Boar Semen Sperm Selection

The boar semen
was provided by Varkens KI Nederland BV. A Ficoll-based separation
technique was used for selection of good-quality spermatozoa. The
semen was centrifuged (300g, 20 min) over a density
gradient of Ficoll-400 (Sigma-Aldrich, Zwijndrecht, The Netherlands),
which separates cells by their motility. The discontinuous density
gradient of Ficoll-400 was obtained with a 40% (v/v) density top layer
and 80% (v/v) density lower layer. The motile spermatozoa formed a
soft pellet at the bottom of the tube, collected, and then dispersed
in the Ringer rinsing solution. After the second centrifugation (500g, 10 min), the pellet was dispersed in noncapacitating
(modified Human Tubal Fluid, mHTF, without BSA, NaHCO₃,
and CaCl₂)^{33 (link)} or capacitating
medium (Human Tubal Fluid, HTF–NaCl 101.6 mM, KCl 4.69 mM,
glucose 2.78 mM, KH₂PO₄ 0.37 mM, MgSO₄ 0.2 mM, sodium lactate 21.4 mM, sodium pyruvate 0.33 mM, BSA 4 mg/mL,
NaHCO₃ 25 mM, CaCl₂ 2.04 mM).^{34 (link)} Sperm cells were counted in a Bürker-Türk
chamber under the microscope. The capacitation status was confirmed
as described in the Supporting Information (see Figure S3).