Characterisation of the immunomodulatory properties of spermatozoa from high and low field fertility bulls

Despite passing all microscopy based quality control (QC) assessments at artificial insemination (AI) centres, bulls with apparently normal semen quality yield unacceptably low pregnancy rates. Based on a statistical animal adjusted model (AAM), bulls can vary in fertility rates by up to 25 % when used in the field. The overall aim of this thesis was to further our understanding of the biology underpinning the causes of sire sub-fertility and to pinpoint where along the pregnancy establishment axis variation occurs. Bulls with divergent fertilities were chosen from a population of 840 Holstein Friesian bulls with > 500 inseminations and with a minimum of 10 % between high fertility (HF) and low fertility (LF) treatment groups. Ex vivo assessments of sperm transport in the female reproductive tract identified a higher curvilinear velocity (VCL) as well as a higher number of spermatozoa bound to oviductal explants (p<0.05). As the uterus is normally the site of semen deposition during AI, using an ex vivo model with heifer follicular phase uterine explants, the inflammatory response to frozen-thawed spermatozoa from HF and LF bulls was characterised. Results identified a significant up-regulation of inflammatory gene expressions that were confirmed at both gene and protein level from frozen-thawed spermatozoa although, there was no difference between HF and LF treatments. Subsequently, sperm - polymorphonuclear neutrophils (PMN) binding was assessed with results reporting a higher number of spermatozoa from the LF group bound per PMN. An in vivo trial was carried out to profile the transcriptome of the uterus of heifer inseminated with HF or LF semen or no insemination (control group). RNA-sequencing revealed a more active transcriptomic response in the uterus of heifers inseminated with HF bulls (845 differentially expressed genes (DEGs)) compared to the control group with just four DEGs between the LF and control group. Uterine transcriptomic analysis showed a prominent role for the immune response with inflammatory marker Interleukin 1 alpha (IL1A) identified as the top DEG between HF and LF treatments. As well as delivering the paternal haploid genome, spermatozoa also deliver an abundance of miRNAs and mRNAs to the oocyte. Transcriptomic profiling of micro RNAs (miRNAs) and messenger RNAs (mRNA) from HF and LF bulls identified differentially expressed miRNAs related to embryonic development and an important involvement in protamine 1 (PRM1) which is a core element of sperm chromatin structure. Overall, this thesis has reported significant advances in the understanding of bull fertility variation and the molecular mechanisms underpinning sire sub-fertility. In particular, it has highlighted differences between semen from HF and LF bulls in relation to the inflammatory response in the uterus with consequences for sperm transport to the site of fertilisation and priming the endometrium for pregnancy as well as identifying differentially expressed miRNA and mRNAs that deliver transcripts to the oocyte that have roles in implantation and embryo development

Characterisation of the immunomodulatory properties of spermatozoa from high and low field fertility bulls

Despite passing all microscopy based quality control (QC) assessments at artificial insemination (AI) centres, bulls with apparently normal semen quality yield unacceptably low pregnancy rates. Based on a statistical animal adjusted model (AAM), bulls can vary in fertility rates by up to 25 % when used in the field. The overall aim of this thesis was to further our understanding of the biology underpinning the causes of sire sub-fertility and to pinpoint where along the pregnancy establishment axis variation occurs. Bulls with divergent fertilities were chosen from a population of 840 Holstein Friesian bulls with > 500 inseminations and with a minimum of 10 % between high fertility (HF) and low fertility (LF) treatment groups. Ex vivo assessments of sperm transport in the female reproductive tract identified a higher curvilinear velocity (VCL) as well as a higher number of spermatozoa bound to oviductal explants (p<0.05). As the uterus is normally the site of semen deposition during AI, using an ex vivo model with heifer follicular phase uterine explants, the inflammatory response to frozen-thawed spermatozoa from HF and LF bulls was characterised. Results identified a significant up-regulation of inflammatory gene expressions that were confirmed at both gene and protein level from frozen-thawed spermatozoa although, there was no difference between HF and LF treatments. Subsequently, sperm - polymorphonuclear neutrophils (PMN) binding was assessed with results reporting a higher number of spermatozoa from the LF group bound per PMN. An in vivo trial was carried out to profile the transcriptome of the uterus of heifer inseminated with HF or LF semen or no insemination (control group). RNA-sequencing revealed a more active transcriptomic response in the uterus of heifers inseminated with HF bulls (845 differentially expressed genes (DEGs)) compared to the control group with just four DEGs between the LF and control group. Uterine transcriptomic analysis showed a prominent role for the immune response with inflammatory marker Interleukin 1 alpha (IL1A) identified as the top DEG between HF and LF treatments. As well as delivering the paternal haploid genome, spermatozoa also deliver an abundance of miRNAs and mRNAs to the oocyte. Transcriptomic profiling of micro RNAs (miRNAs) and messenger RNAs (mRNA) from HF and LF bulls identified differentially expressed miRNAs related to embryonic development and an important involvement in protamine 1 (PRM1) which is a core element of sperm chromatin structure. Overall, this thesis has reported significant advances in the understanding of bull fertility variation and the molecular mechanisms underpinning sire sub-fertility. In particular, it has highlighted differences between semen from HF and LF bulls in relation to the inflammatory response in the uterus with consequences for sperm transport to the site of fertilisation and priming the endometrium for pregnancy as well as identifying differentially expressed miRNA and mRNAs that deliver transcripts to the oocyte that have roles in implantation and embryo development

Comprehensive functional analysis reveals that acrosome integrity and viability are key variables distinguishing artificial insemination bulls of varying fertility

In vitro methods of assessing bull semen quality in artificial insemination (AI) centers are unable to consistently detect individuals of lower fertility, and attempts to reliably predict bull fertility are still ongoing. This highlights the need to identify robust biomarkers that can be readily measured in a practical setting and used to improve current predictions of bull fertility. In this study, we comprehensively analyzed a range of functional, morphological, and intracellular attributes in cryopreserved spermatozoa from a selected cohort of Holstein Friesian AI bulls classified as having either high or low fertility (n = 10 of each fertility phenotype; difference of 11.4% in adjusted pregnancy rate between groups). Here, spermatozoa were assessed for motility and kinematic parameters, morphology, acrosome integrity, plasma membrane lipid packing, viability (or membrane integrity), superoxide production, and DNA integrity. In addition, spermatozoa were used for in vitro fertilization to evaluate their capacity for fertilization and successful embryo development. The information collected from these assessments was then used to phenotypically profile the 2 groups of bulls of divergent fertility status as well as to develop a model to predict bull fertility. According to the results, acrosome integrity and viability were the only sperm attributes that were significantly different between high- and low-fertility bulls. Interestingly, although spermatozoa from low-fertility bulls, on average, had reduced viability and acrosome integrity, this response varied considerably from bull to bull. Principal component analysis revealed a sperm phenotypic profile that represented a high proportion of ejaculates from low-fertility bulls. This was constructed based on the collective influence of several sperm attributes, including the presence of cytoplasmic droplets and superoxide production. Finally, using the combined results as a basis for modeling, we developed a linear model that was able to explain 47% of the variation in bull field fertility in addition to a logistic predictive model that had a 90% chance of distinguishing between fertility groups. Taken together, we conclude that viability and acrosome integrity could serve as fertility biomarkers in the field and, when used alongside other sperm attributes, may be useful in detecting low-fertility bulls. However, the variable nature of low-fertility bulls suggests that additional, in-depth characterization of spermatozoa at a molecular level is required to further understand the etiology of low fertility in dairy bulls

Comprehensive functional analysis reveals that acrosome integrity and viability are key variables distinguishing artificial insemination bulls of varying fertility

In vitro methods of assessing bull semen quality in artificial insemination (AI) centers are unable to consistently detect individuals of lower fertility, and attempts to reliably predict bull fertility are still ongoing. This highlights the need to identify robust biomarkers that can be readily measured in a practical setting and used to improve current predictions of bull fertility. In this study, we comprehensively analyzed a range of functional, morphological, and intracellular attributes in cryopreserved spermatozoa from a selected cohort of Holstein Friesian AI bulls classified as having either high or low fertility (n = 10 of each fertility phenotype; difference of 11.4% in adjusted pregnancy rate between groups). Here, spermatozoa were assessed for motility and kinematic parameters, morphology, acrosome integrity, plasma membrane lipid packing, viability (or membrane integrity), superoxide production, and DNA integrity. In addition, spermatozoa were used for in vitro fertilization to evaluate their capacity for fertilization and successful embryo development. The information collected from these assessments was then used to phenotypically profile the 2 groups of bulls of divergent fertility status as well as to develop a model to predict bull fertility. According to the results, acrosome integrity and viability were the only sperm attributes that were significantly different between high- and low-fertility bulls. Interestingly, although spermatozoa from low-fertility bulls, on average, had reduced viability and acrosome integrity, this response varied considerably from bull to bull. Principal component analysis revealed a sperm phenotypic profile that represented a high proportion of ejaculates from low-fertility bulls. This was constructed based on the collective influence of several sperm attributes, including the presence of cytoplasmic droplets and superoxide production. Finally, using the combined results as a basis for modeling, we developed a linear model that was able to explain 47% of the variation in bull field fertility in addition to a logistic predictive model that had a 90% chance of distinguishing between fertility groups. Taken together, we conclude that viability and acrosome integrity could serve as fertility biomarkers in the field and, when used alongside other sperm attributes, may be useful in detecting low-fertility bulls. However, the variable nature of low-fertility bulls suggests that additional, in-depth characterization of spermatozoa at a molecular level is required to further understand the etiology of low fertility in dairy bulls