Sironen et al. BMC Genomics 2010, 11:699 http://www.biomedcentral.com/1471-2164/11/699 acrosome [7-9] and the knobbed acrosome defect (KAD) in bulls, boars, stallions, rams, and dogs [10-15]. The acrosome is an organelle that develops over the anterior half of the head in the spermatozoa. It is a cap-like structure derived from the Golgi apparatus. The acro- some contains digestive enzymes, which break down the zona pellucida of the ovum, allowing the sperm to deli- ver its haploid nucleus into the ova. Disturbances of acrosomal development and function significantly impair the fertilizing capacity of spermatozoa [16]. Knobbed acrosome defect has been recently described in the Finnish Yorkshire pig population [15]. Testicular weights of boars with KAD did not differ from control boars. However, affected boars had a smaller seminifer- ous tubule diameter and lower number of Sertoli cells relative to control boars [15]. Investigation of the pedi- grees of KA-affected boars suggested an autosomal recessive inheritance of the defect. Generally two com- mon boars were identified in the pedigree of the boars with the KAD. Fertility of KA-affected boars is severely compromised. Depending on the amount of knobbed spermatozoa (25-81%) affected boars had poor non- return rate from no pregnancies to 47%, thus KA- affected boars produced no offspring or on average 2.5 fewer piglets per litter than control boars. Here we have characterized further the severity of the sperm head abnormalities in KA-affected boars. A whole genome scan with microsatellite markers showed increased homozygosity in KA-affected boars in chromosomes 3, 8, 14 and 15 [15]. However, no statisti- cally significant association was detected with available microsatellite markers. In this study we have used the PorcineSNP60 Genotyping BeadChip (Illumina) in order to increase marker density and accurately map the KAD associated region in pigs. All affected boars were homo- zygous for SNPs covering 432 kb on porcine chromo- some 15. The coding region of two genes was located within this homozygous region and sequenced from both a KA-affected and control boar. Results Microscopical analysis of the KAD In the confocal laser scanning microscopy three- dimensional reconstructions of the spermatozoa with acrosomal granules indicated that the granules pro- truded on both sides and contained a vacuolum (Figure 1A). TEM analyses confirmed the three-dimen- sional protrusion of the granules and the occurrence of vacuoles within the granules. The nucleus was also shown to be affected as evident from the Y-shaped form at the apical end (Figure 1B) suggesting that the defect affects both the chromatin and acrosome. These findings highlight that this particular and previously A B Page 2 of 8 Figure 1 Microscopical examination of the KA-defect. A. Confocal laser scanning microscopy images of spermatozoa with acrosomal granule from two KA-affected boars. Yellow lines indicate the perpendicular planes of the 3d-reconstructions. Arrows indicate a vacuolum inside the granule. B. TEM analysis of an acrosomal granule. Two vacuoles are present and the apical part of the nucleus shows a Y-shape. unknown KA-defect appears to be a serious form of the acrosomal granule defect. SNP quality measures Based on all available SNPs and the method to estimate IBDs in the Plink software package (pi_hat) the average relatedness among cases and controls was 0.24 and 0.26, respectively. These levels of relatedness are typical in the studied Finnish Yorkshire pig population. The aver- age sample call rate was 95%. There were 2815 SNPs that did not work for any of the samples analysed. Excluding these SNPs, the average SNP call rate was 0.9982 (s.d. = 0.007) and the average minor allele fre- quency was 0.25 (s.d. = 0.14). Overall, the dataset con- tained 9216 monomorphic SNPs. Observed distribution of P-values in the Hardy-Weinberg equilibrium test sta- tistics did not differ from expectations. In total, 183 SNPs (excluding SNPs on the X-chromosome) had a P-value <1.0E-06 being lower than expected. Genome wide association analysis The association test was performed for 47055 SNPs. The Manhattan plot of the log10 based P-values is presented in Figure 2. The recessive model identified a KAD asso- ciated region covering approximately 3 Mbp between 93 and 96 Mbp (pig genome build 9) on chromosome 15.