Individual bulls differ in their ability to fertilize oocytes due to physiological and molecular characteristics of spermatozoa. The combined use of advanced techniques, as standard semen quality assessment together with sperm molecular investigation, is a promising approach to achieve a better understanding of sperm functions and to predict bull fertility. The aim of this study was to evaluate the relationships among frozen bull sperm parameters, assessed by computer-assisted semen analysis (CASA) and flow cytometry (FCM), and miRNAs expression in high and low fertility bulls. Ten frozen semen doses from 10 Italian Holstein-Friesian bulls, 5 of high and 5 of low fertility according to the estimated relative conception rate (ERCR), were analyzed in two different breeding seasons (Sep-Oct and Feb-Mar), by using CASA system for motility and sperm kinetic parameters assessment and FCM for sperm viability, acrosomal status and DNA integrity evaluation. The semen doses were thawed and pooled; high (HM) and low (LM) motile sperm fractions were isolated by Percoll gradient. For each bull total RNA was extracted from HM fraction and small RNA libraries were generated using Illumina Truseq Small RNA Preparation kit. Libraries were sequenced on a single lane of Illumina Hiseq 2000. Sperm cells were successfully fractionated in HM and LM populations achieving a better quality (P<0.05) in the HM population respect to raw semen and LM population. Fertility level affects raw semen quality for almost all of the variables, significantly for total and progressive motility and for two DNA integrity indicators (degree of chromatin abnormality and percentage of sperm with high green fluorescence), showing a superior quality in high fertile bulls. Season seems not to affect sperm quality. A backward stepwise multiple regression analysis was applied in order to define a model with high relation between semen quality parameters of the HM population and ERCR. A prediction model that explained almost 80% (R2 = 0.78, P < 0.05) of the variation in the conception rate was identified. The model includes five variables: total and progressive motility, curvilinear velocity, degree of chromatin abnormality and percentage of DNA fragmented sperm. miRNA profiling didn't change in sperm collected in different seasons, whereas 9 Bos taurus miRNAs, and 6 new candidate miRNAs were found to be statistically different (False Discovery Rate <0.05) between high and low fertility bulls. Target genes of 9 known miRNAs were predicted, and pathways potentially affecting sperm fertility were identified. Among miRNAs, 5 targeted 469 genes involved in different biological pathways such as regulation of meiosis I and cell division.
Functional characterization and small RNA sequencing of cryopreserved semen in high and low fertility bulls
F Turri;E Capra;P Cremonesi;B Lazzari;TM Gliozzi;A Stella;F Pizzi
2017
Abstract
Individual bulls differ in their ability to fertilize oocytes due to physiological and molecular characteristics of spermatozoa. The combined use of advanced techniques, as standard semen quality assessment together with sperm molecular investigation, is a promising approach to achieve a better understanding of sperm functions and to predict bull fertility. The aim of this study was to evaluate the relationships among frozen bull sperm parameters, assessed by computer-assisted semen analysis (CASA) and flow cytometry (FCM), and miRNAs expression in high and low fertility bulls. Ten frozen semen doses from 10 Italian Holstein-Friesian bulls, 5 of high and 5 of low fertility according to the estimated relative conception rate (ERCR), were analyzed in two different breeding seasons (Sep-Oct and Feb-Mar), by using CASA system for motility and sperm kinetic parameters assessment and FCM for sperm viability, acrosomal status and DNA integrity evaluation. The semen doses were thawed and pooled; high (HM) and low (LM) motile sperm fractions were isolated by Percoll gradient. For each bull total RNA was extracted from HM fraction and small RNA libraries were generated using Illumina Truseq Small RNA Preparation kit. Libraries were sequenced on a single lane of Illumina Hiseq 2000. Sperm cells were successfully fractionated in HM and LM populations achieving a better quality (P<0.05) in the HM population respect to raw semen and LM population. Fertility level affects raw semen quality for almost all of the variables, significantly for total and progressive motility and for two DNA integrity indicators (degree of chromatin abnormality and percentage of sperm with high green fluorescence), showing a superior quality in high fertile bulls. Season seems not to affect sperm quality. A backward stepwise multiple regression analysis was applied in order to define a model with high relation between semen quality parameters of the HM population and ERCR. A prediction model that explained almost 80% (R2 = 0.78, P < 0.05) of the variation in the conception rate was identified. The model includes five variables: total and progressive motility, curvilinear velocity, degree of chromatin abnormality and percentage of DNA fragmented sperm. miRNA profiling didn't change in sperm collected in different seasons, whereas 9 Bos taurus miRNAs, and 6 new candidate miRNAs were found to be statistically different (False Discovery Rate <0.05) between high and low fertility bulls. Target genes of 9 known miRNAs were predicted, and pathways potentially affecting sperm fertility were identified. Among miRNAs, 5 targeted 469 genes involved in different biological pathways such as regulation of meiosis I and cell division.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
