Here, we report that both nano-silica (SiO ₂ -NPs) and reserpine can elicit depression-like behavior in adult zebrafish in a novel tank test.

As an excellent model organism, zebrafish have been widely applied in many fields. The accurate identification and tracking of individuals are crucial for zebrafish shoaling behaviour analysis. However, multi-zebrafish tracking still faces many challenges. It is difficult to keep identified for a long time due to fish overlapping caused by the crossings. Here we proposed an improved Histogram of Oriented Gradient (HOG) algorithm to calculate the stable back texture feature map of zebrafish, then tracked multi-zebrafish in a fully automated fashion with low sample size, high tracking accuracy and wide applicability. The performance of the tracking algorithm was evaluated in 11 videos with different numbers and different sizes of zebrafish. In the Right-tailed hypothesis test of Wilcoxon, our method performed better than idTracker, with significant higher tracking accuracy. Throughout the video of 16 zebrafish, the training sample of each fish had only 200–500 image samples, one-fifth of the idTracker’s sample size. Furthermore, we applied the tracking algorithm to analyse the depression and hypoactivity behaviour of zebrafish shoaling. We achieved correct identification of depressed zebrafish among the fish shoal based on the accurate tracking results that could not be identified by a human.

Somatic cell nuclear transfer (SCNT), which is an important procedure in cloning, has been conducted manually for decades. The operating efficiency drops sharply in batch SCNT because of the long-time observation under microscopy and the time-wasting traditional process. Though the operating time was reduced by robotic SCNT in previous studies, the traditional operating process was still used. In this article, we designed a new robotic batch SCNT process based on a microfluidic groove and two micropipettes in parallel. By using this new SCNT process, the operating area switching, objective lens conversing, and focusing on traditional SCNT process were eliminated, and oocyte localization was simplified, which saved much operating time. Experimental results showed that the new robotic batch process reduced about 50 s (41.7%) compared with the manual process (proposed 70 s versus manual 120 s). A success rate of 93.3% (n = 30) and a survival rate of 96.4% were achieved (n=28), which were similar to manual process. The new robotic batch SCNT method demonstrated a high degree of efficiency and reproducibility.

As there are significant variations of cell elasticity among individual cells, measuring the elasticity of batch cells is required for obtaining statistical results of cell elasticity. At present, the micropipette aspiration (MA) technique is the most widely used cell elasticity measurement method. Due to a lack of effective cell storage and delivery methods, the existing manual and robotic MA methods are only capable of measuring a single cell at a time, making the MA of batch cells low efficiency. To address this problem, we developed a robotic MA system capable of storing multiple cells with a feeder micropipette (FM), picking up cells one-by-one to measure their elasticity with a measurement micropipette (MM). This system involved the following key techniques: Maximum permissible tilt angle of MM and FM determination, automated cell adhesion detection and cell adhesion break, and automated cell aspiration. The experimental results demonstrated that our system was able to continuously measure more than 20 cells with a manipulation speed quadrupled in comparison to existing methods. With the batch cell measurement ability, cell elasticity of pig ovum cultured in different environmental conditions was measured to find optimized culturing protocols for oocyte maturation.

Cell rotation has great significance for cell manipulation, which is applied to intracytoplasmic sperm injection, preimplantation genetic screening and diagnosis, somatic cell nuclear transfer, etc. In this letter, an automatic cell rotation method is proposed based on real-time detection and tracking of the polar body. Image segmentation and visual tracking are combined to detect the polar body accurately and robustly during cell rotation. Both out-of-plane and in-plane rotation are realized by utilizing a pair of standard glass micropipettes. In experiments, two kinds of oocytes, which are quite different in appearance, are used to validate the system. For mouse oocytes, the robotic system achieved a 98% success rate of out-of-plane rotation and a 93% success rate of in-plane rotation. For porcine oocytes, the robotic system achieved an 80% success rate of out-of-plane rotation and a 93% success rate of in-plane rotation. The average time of cell rotation is approximately 9.1s for mouse oocytes and 12.8s for porcine oocytes. The experiment results demonstrate that the proposed method is effective, stable and high speed for automatic cell rotation in 3D.

Objective: The invisibility of domestic oocyte nucleus in bright field currently forces operators to blindly aspirate nucleus out in oocyte enucleation, usually causing large cytoplasm losses and poor developmental competences of cloned embryos. Although fluorescent labeling of nucleus allows for nucleus localization, the involved photobleaching problems and barriers to the execution of enucleation process limit its online-application in oocyte enucleation. This paper reports a novel label-free oocyte enucleation method for precise removal of the nucleus with less cytoplasm loss. Methods: The relative positions between the injection pipette and nucleus for complete removal of nucleus with less cytoplasm loss were determined through a finite element modeling of nucleus aspiration. To position injection pipette to the above positions relative to nucleus, the appropriate oocyte orientation and trajectory of injection pipette inside oocyte were derived according to the offline-calibrated 3-D distribution of nucleus and the simulated dynamic drift of nucleus that occurs as injection pipette is maneuvered inside oocyte. Finally, a robotic label-free precise enucleation procedure was established. Results: The experimental results on more than 1000 porcine oocytes proved that this system is capable of reducing cytoplasm loss by 60% at the same level of enucleation success rate and almost doubling the cleavage rate of clone embryos in comparison to blind aspiration method. Conclusions: The results prove that our method significantly improves the developmental competence of cloned embryos in comparison to manual enucleation method. Significance: Our method is expected to improve the extremely low success rate of animal cloning in the future.