# Robotic Precisely Oocyte Blind Enucleation Method
## 1 Introduction  
Cloning refers to the process in which cells develop into new organisms through asexual reproduction [1; 2]. In 1997, British scientist [PERSON] et al. used sheep mammary cells as donor cells for somatic cell nuclear transfer (SCNT) and obtained the world's first somatic cloned sheep, [PERSON]. Since the birth of [PERSON], many other species, such as cloned mice, cloned dogs, cloned cows, cloned rabbits, cloned horses, etc., have also been born through nuclear transplantation technology [3; 4; 5; 6; 7; 8]. The standard nuclear transplantation procedure is divided into two parts: the enucleation of oocytes and the injection of donor cells. Among them, the enucleation operation needs to tear apart the genetic material and cytoplasm in oocytes, which is the most difficult step in the operation of somatic cell nuclear transfer (SCNT). Yet, the main steps of oocyte enucleation are still manually operated [9], which presents several drawbacks, for example, low precision, high repetition error, and long training time for operators [10]. Therefore, it is necessary to automate the operation process of oocyte enucleation [11; 12; 13], which will improve the operation precision of oocyte enucleation, improve the success rate of oocyte enucleation, and further promote micro-manipulation technology and gene technology [14]. Recently, several studies have been carried out on the automation of oocyte enucleation. However, most of them automate the process by improving the hardware of the system, which will lead to higher cost and device incompatibility issues [15; 16; 17]. For example, [PERSON] et al. designed and fabricated some new operating tools to fulfill the automatic enucleation process, including a magnetically driven micro-gripper for holding the oocyte and a magnetically driven micro-knife for rotating the oocyte and cutting the oocyte into half, then the part without genetic material was used as a somatic cell receptor [17; 18; 19; 20; 21; 22]. However, this method removes too much cytoplasm, which will have an important impact on the developmental potential of oocytes [23; 24]. Therefore, although this method issimple and easy to operate, it has not been widely used at present. The blind enucleation method is most widely used due to its advantages of low cost and relatively low amount of enucleation. It is necessary to design a robotic blind enucleation method, that is, only an enucleation micropipette (EM) and holding micropipette (HM) are used in the whole enucleation process. In this paper, we developed a robotic precise oocyte blind enucleation method to precisely control the removed cytoplasm during enucleation and reduce whole operation time. Firstly, we designed the process of robotic enucleation. Secondly, we introduced the key techniques to solve the problems in the enucleation process, including the oocyte and micropipette detection method, oocyte translation control method, oocyte immobilization and penetration method, and enucleation volume control method. An adaptive slide mode controller (ASMC) was designed to generate enucleation accurately and the controller simulation was conducted to verify the controller's performance. Thirdly, we conducted the experiments of trajectory tracking for extracting cytoplasm. The control results show that the designed controller is able to track the trajectory and extract the specified cytoplasmic volume. Moreover, the proposed robotic enucleation method estimated the overshoot compared with the other control methods. Finally, we conducted the robotic enucleation experiments with porcine oocytes. The experimental results demonstrated that this robotic enucleation method is capable of enucleation with less time-cost, higher enucleation accuracy, higher enucleation success rate, and higher cleavage rate compared with the manual method.