Towards improved boar fertility by genetic characterization and detection of traits important in sperm production and quality (218891)

Two basic prerequisites are needed to do breeding work through artificial insemination: (1) that it is possible to train the male animals up to the ejaculation of semen in an animal-friendly, hygienic and safe manner, and (2) that there is sufficient fertile sperm in the semen dose. In this project we have looked at methods for measuring semen quality from AI-boars and analysed genetic components relative to sperm quality. In addition, we have detected specific genes that contribute to regulation of sperm quality. The results from the genetic association study show that there are significant relationships between total born piglets and SNPs within some of the candidate genes tested. SNPs in four different genes explain each between 0.27 and 1.18% of the genetic variation for the total number of born piglets. This indicates that these genes have a limited effect and that there are other genes that are also important for genetic variation of litter size in pigs. In the project period we also looked at field fertility data (number of born piglets) and the correlations with the CASA and ATP analysis results. Statistical calculations have been made and show that there are differences between breeds in motility, ATP levels and storage capacity. The proportion of motile cells decreased in both breeds after storage for 96 hours. A larger proportion of hyperactivated cells were found in Duroc on the day of collection, but this population decreased after storage. In contrast, fewer hyperactivated cells were found in Landrace, and this population increased after storage. The amount of ATP decreased after storage in both breeds. The proportion of motile sperm cells, curvular velocity and ATP level on the day of collection had a significant effect on the total number of piglets born in Landrace, while the proportion of hyperactivated cells, curative velocity and ATP level on the day of collection had an effect on the total number of born piglets in Duroc. Ejaculated mammalian spermatozoa are not able to fertilize an oocyte before capacitation has occurred, a process where they acquire hyperactivated motility and other modifications that facilitate fertilization. The hyperactive motility is characterized by a vigorous and non-linear swimming pattern, which help the spermatozoa to penetrate the zona pellucida. CASA was used for phenotyping as it recognizes the hyperactive motility pattern as high curvilinear velocity and amplitude of the lateral head movement and low linear trajectory. We sequenced a total of 29 animals divided into two contrasts: 1) high vs. low hyperactivity day 0 in Landrace, and 2) high vs. low hyperactivity day 4 vs. 0 in Landrace. Day 0 is the day of collection and day 4 is the last recommended consumption day of sperm doses in Norsvin. For contrast 1, the average percentage hyperactivity was 14.5% (± 1.73) (n = 4) for the high group and 1.6 (± 0.27) (n = 4) for the low group. Altogether 3219 genes were found differently expressed between the two groups (FDR <0.05) but by using a very strict significance level, we reduced the number of significant genes to 64. The most significant upregulated gene in the high hyperactivity group was the DNL-type zinc finger (DNLZ) (FDR = 1.2e-05), and this was also the highest upregulated gene (logFC = 2.7). The most downregulated gene in the high hyperactivity group was zinc finger DBF-type containing 2 (ZDBF2) (logFC = -1.7). Gene ontology analyses showed that 58 pathways were overrepresented among the differentially expressed genes (p <0.01). For contrast 2) the average percentage hyperactivity was 15.06% (± 2.97) (n=3) for the high group and 2.66 % (± 0.58) (n=4) for the low group. A total of 69 genes were found down-regulated between the groups in this contrast whereas 33 were up-regulated (FDR <0.05). The most significant differentially expressed gene was solute carrier family 40 member 1 (SLC40A1) (FDR = 9.7e-4). The most downregulated gene in the high group was calcium dependent secretion activator (CADPS) (-2.0) whereas the highest upregulated gene was DNLZ (logFC= 1.9). DNLZ is very interesting as this is the same as in contrast 1), indicating an important role in hyperactivity in Landrace boars. SNP detection will be conducted in all contrasts, and this will also show putative genetic variants within DNLZ that can be further investigated for use as genetic markers. Protein analyses have been performed on LC-MS using experienced people at IKBM, NMBU and NOFIMA. Here we look for differentially expressed proteins between samples with high and low DNA fragmentation in sperm, a natural continuation of the transcriptional study on the same contrasts.

Increased variations of sow reproduction is observed between different AI boars and the sperm, especially from Duroc boars, have lower quantity and more variation in quality than demanded from efficient semen boars. Moreover, recent studies show unfavorab le correlations between sperm quality and important production traits, and between major sex hormones and boar taint. Selection of boars has traditionally focused on economically important traits and the sow reproduction, and so far no available parameter s are sufficiently describing the boars reproductive capability. In this project we want to find the traits describing the boar fertility as accurately as possible, and establish the most efficient and cost effective way to measure those traits. Additiona lly, we want to detect associations to other traits in the breeding goal, identify the underlying genes, genetic networks and biochemical pathways of boar fertility, and ultimately use the results and information in practical breeding to improve boar fert ility. A thorough boar breeding strategy will benefit Norwegian pig industry and make Norsvin pigs more attractive internationally. If we are able to genetically increase the boar fertility and decrease the level of boar taint, without reducing other fav orable traits in the breeding goal this would be a huge step in the international pig breeding society. The use of new and highly improved molecular genetics tools will make the data much more applicable and can facilitate effective pre-selection of candi dates entering the boar test. The project will contribute considerably to the development of scientific knowledge on semen quality and genetics in Norsvin, Nortura (owner of the Duroc breed) and our project partners. Economic gain due to increased litter size, increased farrowing rate and reduced number of sperms per dose is estimated to be as much as 140-200 mill NOK per decade.