Standard semen evaluations routinely used in the cattle industry have limited value for fertility prediction. Sperm kinematic assessment is used as a tool to screen poor-quality sperm but cannot accurately discriminate among samples of high and low fertility. High-resolution respirometry assays that capture direct measurements of mitochondrial function may reveal new sperm indices conducive to modeling bull fertility. The aim of the present study was to determine the efficacy and utility of Oroboros Oxygraph-2k (O2k) for quantifying mitochondrial respiration values of frozen-thawed bull sperm and their relationship to sperm kinematics for estimating fertility in beef-on-dairy artificial insemination programs. Cryopreserved sperm from a total of 8 high and 9 low fertility Angus bulls were resuspended to 20 × 106 sperm/mL under non-capacitating conditions and maintained for 24 h at ambient temperature. Sperm were simultaneously evaluated for kinematics via the CASA system and bioenergetics with O2k at 0 and 24 h post-thaw. No significant differences in fertility (high vs low) and fertility × time were detected for any sperm kinematic traits, which were only affected by time at 24 h. However, a new sperm trait identified by O2k (spare respiratory capacity [SCR]) was greater in high fertility bulls regardless of time (P < 0.05). Multiple linear regression using O2k and CASA-derived traits was employed in a fertility model to determine which sperm traits were collectively useful for estimating bull fertility. Four final traits were curated in the model and regressed against a range of fertility (-7.0 - + 3.3). The final model included four indices that collectively presented a high relationship with fertility (R2 = 0.81). These traits included SCR (R2 = 0.53), progressive motility (R2 = 0.24), total motility (R2 = 0.05), and complex-I dependent respiration (R2 = 0.04). In conclusion, high-resolution mitochondrial assessment of frozen-thawed bull sperm reveals new traits that when coupled with sperm kinematic values, provide increased accuracy of fertility estimation that collectively may be useful for adaptation and inclusion with standard semen analyses. These studies reveal the utility of time for identifying differences between high- and low-fertility bulls and a novel relationship of sperm spare respiratory capacity with fertility.
Keywords: artificial insemination; bioenergetics; bovine; cryopreservation; mitochondria; semen.
The quality of bull sperm for artificial insemination is primarily determined by quantifying sperm motility, but sperm movement alone is not very useful for predicting bull fertility. As sperm motility and fertility rely partly upon healthy mitochondrial function, high-resolution respirometry assays that objectively quantify new bioenergetic sperm traits may be more accurate for estimating bull fertility. The aim of this study was to implement the Oroboros Oxygraph-2k (O2k) system for assessing sperm mitochondrial respiration towards developing a predictive model that can estimate bull fertility prior to artificial insemination. Frozen-thawed bull sperm from 8 high and 9 low-fertility bulls were used to determine different movement parameters and mitochondrial bioenergetics. Multiple linear regression using these sperm traits was employed in a prediction model for estimating bull fertility. Using the O2k assay, we identified a new sperm trait (spare respiratory capacity) that was related to fertility. When spare respiratory capacity was combined with three other measurements of sperm movement and mitochondrial bioenergetics, a statistical model explained 81% of observed fertility differences among bulls. In conclusion, quantifying mitochondrial respiration of bull sperm allows analyses of new sperm traits that can be included in statistical models to predict bull fertility for commercial artificial insemination programs.
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