RESUMEN
The postpartum period in dairy cows is associated with a state of temporary negative energy balance and could induce functional changes into ovarian granulosa cells (GC) resulting in significant impact on the ovarian function and fertility. Yet, the regulation of interleukin receptors (ILRs) in GC as well as ILs expression profile during the postpartum period have not been fully investigated. We hypothesized that the postpartum period is associated with changes in ILs expression profile that could affect follicular development and ovulation rate. First, we aimed to investigate the expression and regulation of different IL and IL receptors in GC at different stages of follicular development and then analyse the changes in target ILs expression profile induced during the postpartum period. In the first objective, normal cycling cows were selected and GC were collected from small follicles (SF), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 h following hCG injection (OF). In the second objective, dairy cows between 50 and 70 days postpartum were randomly selected, and ß-hydroxybutyrate (BHB) concentrations were measured in blood samples in order to assign cows to the BHB+ group (>1.4 mmol/L) or BHB- group (<1.2 mmol/L). GC were collected from preovulatory follicles by transvaginal aspiration. Total RNA was extracted from GC of all groups for analysis of target ILs and ILRs expression. Steady-state mRNA levels of IL4R was strongest in the DF, while IL15R expression was greatest in the OF, and IL21R showed increased steady-state mRNA levels in the corpus luteum as compared to the different groups of follicles. Overall, expression of IL1A, IL1B, IL8, IL15, IL23 and TNFα was stronger in OF as compared to DF, while IL4 and IL10 expression was stronger in SF than in DF. Similarly, expression of IL1A, IL1B, IL8, IL15, IL23, and TNFα were significantly stronger in GC of BHB+ cows than in the control, while IL4 expression was significantly reduced in BHB+ as compared to control cows. We have established an IL expression profile, which suggest a correlation with BHB levels during the postpartum period. Additionally, we have demonstrated a differential regulation of target ILRs in GC at different stages of follicular development. Overall, these data provide a better understanding of the changes that could affect follicular development and ovulation during the postpartum period and lay the ground for further investigations.
RESUMEN
This paper describes the development of an on-chip nucleic acid (NA) extraction assay from whole blood using a centrifugal microfluidic platform that allows for pneumatic actuation of liquids during rotation. The combination of pneumatic and centrifugal forces makes it possible to perform fluidic operations without the need for integrating active control elements on the microfluidic cartridge. The cartridge is fabricated from thermoplastic polymers (e.g., Zeonor 1060R) and features a simple design that is compatible with injection molding. In addition, the cartridge is interfaced with two external vials for off-chip storage of the blood sample and retrieval of the eluted NA solution, respectively. On-chip capture of NAs is performed using an embedded solid-phase extraction matrix composed of commercial glass microfiber filters (Whatman GF/D and GF/F). The yield of the automated, on-chip extraction protocol, determined by measuring absorbance at 260 nm, is comparable to some of the best manually operated kits (e.g., Qiagen QIAamp DNA Mini Kit) while providing low assay-to-assay variability due to the high level of control provided by the platform for each processing step. The A260/A280 and A260/A230 ratios of the absorbance spectra also reveal that protein contamination of the sample is negligible. The capability of the pneumatic platform to circulate air flux through the microfluidic conduit was used to dry leftover ethanol residues retained in the capture matrix during washing. This method, applied in combination with localized heating, proved effective for reducing ethanol contamination in eluted samples from â¼12% to 1% (v/v).
