Cholesterol supplementation of bovine sperm

Abstract

Bull sperm were treated with a range of cholesterol-loaded cyclodextrin (CLC) concentrations and frozen in egg yolk Tris or sodium citrate diluent. After thawing, the percentages of motile sperm in the 1.5mg CLC treatment (Tris) were higher (60%) than control (0mg CLC) sperm (42%; P<0.05) but no differences were detected with the sodium citrate diluent (P>0.05). Bull sperm treated with 3.0mg CLC and frozen in egg yolk Tris diluent had more viable cells (66%) compared to the control (48%; P<0.05). Analysis of cholesterol incorporation by spectrofluorometry and HPLC demonstrated that cholesterol content of bull sperm increases with CLC treatment (R2=0.824) and flow cytometry illustrated uniform cholesterol incorporation (CV=12.9% ± 0.73). Bull sperm were analyzed to for calcium levels and ability to acrosome react when treated with dilauroylphosphatidylcholine (PC-12), calcium ionophore A23187 (A23187) or heparin. Fresh sperm treated with 1.5mg CLC accumulated calcium at lower rates compared to control sperm when cells were capacitated with PC-12 or A23187 (P<0.01). After cryopreservation, CLC treated sperm accumulated calcium at slower when capacitated with PC-12 only. CLC treatment also decreased the rate fresh sperm underwent the acrosome reaction, compared to control sperm, when cells were capacitated with A23187 or heparin (P<0.05). No differences were detected in the rate cryopreserved sperm acrosome reacted with any of the compounds tested. In vitro fertilization using frozen thawed bull sperm treated with either 0 or 1.5mg CLC prior to cryopreservation resulted in no differences in fertility (P>0.05). A breeding trial using dairy heifers resulted in similar pregnancy rates for control (50%) sperm and for sperm treated with 1.5mg CLC (59%; P>0.05. The membrane fluidity of Chinese hamster ovary cells and bull sperm was measured following treatment with CLC. Addition of CLC to CHO cells analyzed at 23°C or at 5°C resulted in decreased membrane fluidity compared to control cells. When CHO cells were cryopreserved (control and CLC treatments) membrane fluidity decreased. Treating CHO cells with PC-12 caused an increase in membrane fluidity. Bull sperm form 2 distinct populations of cells with different membrane fluidity, compared to the single population in CHO cell analyses. At 23°C, bull sperm treated with CLC showed reduced membrane fluidity, in a dose dependent manner. Analysis of percentages of sperm cells in the 2 populations demonstrated that cells from the high fluidity population were converting to the low fluidity population, as CLC concentration increased (P<0.05). After cooling to 5°C, control samples had significantly fewer viable cells (59%) than sperm samples treated with 1.5 or 5.0mg CLC (89 and 95%, respectively; P<0.05). Furthermore, at 5°C, cells shifted to the more fluid population in a dose dependent manner (P<0.05). Following cryopreservation, both populations of bull sperm were significantly less fluid than unfrozen cells (P<0.05). Addition of cholesterol resulted in higher percentages of cells surviving cryopreservation (>76%) compared to control sperm (60%; P<0.05). Treating bull sperm with PC-12 resulted in a single population of cells that was significantly different from all populations at 23°C (P<0.05).