Melatonin treatment can improve quality and in vitro development of porcine oocytes, but the mechanism of improving quality and developmental competence is not fully understood. In this study, porcine cumulus–oocyte complexes were cultured in TCM199 medium with non-treated (control), 10⁻⁵ M luzindole (melatonin receptor antagonist), 10⁻⁵ M melatonin, and melatonin + luzindole during in vitro maturation, and parthenogenetically activated (PA) embryos were treated with nothing (control), or 10⁻⁵ M melatonin. Cumulus oophorus expansion, oocyte survival rate, first polar body extrusion rate, mitochondrial distribution, and intracellular levels of reactive oxygen species (ROS) and glutathione of oocytes, and cleavage rate and blastocyst rate of the PA embryos were assessed. In addition, expression of growth differentiation factor 9 (GDF9), tumor protein p53 (P53), BCL2 associated X protein (BAX), catalase (CAT), and bone morphogenetic protein 15 (BMP15) were analyzed by real-time quantitative PCR. The results revealed that melatonin treatment not only improved the first polar body extrusion rate and cumulus expansion of oocytes via melatonin receptors, but also enhanced the rates of cleavage and blastocyst formation of PA embryos. Additionally, melatonin treatment significantly increased intraooplasmic level of glutathione independently of melatonin receptors. Furthermore, melatonin supplementation not only significantly enhanced mitochondrial distribution and relative abundances of BMP15 and CAT mRNA, but also decreased intracellular level of ROS and relative abundances of P53 and BAX mRNA of the oocytes. In conclusion, melatonin enhanced the quality and in vitro development of porcine oocytes, which may be related to antioxidant and anti-apoptotic mechanisms.

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.

The MPF and MAPK genes play crucial roles during oocyte maturation processes. However, the pattern of MPF and MAPK gene expression induced by melatonin (MT) and its correlation to oocyte maturation quality during the process of porcine oocyte maturation in vitro remains unexplored. To unravel it, in this study, we cultured the porcine oocytes in maturation medium supplemented with 0, 10⁻⁶, 10⁻⁹, and 10⁻¹² mol/L melatonin. Later, we analyzed the MPF and MAPK gene expression levels by RT-PCR and determined the maturation index (survival and maturation rate of oocytes). The GSH content in the single oocyte, and cytoplasmic mitochondrial maturation distribution after porcine oocyte maturation in vitro was also evaluated. We also assessed the effects of these changes on parthenogenetic embryonic developmental potential. The oocytes cultured with 10⁻⁹mol/L melatonin concentration showed higher oocyte maturation rate, and MPF and MAPK genes expression levels along with better mitochondrial distribution than the 0, 10⁻⁶, and 10⁻¹² mol/L melatonin concentrations (p < 0.05). No significant difference was observed in the survival rates when the oocytes were cultured with different melatonin concentrations. The expression of the MPF gene in the oocytes cultured with 10⁻⁶ mol/L melatonin was higher than with 10⁻¹² and 0 mol/L melatonin, and the expression of the MAPK gene in 10⁻⁶ and 10⁻¹² group was higher than the control (p < 0.05). As far as the embryonic developmental potential is concerned, the cleavage and blastocyst rate of oocytes cultured with 10⁻⁶ and 10⁻⁹ mol/L melatonin was significantly higher than the 10⁻¹² mol/L melatonin and control. In conclusion, 10⁻⁹–10⁻⁶ mol/L melatonin significantly induced the MPF and MAPK gene expression; besides, it could also be correlated with GSH content of single oocyte, mitochondrial maturation distribution, and the first polar body expulsion. These changes were also found to be associated with parthenogenetic embryo developmental potential in vitro.

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.

Melatonin enhances the quality and in vitro maturation (IVM) of oocytes under heat stress (HS), but the mechanism of melatonin in reducing HS injury on oocytes is not fully understood. In this study, porcine cumulus-oocyte complexes (COCs) were randomly divided into three groups. The COCs of the control group were cultured at 38.5 °C for 42 h, and the COCs of the HS group were cultured at 41.5 °C for 4 h, and then transferred into 38.5 °C for 38 h. The COCs of the HS + melatonin group were cultured with 10⁻⁹ M melatonin under the same conditions as the HS group. The survival rate, maturation rate, distribution of α-tubulin and F-actin of the oocytes were assessed. In addition, the expression profiles for genes related to the oocyte maturation, including heat shock protein 70 (HSP70), nuclear factor erythroid 2-related factor 2 (NRF2), cyclin-dependent kinase 1 (CDK1), growth differentiation factor 9 (GDF9) were analyzed by real-time quantitative PCR. The results showed that HS decreased the survival rate and maturation rate, distribution of α-tubulin and F-actin, but melatonin treatment could partly counteract these adverse effects. In addition, HS increased expression of HSP70 and NRF2 mRNA, and melatonin treatment had a similar effect on HSP70 expression, but had a contrary effect on NRF2 expression. Furthermore, HS inhibited expression of CDK1 and GDF9 mRNA, but melatonin treatment could weaken the effect on GDF9 expression induced by HS. In summary, melatonin treatment could attenuate the unfavorable effects induced by HS to enhance developmental competence of porcine oocytes during IVM.

Cell-laden hydrogel fibers are widely used as the fundamental building blocks to fabricate more complex functional three-dimensional (3D) structures that could mimic biological tissues. The control on the diameter of the hydrogel fibers is important so as to precisely construct structures in the above 3D bio-fabrication. In this paper, a pneumatic-actuated micro-extrusion system is developed to produce hydrogel fibers based on the crosslinking behavior of sodium alginate with calcium ions. Excellent uniformity has been obtained in the diameters of the fabricated hydrogel fibers as a proportional-integral-derivative (PID) control algorithm is applied on the driving pressure control. More importantly, a linear relationship has been obtained between the diameter of hydrogel fiber and the driving pressure. With the help of the identified linear model, we can precisely control the diameter of the hydrogel fiber via the control of the driving pressure. The differences between the measured and designed diameters are within ±2.5%. Finally, the influence of the calcium ions on the viability of the encapsulated cells is also investigated by immersing the cell-laden hydrogel fibers into the CaCl₂ bath for different periods of time. LIVE/DEAD assays show that there is little difference among the cell viabilities in each sample. Therefore, the calcium ions utilized in the fabrication process have no impact on the cells encapsulated in the hydrogel fiber. Experimental results also show that the cell viability is 83 ± 2% for each sample after 24 h of culturing.