BackgroundSmall RNAs present in bovine ejaculate can be linked to sperm abnormalities and fertility disorders. At present, quality parameters routinely used in semen evaluation are not fully reliable to predict bull fertility. In order to provide additional quality measurements for cryopreserved semen used for breeding, a method based on deep sequencing of sperm microRNA (miRNA) and Piwi-interacting RNA (piRNA) from individual bulls was developed.To validate our method, two populations of spermatozoa isolated from high and low motile fractions separated by Percoll were sequenced, and their small RNAs content characterized.ResultsSperm cells from frozen thawed semen samples of 4 bulls were successfully separated in two fractions. We identified 83 miRNAs and 79 putative piRNAs clusters that were differentially expressed in both fractions. Gene pathways targeted by 40 known differentially expressed miRNAs were related to apoptosis. Dysregulation of miR-17-5p, miR-26a-5p, miR-486-5p, miR-122-5p, miR-184 and miR-20a-5p was found to target three pathways (PTEN, PI3K/AKT and STAT).ConclusionsSmall RNAs sequencing data obtained from single bulls are consistent with previous findings. Specific miRNAs are differentially represented in low versus high motile sperm, suggesting an alteration of cell functions and increased germ cell apoptosis in the low motile fraction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3394-7) contains supplementary material, which is available to authorized users.

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The effect of cryopreservation on boar sperm viability, motility, lipid content and antioxidant enzymatic activities was studied. Three classes of semen were determined according to a cluster analysis on the basis of the proportion of live and dead cells after freezing and thawing. The classes identified were: high (H, n = 4), average (A, n = 12) and low (L, n = 3) viability. The concentration of sperm cells decreased from class H to A to L. Fresh semen samples with higher viability and a higher proportion of motile cells also maintained better quality after the freezing and thawing procedure. Sperm viability and motility in both fresh and thawed samples were similar in classes H and A, while significantly lower values were measured in class L. The relative decrease in sperm viability and motility after cryopreservation increased from class H to A to L. The lipid content of spermatozoa (micrograms per 10(9) cells) increased significantly after freezing and thawing in classes H and A but not in class L. This result indicated that active sperm lipid metabolism might be responsible for the increase in lipid content. Phospholipid and triacylglycerol contents increased whereas free cholesterol content decreased after thawing. The fatty acid composition of fresh spermatozoa was similar in all three classes. The proportion of polyunsaturated fatty acids decreased significantly after freezing and thawing, indicating contamination from the diluent or peroxidation. After freezing and thawing, superoxide dismutase activity in spermatozoa was significantly higher in class L than in classes H and A, which did not differ from each other.

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Within recent years, there has been growing interest in the prediction of bull fertility through in vitro assessment of semen quality. A model for fertility prediction based on early evaluation of semen quality parameters, to exclude sires with potentially low fertility from breeding programs, would therefore be useful. The aim of the present study was to identify the most suitable parameters that would provide reliable prediction of fertility. Frozen semen from 18 Italian Holstein-Friesian proven bulls was analyzed using computer-assisted semen analysis (CASA) (motility and kinetic parameters) and flow cytometry (FCM) (viability, acrosomal integrity, mitochondrial function, lipid peroxidation, plasma membrane stability and DNA integrity). Bulls were divided into two groups (low and high fertility) based on the estimated relative conception rate (ERCR). Significant differences were found between fertility groups for total motility, active cells, straightness, linearity, viability and percentage of DNA fragmented sperm. Correlations were observed between ERCR and some kinetic parameters, and membrane instability and some DNA integrity indicators. In order to define a model with high relation between semen quality parameters and ERCR, backward stepwise multiple regression analysis was applied. Thus, we obtained a prediction model that explained almost half (R 2=0.47, P<0.05) of the variation in the conception rate and included nine variables: five kinetic parameters measured by CASA (total motility, active cells, beat cross frequency, curvilinear velocity and amplitude of lateral head displacement) and four parameters related to DNA integrity evaluated by FCM (degree of chromatin structure abnormality Alpha-T, extent of chromatin structure abnormality (Alpha-T standard deviation), percentage of DNA fragmented sperm and percentage of sperm with high green fluorescence representative of immature cells). A significant relationship (R 2=0.84, P<0.05) was observed between real and predicted fertility. Once the accuracy of fertility prediction has been confirmed, the model developed in the present study could be used by artificial insemination centers for bull selection or for elimination of poor fertility ejaculates.

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and %tituto per la Difesa e Valorizzazione del Germoplasma Animale, Consiglio Nazionale delle Ricerche, via Celoria 10,20133 Milano, ItalyThe major polyunsaturate in phospholipids of chicken spermatozoa is docosatetraenoic acid, 22:4n-6, which is positively correlatable with sperm motility and fertility. The potential for dietary manipulation of sperm fatty acid in order to improve male fertility has been extensively studied in the chicken. The effects of diets enriched in n-3 and n-6 long chain polyunsaturates have been investigated in different trials using different oil sources and levels of oil inclusion. The 22:6n-3 and 22:Sn-3 content of avian spermatozoa is increased by supplementing the feed with fish oil (rich in 22:6n-3) and linseed oil (rich in 18:3n-3) respectively. The 22:4n-6 content is also increased by supplementing the feed with evening primrose oil (rich in 18:3n-6) in association with high level of vitamin E (200 mgkg) and with arasco oil (rich in 20:4n-6). The effects of these fatty acid manipulations on sperm quality and lor fertility are reviewed. Both n-3 and n-6 rich diets showed a positive effect on sperm movement during the reproductive period and an age-dependent positive effect on fertility. Reported effects of n-6 rich diets on semen production have been variable with 20:4n-6 rich diet having a positive effect on semen volume and thus on total sperm number and 18:3n-6 rich diets having a negative effect on semen concentration. Spermatozoa enriched in 22511-3, or 22:6n-3 or 22:4n-6 result in significantly higher fertility values following artificial insemination compared to control spermatozoa; however, such a positive effect is age dependent and observed at 37 to 42 weeks, but not in older birds. The n-6 fatty acid composition of chicken spermatozoa is recognised as a speciespecific characteristic. The fundamental relation between dietary lipid, spermatozoa fatty acid composition and thus sperm quality and fertility can be seen as having a potential commercial application. Dietary lipid manipulation of chicken semen: S. Cerolini et al. Abbreviation Key: 20:4n-6 arachidonic acid = AA; 22:4n-6 docosatetraenoic acid = DTA; 22:6n-3 docosahexaenoic acid =DHA; long chain polyunsaturated fatty acids = LCPUFA; phosphatidylethanolamine = PE; phosphatidylserine = PS. Dietary lipid manipulation of chicken semen: S. Cerolini et al.