Deletion of porcine BOLL is associated with defective acrosomes and subfertility in Yorkshire boars.

A recessive sperm defect of Yorkshire boars was detected more than a decade ago. Affected boars produce ejaculates that contain spermatozoa with defective acrosomes, resulting in low fertility. The acrosome defect was mapped to porcine chromosome 15 but the causal mutation has not been identified. We re-analyzed microarray-derived genotypes of affected boars and confirmed that the acrosome defect maps to a 12.24 Mb segment of porcine chromosome 15. To detect the mutation causing defective acrosomes, we sequenced the genomes of two affected and three unaffected boars to an average coverage of 11-fold. Read depth analysis revealed a 55 kb deletion that is associated with the acrosome defect. The deletion encompasses the BOLL gene encoding the boule homolog, an RNA binding protein which is an evolutionarily conserved member of the DAZ (Deleted in AZoospermia) gene family. Lack of BOLL expression causes spermatogenic arrest and sperm maturation failure in many species. Boars that carry the deletion in the homozygous state produce sperm but their acrosomes are defective, suggesting that lack of porcine BOLL compromises acrosome formation. Our findings warrant further research to investigate the role of BOLL during spermatogenesis and sperm maturation in pigs.

Estimation of intrachromosomal inbreeding depression on female fertility using runs of homozygosity in Finnish Ayrshire cattle.

Inbreeding increases homozygosity, which in turn increases the frequency of harmful recessive alleles, resulting in inbreeding depression. Inbreeding depression on fertility reduces the profitability of dairy farming by decreasing the lifetime milk production of cows and by increasing insemination and veterinary costs. Continuous homozygous segments, called runs of homozygosity (ROH), are currently considered to provide an effective measure of genomic inbreeding. The aim of this study was to estimate the effect of increased intrachromosomal homozygosity for female fertility in the Finnish Ayrshire population using ROH and haplotype analysis. Genotypes were obtained from 13,712 females with the Illumina BovineLD v.2 BeadChip low-density panel (Illumina Inc., San Diego, CA) and imputed to 50K density. After quality control, 40,554 single nucleotide polymorphisms remained for the analysis. Phenotypic data consisted of records for nonreturn rate, intervals from first to last insemination (IFL), and intervals from calving to first insemination. The raw phenotypic values were preadjusted for systematic effects before statistical analyses. The ROH-based inbreeding coefficients (FROH) were used as covariates in the mixed model equation to estimate the association between inbreeding and inbreeding depression on female fertility. First, we estimated the effect of increased chromosomal FROH. We detected significant inbreeding depression on IFL. Based on our results, a 10% increase in FROH on chromosomes 2, 18, and 22 were associated with IFL of heifers lengthening by 1.6, 0.9, and 0.7 d, respectively. Similarly, a 10% increase in FROH on chromosome 15 was associated with IFL of second-parity cows increasing by 2.3 d. Next, we located the regions within the chromosomes showing inbreeding depression. Our analysis revealed regions near the beginning of chromosome 2 and toward the ends of chromosomes 15, 18, and 22 that were associated with inbreeding depression on IFL. Last, we performed a haplotype analysis for the detected regions. The most promising haplotypes of each region were associated with IFL of heifers increasing by 4.4, 3.2, and 4.1 d on chromosomes 2, 18, and 22, respectively. The haplotype on chromosome 15 associated with IFL of second-parity cows increasing by 7.6 d. Overall, the breeding program requires inbreeding control, as increased genomic inbreeding in our study was associated with reduced reproductive ability in Finnish Ayrshire cattle.

Formation and function of the manchette and flagellum during spermatogenesis.

The last phase of spermatogenesis involves spermatid elongation (spermiogenesis), where the nucleus is remodeled by chromatin condensation, the excess cytoplasm is removed and the acrosome and sperm tail are formed. Protein transport during spermatid elongation is required for correct formation of the sperm tail and acrosome and shaping of the head. Two microtubular-based protein delivery platforms transport proteins to the developing head and tail: the manchette and the sperm tail axoneme. The manchette is a transient skirt-like structure surrounding the elongating spermatid head and is only present during spermatid elongation. In this review, we consider current understanding of the assembly, disassembly and function of the manchette and the roles of these processes in spermatid head shaping and sperm tail formation. Recent studies have shown that at least some of the structural proteins of the sperm tail are transported through the intra-manchette transport to the basal body at the base of the developing sperm tail and through the intra-flagellar transport to the construction site in the flagellum. This review focuses on the microtubule-based mechanisms involved and the consequences of their disruption in spermatid elongation.

A recent L1 insertion within SPEF2 gene is associated with changes in PRLR expression in sow reproductive organs.

The immotile short tail sperm (ISTS) defect was recognized in the Finnish Yorkshire population at the end of the 1990s when several affected boars were identified. The causal mutation for this defect is a recent L1 insertion within the SPEF2 gene. In 2001, the insertion frequency was already 0.23. Even though all homozygous boars are eliminated from the population due to infertility, the amount of affected boars increased rapidly until marker-assisted selection against the defect was established. Previously we identified an association between the L1 insertion and litter size in the first parity. In this study, we analyzed the expression of the genomic region adjacent to the L1 insertion on porcine chromosome 16. Based on the RNA-seq data analysis, prolactin receptor (PRLR) was identified as down-regulated in the oviduct of ISTS homozygous sows. Quantitative PCR (qPCR) analysis confirmed the significant down-regulation of PRLR in the ovary, oviduct, and uterus of ISTS homozygous and carrier sows compared with controls. In addition, three unannotated loci between PRLR and SPEF2 showed some transcription activity in the analyzed samples. We further investigated the possible mechanisms of the L1 influence on the decrease in the identified gene expression. The methylation pattern of the PRLR gene region appeared unaffected. However, reads mapping to the L1 sequence indicated an increase in L1 antisense promoter expression in the ISTS homozygous animals. The current data suggest that the presence of the L1 affects by some mechanism the expression patterns upstream of the insertion site.

L1 insertion within SPEF2 gene is associated with increased litter size in the Finnish Yorkshire population.

Immotile, short-tail sperm defect (ISTS) expanded in the Finnish Yorkshire population in the end of 1990s. The causal mutation for this defect is a recent L1 insertion within the SPEF2 gene in chromosome 16. Even though all homozygous boars are eliminated from the population because of infertility, the amount of affected boars increased rapidly until marker-assisted selection against the defect was established. To elucidate the associated effects of the ISTS defect on production traits, we have investigated the association of the L1 insertion and PRLR haplotype with reproduction traits in the Finnish Yorkshire population. Two data sets including 357 sows and 491 AI-boars were genotyped for the presence of the L1 insertion and analysed for association with reproduction traits. A Proc Mixed procedure (SAS Inc) and a software package for analysing multivariate mixed models (DMU) were used to study the effect of polymorphisms on reproduction traits. The L1-insertion within SPEF2 gene was associated with litter size in the first parity. The SPEF2 gene is located adjacent to a candidate gene for litter size in the pig, PRLR. Haplotypes within PRLR exon 10 were analysed in data set of 93 AI-boars for the association with reproduction traits. However, no associations were detected within the analysed data set indicating that PRLR sequence variants are not the causal cause for the identified effect on litter size.

Infertile boars with knobbed and immotile short-tail sperm defects in the Finnish Yorkshire breed.

In the period 1996-2006 two specific sperm defects, the knobbed acrosome (KA) defect and the immotile short-tail sperm (ISTS) defect, showed a strong negative association with fertility in Finnish breeding boars. In this study, we examined the incidence of these two sperm defects in two pig breeds, their effects on fertility and their associations with sperm morphology and testicular histology. Semen samples from 2048 (1097 Yorkshire, 951 Landrace) boars were collected. None of the Landrace boars revealed either the KA defect or the ISTS defect. Of the Yorkshire boars, 0.8% were afflicted with the KA defect and 7.6% with the ISTS defect. Boars diagnosed with the ISTS defect produced no litters. Fertility data were available from two artificially inseminated (AI) boars and six farm breeding boars affected with the KA defect. Breeding boars with 45-81% knobbed spermatozoa (n = 6) did not produce any litters out of 71 sows bred. AI boars with 25-30% knobbed spermatozoa had a poor non-return rate (on average 47% compared with 85% for normal control boars) and produced small litters, on average 2.5 piglets less than other boars of the same breed. Morphometry of testicular tissue and distribution of different cells in the seminiferous tubules were examined in nine boars. Boars with the KA defect had a smaller diameter of the seminiferous tubules (p < 0.05) and a lower number of Sertoli cells (p < 0.05) than controls. ISTS boars, in turn, had a significantly lower number of elongated spermatids (p < 0.05), and they also produced on average only 12% of the spermatozoa of normal boars. The ISTS defect is a manifestation of an autosomal recessive disease caused by an insertion in the KPL2 gene in porcine chromosome 16. Although we tried to map the KA defect, its aetiology remains unclear.

Evidence for three highly significant QTL for meat quality traits in the Finnish Yorkshire pig breed.

Meat quality is important both to consumers and to the meat processing industry. Commonly used measures of porcine meat quality are the pH and color of the meat. The purpose of this study was to identify SNP associated with these meat quality traits in Finnish Yorkshire using the Illumina PorcineSNP60 BeadChip. The association of each SNP with the quality traits was tested with a weighted linear model. The relatedness of samples was accounted for by a random polygenic genetic effect with the accompanying full relationship matrix. The original EBV from single-trait evaluations were deregressed before analysis. The statistical significance of SNP was established using the Bonferroni correction to adjust for multiple testing. Three genomic regions were significant for the meat quality traits. The PRKAG3 region on chromosome 15 was significant for pH measured from loin and ham and for a* (redness) measured from loin. The smallest P-value in the region was obtained for pH measured from loin (ASGA0070634, P-value = 3.8 × 10(-13)). The allele substitution effect (-0.047) of the unfavorable allele A corresponds to 1 SD of the polygenic effect. The second significant region, on chromosome 2 at around 31 megabases (Mb), was associated with pH and L* (lightness) measured from loin. The most significant SNP (ASGA0009814, P-value = 3.89 × 10(-10)) had an allele substitution effect of 0.86, corresponding to 0.7 SD of the polygenic effect of L*. The third region, located on chromosome 6 at around 83 Mb, was significant for a* measured from ham. The P-value of the best SNP (ALGA0035896) was 8.71 × 10(-7) and the allele substitution effect -0.38, corresponding to 0.5 SD of the polygenic effect of a*. The significant association of PRKAG3 with pH was not due to the known AA substitutions. The candidate gene on chromosome 2 associated with color L* is RCN1, which has a high affinity Ca(2+)-binding motif, the EF hand. The significant region on chromosome 6 for color a* contains several genes, so more data are needed to identify the causative gene. Our results indicate that instead of the known AA substitutions of PRKAG3, some yet-unknown AA substitutions are causative for the pH variation in Finnish Yorkshire. Also, a new major QLT for L* was found on chromosome 2. The significant SNP identified in this study can be used in marker-assisted selection.

Effect of polymorphisms in candidate genes on reproduction traits in Finnish pig populations.

Reproduction traits play an important role in economically viable piglet production and are closely related to the quality and length of the productive life of the sow. A increased removal rate of young sows is undesirable not only because of the associated financial penalties incurred, but also because of ethical concerns. Candidate genes and gene pathways have been identified for fertility in model species, and recent studies have provided evidence that polymorphisms within these genes are associated with reproduction traits in American Yorkshire/Large White and Landrace populations. In this study we evaluated the impact of single polymorphisms (n = 7) in 7 candidate genes on reproductive efficiency in Finnish Yorkshire (n = 280) and Landrace (n = 271) populations: IGFBP1, IGFBP2, IGFBP3, IGFBP5, CPTIA (carnitine O-palmitoyltransferase I), COX2 (PG-endoperoxide synthase 2, also known as cyclooxygenase-2), and SLC22A5 [organic cation/carnitine transporter 2 (solute carrier family member I), OCTN2]. In the Finnish Yorkshire population, only 4 of the analyzed markers were polymorphic. Significant effects on farrowing time were detected from the Yorkshire data, with polymorphisms within the genes CPT1A [a (allele substitution effect of allele A) = 2.97 d for age at first farrowing)], IGFBP3 (a = 0.54 d for farrowing interval of parities >1), and IGFBP5 (a = 3.22, 1.27, and 0.85 d for age at first farrowing and farrowing interval in the first and later parities, respectively). For the Landrace population, 6 markers were polymorphic, and significant effects were detected for traits affecting litter size. The polymorphism within the COX2 gene had an additive effect of 0.3 piglets for litter size in parities >1, and the IGFBP1 gene had an additive effect of 0.21, 0.26, and 0.11 piglets for litter size in the first parity, parities >1, and stillborn in parities >1, respectively. The additive effect of the SNP within the IGFBP2 gene was 0.16, 0.09, and 0.09 piglets for litter size in parities >1 and stillborn in the first and later parities, respectively. Finally, the IGFBP5 gene had an additive effect of 0.18, 0.07, and 0.07 piglets for litter size in the first parity, stillborn in parities >1, and mortality between farrowing and weaning in the first parity, respectively. These results support the suitability of the candidate gene approach for identification of markers to improve the reproductive performance of sows and to provide potential markers for marker-assisted selection.

Transplantation of normal boar testicular cells resulted in complete focal spermatogenesis in a boar affected by the immotile short-tail sperm defect.

Transplantation of testicular cells, also known as spermatogonial stem cell transplantation, is a relatively new approach in the field of male infertility. We used this technique to determine whether donor-derived sperm production in unrelated porcine recipients is possible following ultrasound-guided transfer of testicular cells. This study was undertaken because we had a strain of Finnish Yorkshire boars with a hereditary recessive gene defect rendering all spermatozoa immotile and anatomically abnormal in homozygous boars. Thus, monitoring of the focal success of colonization of donor spermatogonia with subsequent production of progressively motile spermatozoa was extremely sensitive. Testicular cells from young normal crossbred boars were transplanted into the testes of two boars affected with the immotile short-tail sperm (ISTS) defect. Prior to the transplantations, busulfan was used to suppress recipients' endogenous spermatogenesis. The ejaculates were collected and analysed for the presence of motile spermatozoa. In one of the two recipient boars transplanted with testicular cells from normal donors, motile spermatozoa appeared in the ejaculates 12 weeks after the transplantation. Spermatozoa manually selected under a microscope from a frozen aliquot of ejaculate collected 27 weeks after transplantation were genotyped. In two of the 20 vials the donor-derived genotype was visible. The genotyping results substantiated the success - as indicated by the appearance of motile spermatozoa after the spermatogonial transfer. Thus, donor-derived sperm production in unrelated recipients is possible. In addition, the production after transplantation of progressively motile spermatozoa with normal tail lengths shows that the ISTS defect in Finnish Yorkshire boars apparently results from defective transcription of an essential gene for sperm motility in germline cells. To conclude, the transplantation of donor testicular cells can, at least in boars with the ISTS defect, result in complete focal spermatogenesis.

Effects of pentachlorophenol and biotic interactions on soil fauna and decomposition in humus soil.

In a laboratory experiment, effects of chemical stress (pentachlorophenol, PCP, at concentrations of 0, 50, and 500 mg/kg) and biotic interactions (nematodes in the presence or absence of collembolas and enchytraeids) on the community structure of soil animals and decomposition processes were studied. PCP was strongly adsorbed to humus that contained 65% organic matter. Numbers of fungal-feeding nematodes decreased significantly at the highest PCP concentration, while no effects were found in bacterial feeders. There were differences in the numbers of nematodes between different animal combinations, but at the highest PCP concentration, collembolas and enchytraeids had no effect on them. Numbers of collembola Willemia anophtalma were lowered at the highest PCP concentration, although PCP was not acutely toxic at this concentration. The highest PCP concentration was acutely toxic to enchytraeids, and for an unknown reason all of them died in the main experiment. Both ATP content of the soil and soil respiration were reduced at the highest PCP concentration, while no differences were found between animal treatments. Amounts of NH4-N and PO4-P in the soil increased with increasing PCP concentration. It was concluded that in the presence of simple animal communities, harmful chemicals like PCP regulate the community structure of soil animals as well as decomposition and nutrient mobilization.