. It has been reported that the cranial vagina is < 2 mm in diameter in most queens during all stages of the estrous cycle. Moreover, the thickening of the dorsal medial fold during or close to the follicular phases contributes to the reduction of the width of the cranial vagina [7]. Therefore, development of a suitable catheter has been proposed. Hurlbut et al. developed a 2 mm glass speculum [8], and a modified polypropylene urinary catheter (2.7–2.8 mm) had also been used as a speculum [9] to aid the insertion of a 3-Fr tomcat catheter through the cervix. However, these devices are not suitable for some queens with a narrower cranial vagina lumen, especially in virgin cats. Zambelli et al. developed a new method for endoscopically guided cervical catheterization and a new specially designed transcervical catheter [7]. The new device is a modification of a 3-Fr 11 cm-long tomcat urinary catheter with a 100 mm stainless-steel rounded tip needle (0.65 mm in diameter) inserted into its open end. Using these methods, the same team performed transcervical insemination in queens and received first deliveries in 2015 [10]. However, these methods require strict training and expensive devices, limiting their application in practice. To date, offspring delivery after transcervical ET has not been reported.

The CRX gene is expressed to produce a protein called the cone-rod homeobox protein that is found in the eyes, specifically in the retina [11]. CRX controls the expression of rod and cone photoreceptor genes through its interaction with transcription factors and/or a network of regulatory proteins [12]. Deletion of the CRX gene causes the abnormal development of light detecting cells [13].

This study aimed to develop a simple method for nonsurgical ET in queens for breeding and commercial pet cloning, especially for large felid reproduction. In the present study, using 3D printing technology, a simple device was optimized to allow the insertion of a tomcat 3-Fr catheter through the cervix. The gene-edited embryos were cultured in vitro and transferred into a surrogate mother by nonsurgical ET, and healthy offspring were delivered successfully.