Cholesterol addition protects membrane intactness during cryopreservation of stallion sperm

Addition of cholesterol to sperm membranes improved equine sperm stability during semen cryopreservation; however, it also reduced in vivo fertility. The objective of the present study was to determine the effects of adding cholesterol to stallion sperm prior to freezing, and subsequently removing it from frozen–thawed sperm. Semen from 12 stallions was subjected to four treatments: (T1) control, semen was diluted with Kenney extender, centrifuged, and resuspended to 100 × 106 spermatozoa/mL in INRA 82 freezing extender, packaged into 0.5-mL straws, cooled to 5 °C, and cryopreserved in liquid nitrogen; (T2) T1 with the addition of cholesterol before cooling (the cholesterol was incorporated to the sperm membranes with the methyl-β-cyclodextrin-cholesterol complex); (T3) T2 with post-thaw removal of the cholesterol with 0.052 mg methyl-β-cyclodextrin/50 × 106 sperm; and (T4) T3 with 0.156 mg methyl-β-cyclodextrin/50 × 106 sperm. Sperm progressive motility and functional integrity of sperm plasma membranes were evaluated microscopically and by the hyposmotic swelling test, respectively. Using flow cytometry, physical integrity of sperm plasma membranes was assessed with propidium iodide, acrosomal integrity with fluoresceinated lectin peanut agglutinin, and rate of sperm acrosome reaction induced with of the calcium ionophore A23187. Cholesterol inclusion (T2) increased the proportion of frozen–thawed sperm with intact plasma membrane. Nevertheless, sperm from T2 (9.3 ± 5.9%) had a lower rate of acrosome reaction after induction, compared to the control group (16.5 ± 11.0%). After cholesterol removal, there was no increase in the induced acrosome reaction rate (T3: 11.3 ± 7.1% and T4: 11.8 ± 9.9%). Perhaps the cyclodextrin concentrations used were too low to remove sufficient cholesterol from sperm membranes to restore the ability of cryopreserved sperm to undergo an acrosome reaction. Regardless, the addition of cholesterol to improve post-thaw sperm integrity, and its subsequent removal, still has potential for cryopreservation of stallion sperm.