Assessment of sperm attributes of frozen-thawed AI doses from Swedish and Estonian dairy bull sires

The fertility of bull semen used in artificial insemination (AI) is essential for the effective use of this technology in bovine breeding. The laboratory evaluation of semen from healthy bulls, before and after freezing for AI, is largely based on subjectively scoring sperm motility, and on measurement of sperm concentration. Since the relationship between these parameters is barely indicative of the fertility of the semen samples, other, often more complicated methods are used to evaluate a battery of sperm attributes of importance for their fertilizing capacity. This thesis evaluated the usefulness of various conventional and novel sperm quality tests in assessing frozen-thawed (FT) AI doses produced from semen of young, unproven and older, progeny-tested dairy bulls of the Estonian Holstein Friesian (EHF) and Swedish Red and White (SRB) breeds and their relationship with field fertility after AI. In addition, the influence of sperm preparation methods prior to quality measurements was investigated. Semen AI doses were evaluated immediately post-thaw (PT) and after cleansing through washing/resuspension (W) and swim-up (SU). Special attention was given to age-related changes in semen from 1- v. 4-, and 3- v. 5- and 7-year-old bulls. Spermatozoa were evaluated for their morphology (microscopy on wet and dried, stained smears), motility (assessed subjectively and by computer-assisted sperm analysis [CASA]) and membrane integrity (using SYBR-14/propidium iodide [PI] fluorophores and microscopy). Mitochondrial activity (with MitoTracker Deep Red), membrane fluidity (using Merocyanine 540 lipid dye/Yo-Pro-1/H332) and deoxyribonucleic acid (DNA) integrity (using acridine orange [AO] staining) were assayed using flow cytometry (FC). Use of SU provided spermatozoa with significantly better motility, morphology, membrane integrity, mitochondrial activity and chromatin stability compared with either PT or W treatment. Although the SU selection step increased the proportion of spermatozoa with a stable plasma membrane, it also initiated membrane destabilization. Age differences in sperm quality (motility, membrane integrity and mitochondrial function) were seen PT (at 4 years of age for SRB and at 3–7 years for EHF bulls) and were accentuated when using SU, but not W, as pre-selection procedure. No changes in chromatin stability were, however, registered for either breed. Only few FT sperm quality attributes (e.g. average path velocity (VAP), proportion of cells with tail abnormalities, and non-linear motility) showed significant correlation to fertility after AI when evaluated PT or after W. More attributes (e.g. CASA total sperm motility, concentration of motile and linearly motile spermatozoa, VAP, as well as the percentage of spermatozoa with unstable plasmalemma) had a significant relationship with non-return rate (NRR) when spermatozoa were examined after SU. In conclusion, SU was superior to W in harvesting intact spermatozoa with attributes essential for fertilization, distinguishing bulls and revealing age-dependent changes. Also, the use of CASA and FC of fluorophore-loaded spermatozoa increased the objectivity of the tests assayed. Combining SU and CASA, methods considered to be easily applicable at the semen-producing enterprises, indicated that the overall semen quality of proven bulls was predictable from measurements done at an early age. While sperm membrane stability and mitochondrial activity were seen as suitable markers for monitoring semen quality, measurements of chromatin intactness could not yield additional information.