The primary objective of this research was to determine whether an automated electronic cell counter using microfluidic technology (Moxi Z) could be effective for determining sperm concentrations. Validation consisted of comparing frozen-thawed conventional bovine sperm counts using the Moxi Z to Coulter Z2 counter results. Specific goals were to determine (1) if the Moxi Z could count intact motile sperm, (2) if differences in media containing egg yolk lipids would affect counting, and (3) evaluate sample concentration requirements for Moxi Z accuracy. Cryopreserved straws (n = 20) of conventional bovine semen (Brangus), were obtained from Stroud Veterinary Embryo Services Inc. Straws were thawed (~30 s, 38°C) in a water bath, dried, and cut open. Semen was expelled into 15-mL conical tubes (BD Falcon) using a straw plunger. Thereafter, samples were centrifuged (400 × g, 10 min). The supernatant was removed and replaced with PBS (media 1) or Tris A semen extender (20% vol/vol egg yolk; media 2). Sperm samples exhibited ~70% motility for the experiments, based on subjective microscopy by placing 10-μL sample volumes on 25 × 75 × 1-mm microscope slides (#2951–001, Thermo Scientific, Waltham, MA, USA) with 24 × 50-mm #1.5 coverslips (#M6047–9, Baxter, McGraw Park, IL, USA) and viewing with a Axio Scope.A1 microscope (Zeiss, Thornwood, NY, USA) using 10× (M Plan Apo NIR, Mitutoyo Am, Aurora, IL, USA) and 40× (LD-Plan NeoFluor, Zeiss) lenses (i.e. 100–400×). The protocol for each sample was to establish an initial concentration of ~2.5 × 106 cells mL–1 and perform a serial dilution assay, using modified Hank's Balanced Salt Solution (Sigma, St. Louis, MO, USA). Counts (n = 3) for each dilution level (e.g. 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05, 0.025) were made simultaneously on each instrument, Moxi Z (v3.6 OS, Orflo) and Coulter Z2 (Beckman Coulter, Fullerton, CA, USA). Data points were compared to linear fitted curves. The Moxi Z gated range was set at 2.88 to 6.54 μ to approximate the count range of the Z2 system (2.91–6.46 μ). The sperm samples provided similar results on both the Moxi Z and the Z2 instruments. Specifically, data for the 2 media using serial dilutions of the sperm samples were analysed, and the Moxi Z counts correlated well with the Z2 counts for media 1 (R2 > 0.9469) and media 2 (R2 > 0.9771). Overall the Moxi Z can provide rapid bovine sperm counts (<15 s) using small sample volumes. The degree of precision and accuracy depends strongly on proper gate setting and on the ability to reduce sample debris. Increased size resolution will be realised by future improvements to the Moxi Z signal processing (v 4.0), as well as by slowing the system flow rate; thereby improving cell peak discrimination. The portability of the Moxi Z may be an asset for field work and will be tested in future research. The data and dilution ranges herein demonstrate the utility of the Moxi Z for diluted conventional semen and lower dose concentrations, such as that of sexed semen.
Identification of the Capacitating Agent for Bovine Sperm in Egg Yolk-TEST Semen Extender
