Fabrication of spectroscopic microfluidic chips for mastitis detection in raw milk.

Mastitis is a disease that directly affects the quantity and quality of milk produced by dairy cows, which can have a negative impact on the income generated from selling the milk. Severe inflammation caused by this mammary disease can result in up to 1 × 106 white blood cells per milliliter of cow milk. Currently, the California mastitis test is a popular chemical inspection test, but its error rate of over 40% is a significant factor in the ongoing spread of mastitis. In this study, a new microfluidic device was designed and fabricated to identify normal, sub-clinical, and clinical mastitis. This portable device allows for precise and analysis of results within a second. The device was designed to screen somatic cells and a staining process was added to identify somatic cells using single-cell process analysis. The fluorescence principle was used to identify the infection status of the milk, which was analyzed using a mini-spectrometer. The accuracy of the device was tested, and it was found to determine the infection status with 95% accuracy, compared to the accuracy obtained using the Fossomatic machine. By introducing this new microfluidic device, it is believed that the spread of mastitis in dairy cows can be significantly reduced, leading to higher quality and more profitable milk production.

Antibody-Conjugated Magnetic Beads for Sperm Sexing Using a Multi-Wall Carbon Nanotube Microfluidic Device.

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.

Association of IFNA16 and TNFRSF19 Polymorphisms with Intramuscular Fat Content and Fatty Acid Composition in Pigs.

Interferon-alpha-16 (IFNA16) and tumor necrosis factor receptor superfamily member 19 (TNFRSF19) are cytokines that may play a role in adipogenesis and fatness. Single nucleotide polymorphisms (SNPs) of the porcine IFNA16 and TNFRSF19 genes were verified and their association with intramuscular fat (IMF) content and fatty acid (FA) composition were evaluated in commercial crossbred pigs. Two non-synonymous SNPs of the porcine IFNA16 c.413G > A and TNFRSF19 c.860G > C loci were detected in commercial crossbred pigs. The porcine IFNA16 c.413G >A polymorphism was significantly associated with stearic acid, total saturated FAs (SFAs), and the ratio of monounsaturated FAs (MUFAs) to SFAs (p < 0.05). Furthermore, the porcine TNFRSF19 c.860G > C polymorphism was found to be significantly associated with IMF content and arachidic acid levels (p < 0.05). The results revealed that porcine IFNA16 and TNFRSF19 polymorphisms are related to IMF content and/or FA composition and affirmed the importance of these cytokine genes as potential candidate genes for lipid deposition and FA composition in the muscle tissue of pigs.

Association of adipocytokine IL-1A and IL-6 genes with intramuscular fat content and fatty acid composition in pigs.

Several adipocytokines are involved in inflammatory and immune responses as well as regulated fat deposition and lipid metabolism in mammals. This study aimed to verify the polymorphisms of the porcine interleukin 1A (IL-1A) and interleukin 6 (IL-6) genes and to assess their association with intramuscular fat (IMF) content and fatty acid (FA) composition in commercial crossbred pigs. Two single nucleotide polymorphisms (SNPs) of the porcine IL-1A g.43722547A>G and IL-6 g.91508173C>T loci were found to be segregating in these crossbred pigs. Furthermore, the porcine IL-1A g.43722547A>G polymorphism was found to be significantly associated with myristic, palmitic, palmitoleic, and eicosadienoic acid levels. Moreover, the porcine IL-6 g.91508173C>T polymorphism was significantly associated with IMF content and homolinolenic acid levels. These results suggest that the polymorphisms of the porcine IL-1A and IL-6 genes correlated with lipid content and FA composition and confirmed the importance of the adipocytokine IL-1A and IL-6 genes as candidate genes for fatty acid composition in the muscles of pigs.

Impacts of pineapple peel powder on growth performance, innate immunity, disease resistance, and relative immune gene expression of Nile tilapia, Oreochromis niloticus.

An 8-week growth trial was conducted to examine the efficacy of pineapple peel powder (PAPP) on growth rate and immunity of Nile tilapia, O. niloticus. Three hundred Nile tilapia (20.91 ± 0.11 g) were fed five diets containing different levels of PAPP at 0, 10, 20, 30 and 40 g kg-1 PAPP, respectively. After four and eight weeks of the feeding trial, growth rates, and immune responses were tested. A challenge test using Streptococcus agalactiae and relative immune gene expression were performed after eight weeks of PAPP feeding. It was found that skin mucus and serum lysozyme, skin mucus and serum peroxidase, alternative complement, phagocytosis, and respiratory burst activities were significantly increased with the addition of PAPP. The maximum (P ≤ 0.05) innate immune values were noted in fish fed 10 g kg-1 PAPP. Similarly, the up-regulation of IL1, IL8, and LBP gene expressions were also detected in fish fed PAPP diets, with the maximum value was found in 10 g kg-1 PAPP fed fish. The relative percentage of survival (RPS) of Oreochromis niloticus after the challenge test were (56.00%, 72.00%, 60.00%, and 44.00%) for the 5, 10, 20 and 40 g kg-1 PAPP diets, respectively. Fish fed the 10 g kg-1 PAPP supplemented diet achieved the highest (P < 0.05) survival rate against S. agalactiae. Growth and feed efficiency were outstandingly (P < 0.05) enhanced in the PAPP groups. In conclusion, PAPP can be potentially used as a feed additive in Nile tilapia culture under Biofloc system.

Association of IL-4 and IL-4R Polymorphisms with Litter Size Traits in Pigs.

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.

A novel microfluidic chip-based sperm-sorting device constructed using design of experiment method.

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.

Effects of elephant's foot (Elephantopus scaber) extract on growth performance, immune response, and disease resistance of nile tilapia (Oreochromis niloticus) fingerlings.

Medicinal plant has been applied as an alternative strategy for antibiotics and chemotherapeutics for controlling the outbreak of diseases in tilapia farming. In this study, five doses of Elephantopus scaber extract (ESE) were added to the basal diet at 0, 2.5, 5, 10, and 20 g kg-1 feed of Nile tilapia fingerlings (13.92 ±â€¯0.06 g initial weight) in triplicate. After 4- and 8- weeks post-feeding, fish were sampled to determine the effects of the ESE supplemented on fish's growth performance, humoral, and skin mucus immune response. After 8 weeks post-feeding, a challenge test against Streptococcus agalactiae was carried out using 10 fish from each tank. Fish fed ESE showed significantly increased serum lysozyme (SL), serum peroxidase (SP), alternative complement (ACH50), phagocytosis (PI), and respiratory burst (RB) compared to the control group (P < 0.05). The skin mucus lysozyme (SMLA) and skin peroxidase (SMPA) were stimulated in fish fed ESE diets. Dietary inclusion of ESE significantly (P < 0.05) promoted final body weight (FW), weight gain (WG), and specific growth rate (SGR); while a reduction in feed conversion ratio (FCR) was observed in fish fed 5 g kg-1 ESE, after 8 weeks post-feeding. The challenge study indicated that the relative percent survival (RSP) was 38.10%, 76.19%, 66.67%, and 47.62% in Diet 2, Diet 3, Diet 4, and Diet 5, respectively. Among the supplemented groups, dietary of 5 g kg-1 ESE showed significantly higher RPS and the highest resistance to S. agalactiae in comparison with other groups. In conclusion, supplementation of ESE (5 g kg-1) enhanced the humoral and mucosal immunity, promoted growth performance, and improved disease resistance of Nile tilapia against Streptococcus agalactiae.

Bovine embryo sex determination by multiplex loop-mediated isothermal amplification.

In cattle, the ability to determine the sex of embryos before embryo transfer is beneficial for increasing the number of animals with the desired sex. This study therefore developed a new modification of loop-mediated isothermal amplification in a multiplex format (multiplex LAMP) for highly efficient bovine embryo sexing. Two chromosomal regions, one specific for males (Y chromosome, S4 region) and the other common to both males and females (1.715 satellite DNA), were amplified in the same reaction tube. Each target was amplified by specifically designed inner primers, outer primers, and loop primers, where one of the S4 loop primers was labeled with the fluorescent dye 6-carboxyl-X-rhodamine (emitting a red color), whereas both satellite loop primers were labeled with the fluorescent dye fluorescein isothiocyanate (emitting a green color). After amplification at 63 °C for 1 hour, the amplified products were precipitated by a small volume of cationic polymer predispensed inside the reaction tube cap. Green precipitate indicated the presence of only control DNA without the Y chromosome, whereas orange precipitate indicated the presence of both target DNAs, enabling interpretation as female and male, respectively. Accuracy of the multiplex LAMP assay was evaluated using 46 bovine embryos with known sex (25 male and 21 female) generated by somatic cell nuclear transfer and confirmed by multiplex polymerase chain reaction. The multiplex LAMP showed 100% accuracy in identifying the actual sex of the embryos and provides a fast, simple, and cost-effective tool for bovine embryo sexing.

Determination of Sperm Sex Ratio in Bovine Semen Using Multiplex Real-time Polymerase Chain Reaction.

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.