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Microfluidics is proposed as a technique for efficient sperm sorting, to achieve the ultimate goal of resolving infertility problems in livestock industry. Our study aimed to design a microfluidic sperm-sorting device (SSD) through a high-efficacy and cost- and time-effective fabrication process, by using COMSOL Multiphysics simulation and modeling software, and the design of experiment (DOE) method. The eight factors affecting SSD performance were established. The simulation was then run, and statistically significant factors were analyzed. Minitab16 was used to optimize the design modulus factor. By setting the statistical significance at p < 0.05, the factors affecting experimental structure were analyzed. At a desirability of 97.99, the optimal parameters for the microfluidic chip were: angle between sperm and medium inlet chambers (A = 43°), sperm inlet flow rate (B = 0.24 µL min−1), medium inlet flow rate (C = 0.34 µL min−1), and inlet and outlet chamber lengths (D = 5000 µm). These optima were then applied to microfluidics device construction. The device was produced using soft lithographic microfabrication techniques and tested on Holstein–Friesian bull sperm. The highest bull sperm-sorting performance for this microfluidic device prototype was 96%. The error between the simulation and the actual microfluidic device was 2.72%. Fluid viscosity ranges analysis-based simulations revealed acceptable fluid viscosity tolerances for the SSD. The simulation results revealed that the acceptable tolerance range for fluid viscosity was 0.00001–0.003 kg m−1 s−1. This optimally designed microfluidic chip-based SSD may be integrated into sperm x/y separation micro devices.

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Gender selection is important in livestock industries; for example, female calves are required in the dairy industry. Sex-sorted semen is commonly used for the production of calves of the desired gender. However, assessment of the sex ratio of the sorted semen is tedious and expensive. In this study, a rapid, cost effective and reliable method for determining the sex ratio was developed using a multiplex real-time polymerase chain reaction (PCR) assay. In this assay, the X and Y chromosome-specific markers, i.e., bovine proteolipid protein (PLP) gene and sex-determining region Y (SRY) were simultaneously quantified in a single tube. The multiplex real-time PCR assay was shown to have high amplification efficiencies (97% to 99%) comparable to the separated-tube simplex real-time PCR assay. The results obtained from both assays were not significantly different (p>0.05). The multiplex assay was validated using reference DNA of known X ratio (10%, 50%, and 90%) as templates. The measured %X in semen samples were the same within 95% confidence intervals as the expected values, i.e., >90% in X-sorted semen, <10% in Y-sorted semen and close to 50% in the unsorted semen. The multiplex real-time PCR assay as shown in this study can thus be used to assess purity of sex-sorted semen.

This study proposes a microfluidic device used for X-/Y-sperm separation based on monoclonal antibody-conjugated magnetic beads, which become positively charged in the flow system. Y-sperms were selectively captured via a monoclonal antibody and transferred onto the microfluidic device and were discarded, so that X-sperms can be isolated and commercially exploited for fertilization demands of female cattle in dairy industry. Therefore, the research team used monoclonal antibody-conjugated magnetic beads to increase the force that causes the Y-sperm to be pulled out of the system, leaving only the X-sperm for further use. The experimental design was divided into the following: Model 1, the microfluid system for sorting positive magnetic beads, which yielded 100% separation; Model 2, the sorting of monoclonal antibody-conjugated magnetic beads in the fluid system, yielding 98.84% microcirculation; Model 3, the sorting of monoclonal antibody-conjugated magnetic beads with sperm in the microfluid system, yielding 80.12% microcirculation. Moreover, the fabrication microfluidic system had thin film electrodes created via UV lithography and MWCNTs electrode structure capable of erecting an electrode wall 1500 µm above the floor with a flow channel width of only 100 µm. The system was tested using a constant flow rate of 2 µL/min and X-/Y-sperm were separated using carbon nanotube electrodes at 2.5 V. The structure created with the use of vertical electrodes and monoclonal antibody-conjugated magnetic beads technique produced a higher effective rejection effect and was able to remove a large number of unwanted sperm from the system with 80.12% efficiency.

The interleukin-4 (IL-4) and interleukin-4 receptor (IL-4R) are cytokines that are involved in the immune and reproductive systems. This study aimed to verify the polymorphisms in the porcine IL-4 and IL-4R genes and to assess their effects on litter size traits in commercial pigs. Single nucleotide polymorphisms (SNPs) in the porcine IL-4 and IL-4R genes were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. A non-coding SNP of IL-4 g.134993898T > C and a non-synonymous SNP of IL-4R c.1577A > T (amino acid change at position 526, Q526L) were found to be segregating in Landrace sows. The IL-4 g.134993898T > C polymorphism was significantly associated with the number of piglets weaned alive (NWA) trait. The IL-4R c.1577A > T polymorphism was significantly associated with the number born alive (NBA) and NWA traits. Moreover, the accumulation of favorable alleles of these two SNP markers revealed significant associations with the NBA, NWA, and mean weight of piglets at weaning (MWW) traits. These findings indicate that the porcine IL-4 and IL-4R genes may contribute to the reproductive traits of pigs and could be used as candidate genes to improve litter size traits in the pig breeding industry.