The use of frozen-thawed boar sperm is not widespread, owing to reduced fertility rates and high cost per dose (Eriksson et al. 2004 Proc. Aust. Assoc. Pig Vet., 61–69). Improvements in post-thaw sperm survival are required for commercialization. Platelet-activating factor (PAF) is a phospholipid involved in regulating sperm function. PAF:acetylhydrolase (PAF:AH) regulates PAF activity by conversion to its inactive isoform. Both occur naturally in boar semen (Kordan et al. 2003 Pol. J. Vet. Sci. 6, 55–60). Removal of PAF and PAF:AH along with seminal plasma during the cryopreservation process may inhibit the ability of sperm to withstand the freeze-thawing process. The aim of this study was to assess the effect of PAF and PAF:AH, added to boar semen pre-freeze, on the post-thaw motility and acrosome integrity of sperm. The sperm rich fraction was collected from a mature Large White × Landrace boar, diluted with Androhep (1:2, semen:Androhep; Minitube, Verona, WI, USA), cooled to 17°C over 2 h, and then centrifuged (10 min, 800g). The sperm pellet was resuspended in cooling extender (11% (w/v) lactose solution with 20% (v/v) egg yolk; control), cooling extender plus 100 ng/mL PAF (PAF), or cooling extender plus 0.4% (v/v) PAF:AH (Pafase; ICOS Corporation, Seattle, Washington, USA), and cooled to 5°C over 2.5 h. Sperm were further diluted with cooling extender plus 9% (v/v) glycerol and 1.5% (v/v) Equex STM (freezing extender), loaded into 0.5-mL straws, and frozen. Straws were thawed (20 s, 42°C) and the motility and acrosome integrity (FITC-PNA; Mortimer etal. 1990 Hum. Reprod. 5, 99–103) assessed at 0, 3, and 6 h post-thaw after incubation at 37°C. Data from three replicates were analyzed by ANOVA and a Tukey test applied where significant differences were found. Post-thaw motility (0 and 3 h) was higher for PAF (60.0 ± 0.0% and 25.0 ± 2.9%) than for control (41.7 ± 1.7% and 10.0 ± 2.9%; P < 0.05), but was similar for Pafase (41.7 ± 1.7% and 16.7 ± 1.7%; P > 0.05). By 6 h post-thaw, motility was similar for PAF (1.7 ± 1.7%), Pafase (6.7 ± 6.8%), and control (1.7 ± 1.7%, all respectively; P > 0.05). Acrosome integrity was higher at 0, 3 and 6 h post-thaw for Pafase (55.7 ± 3.2%, 45.7 ± 3.7% and 23.0 ± 3.1%) than for control (42.7 ± 1.5%, 25.7 ± 5.7% and 12.3 ± 2.7%) and PAF (33.0 ± 3.7%, 26.3 ± 2.2% and 11.7 ± 0.3%, all respectively; P < 0.05), but was similar between control and PAF (P > 0.05). Supplementation of cooling extender with 100 ng/mL PAF increased initial post-thaw motility, but this benefit was lost after 6 h post-thaw. Pafase in the cooling extender improved the proportion of intact acrosomes, even after 6 h post-thaw. In vitro studies investigating the interaction between Pafase-treated frozen-thawed sperm and oviducal epithelial cells would be of interest to further establish the potential benefits of pre-freeze addition of Pafase on the fertilizing potential of frozen-thawed boar sperm.
Flow cytometric fluorescence emission spectrum analysis of hoechst-33342-stained DNA in chicken thymocytes
