Collecting and measuring fluid pH and calcium
Due to the limited quantity of fluids collected from each female reproductive tract for our viscosity measurements, we were not able to also measure pH and calcium using these same individuals. Because we were further limited by the number of available animals in our lab colony, we focused on two species in which ten females were available for this study – the polyandrous P. maniculatus and its monogamous sister-species P. polionotus . To extract reproductive fluid for these measurements, we dissected the reproductive tract, reserved the right side of each tract for the pH measurements and submersed the left side of each tract into phosphate buffer solution (PBS) at 4ºC for the calcium measurements.
To measure the pH of reproductive fluids, we placed pH strips (Hydrion [9400] Spectral 5.0-9.0) on microscope slides (VWR Plastic Microslides, USA) on a 37ºC warmer (Fisherbrand Isotemp Digital Dry Bath/Block Heater, USA) and under 0.63X magnification (Zeiss Stemi 2000, USA). We trimmed the fat, unraveled the coiled oviduct, placed the elongated tract on a pH strip, and severed the uterus from the oviduct at the UTJ. We then placed the oviduct on another pH strip, cut it in half, pinched the open end of the lower oviduct closed with forceps, and placed it on a third pH strip. We used separate u-shaped glass pipettes to push from one end of each region of the reproductive tract to the other end to squeeze fluid out onto the pH strip to measure pH immediately after release from the tissue (Yeung et al. 2004).
To measure calcium in reproductive fluids, we used a calcium assay kit (Abcam Calcium Assay Kit ab102505, USA). After removing the tissue from PBS, we severed the uterus from the oviduct at the UTJ, cut the oviduct in half to separate the lower and upper oviducts, placed each tissue region in a separate tube containing calcium assay buffer, and ground the tissues with disposable plastic pestles (ThermoFisher, USA). We separated the tissue from the buffer and reproductive fluid by centrifuging at 4ºC for 5 min at 3500 rpm (Eppendorf Centrifuge 5702RH, USA), pipetted the supernatant from each tract region into microplate wells (CellStar 96 Well Cell Culture Plate, USA), added the chromogenic reagent, calcium assay buffer, and calcium standard, and immediately measured the mixture’s absorbance at 575nm using a microplate reader (Thermo Scientific Multiskan FC, USA).