Hyperactivated stallion spermatozoa fail to exhibit a rheotaxis-like behaviour, unlike other species

Abstract

The journey of spermatozoa through the female genital tract is facilitated by rheotaxis, or the cell's preference to swim against a flow, as well as thigmotaxis, the wall tracking behaviour, which guides them to the site of fertilisation. The aim of this study was to characterise the rheotactic and thigmotactic response of stallion sperm within a microfluidic channel. Stallion sperm rheotaxis was assessed within the microfluidic channel with regard to: (i) A range of flow velocities, (ii) Varying media viscosity and (iii) Sperm hyperactivation. Sperm distribution across the microfluidic channel was also studied and compared to human and ram sperm. Stallion sperm progressed furthest at a velocity range of 10-30 µm/s, with an optimum velocity of 20 µm/s. A flow viscosity of 2.5cP or greater reduced sperm rheotaxis (P < 0.05). Stallion sperm that were hyperactivated were unable to exhibit rheotaxis within the microfluidic channel, whereas, both hyperactivated human and ram sperm did exhibit positive rheotaxis under the same conditions. The number of sperm swimming near the microfluidic channel walls was higher than in the microfluidic channel centre (P < 0.05). This is the first study to illustrate that stallion sperm are rheotactically responsive and increasing viscosity reduces this response. We also demonstrated that sperm are predominantly inclined to swim along a surface and uniquely, hyperactivated stallion sperm are non-progressive and do not exhibit a rheotactic response unlike other species.

Figure 1

Number of stallion sperm progressing passed 15 mm in a microfluidic channel after 10 mins at varying flow velocities in tyrosine, albumin, lactate and pyruvate media (TALP; 0.9cP; Experiment 1). Vertical bars represent s.e.m. (n = 4 replicates). ^abcDiffering superscripts differ significantly (P < 0.05).

Figure 2

Kinematic parameters for frozen-thawed stallion sperm within a static chamber in a solution of varying viscosities (Experiment 2a). (a) Curvilinear-Velocity (VCL; µm/s); (b) Linearity (LIN, %); (c) Amplitude of Lateral Head Displacement (ALH; µm). Vertical bars represent s.e.m. (n = 5 replicates).

Figure 3

Number of frozen-thawed stallion sperm progressing passed 15 mm in a microfluidic channel after 10 mins against differing media viscosities at varying flow velocities (0–100 µm/s; Experiment 2b). Vertical bars represent the s.e.m. (n = 3 replicates).

Figure 4

The percentage of sperm swimming at the microfluidic channel wall or in the centre position (at 15 mm along the micro-channel) in a range of viscosities (0.9–6cP; Experiment 2c). Vertical bars represent the s.e.m. (n = 3 replicates). ^abDiffering subscripts differ significantly within viscosity (P < 0.05).

Figure 5

Number of stallion, human and ram sperm progressing passed 15 mm in a microfluidic channel after 10 mins against a flow (20 µm/sec) following treatment with hyperactivation agonists or not (Control; Experiment 4). Vertical bars represent the s.e.m. (n = 3 replicates). *Represents statistical significance.

Figure 6

Computer assisted sperm analysis showing the swimming patterns of hyperactivated stallion (a), human (b) and ram (c) sperm (Experiment 3). The different coloured lines represent the curvilinear velocity (VCL, red), average path velocity (VAP, green) and straight line velocity (VSL, blue).