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).