Impact of bull age, sperm processing, and microclimatic conditions on the viability and DNA integrity of cryopreserved bovine sperm.

Context Seasonal microclimatic fluctuations can cause changes in sperm quality even in dairy bulls bred under temperate climate. These changes can vary between sires of different age and affect sperm freezability. Aims We aimed to evaluate the modulating effect of bull age and equilibration time before freezing on the seasonal pattern of sperm viability and DNA integrity post-thaw. Methods In the frame of systematic sperm quality control, we assessed the integrity of sperm plasma membrane and acrosome (PMAI) in 15,496 cryopreserved bovine batches, and the percentage of sperm with high DNA fragmentation index (%DFI) after 0h and 3h incubation at 38°C post-thaw (3h) in 3422 batches. Semen was equilibrated for 24h before freezing if collected on Monday or Wednesday and 72h if produced on Friday. We investigated the effect of season, bull age, equilibration, and temperature-humidity index (THI) on the day of semen collection on sperm traits using mixed-effects linear models. Key results PMAI and %DFI (0h and 3h) deteriorated with increasing THI. The effect of THI on %DFI was detected with a 30-day time lag. Seasonal fluctuations of sperm quality were similar between young, mature, and older sires. Prolonged equilibration did not affect PMAI but was linked to elevated %DFI (3h) in summer. Conclusions Extending equilibration from 24 to 72h is compatible with commercial standards of bovine sperm quality post-thaw; however, it could interfere with the seasonal pattern of the latter. Implications Systematic monitoring of bovine sperm quality enables the prompt detection of stress factors related to microclimate and semen processing.

Mitoquinone does not improve sperm cryo-resistance in bulls.

Oxidative stress is associated with impaired post-thaw sperm quality. As mitochondria are the main source of reactive oxygen species (ROS) in sperm, the goal of this study was to evaluate effects of the mitochondria-targeting antioxidant Mitoquinone (MitoQ) during cryopreservation of bull sperm. Semen was collected from 11 Simmental bulls (two ejaculates per bull) and diluted in Triladyl® supplemented with various concentrations of MitoQ (0, 0.2, 2, and 20 nM) to a final concentration of 65 × 106  sperm/ml. After thawing (0 and 3 hr), we assessed the following sperm traits: sperm motility by computer-assisted sperm analysis (CASA), DNA fragmentation index by SCSA® and plasma and acrosome membrane integrity, intracellular calcium concentration, esterase activity, mitochondrial membrane potential and synthesis of ROS using two multicolour flow cytometric assays. After 3 hr of incubation, 20 nM MitoQ increased (p < .05) sperm ROS synthesis compared to Control, whereas none of the other quality parameters were altered (p > .05). Therefore, we concluded that addition of MitoQ to semen extender before cryopreservation of bull sperm was unable to improve post-thaw sperm quality. Furthermore, 20 nM of MitoQ increased frozen-thawed sperm ROS synthesis, without apparent negative effects on the evaluated sperm traits.

The micro-RNA content of unsorted cryopreserved bovine sperm and its relation to the fertility of sperm after sex-sorting.

BACKGROUND: The use of sex-sorted sperm in cattle assisted reproduction is constantly increasing. However, sperm fertility can substantially differ between unsorted (conventional) and sex-sorted semen batches of the same sire. Sperm microRNAs (miRNA) have been suggested as promising biomarkers of bull fertility the last years. In this study, we hypothesized that the miRNA profile of cryopreserved conventional sperm is related to bull fertility after artificial insemination with X-bearing sperm. For this purpose, we analyzed the miRNA profile of 18 conventional sperm samples obtained from nine high- (HF) and nine low-fertility (LF) bulls that were contemporaneously used to produce conventional and sex-sorted semen batches. The annual 56-day non-return rate for each semen type (NRRconv and NRRss, respectively) was recorded for each bull. RESULTS: In total, 85 miRNAs were detected. MiR-34b-3p and miR-100-5p were the two most highly expressed miRNAs with their relative abundance reaching 30% in total. MiR-10a-5p and miR-9-5p were differentially expressed in LF and HF samples (false discovery rate < 10%). The expression levels of miR-9-5p, miR-34c, miR-423-5p, miR-449a, miR-5193-5p, miR-1246, miR-2483-5p, miR-92a, miR-21-5p were significantly correlated to NRRss but not to NRRconv. Based on robust regression analysis, miR-34c, miR-7859 and miR-342 showed the highest contribution to the prediction of NRRss. CONCLUSIONS: A set of miRNAs detected in conventionally produced semen batches were linked to the fertilizing potential of bovine sperm after sex-sorting. These miRNAs should be further evaluated as potential biomarkers of a sire's suitability for the production of sex-sorted sperm.

Motile sperm subpopulations in bull semen using different clustering approaches - Associations with flow cytometric sperm characteristics and fertility.

There are sperm subpopulations (SPs) with different kinematic characteristics in various species, however, biological relevance of these SPs is still uncertain. The objective of the present study was to investigate associations of motile sperm SPs with sperm characteristics determined by evaluations with flow cytometry and assessment of bull fertility, using multiple approaches for sperm clustering. Semen from 24 bulls was evaluated concomitantly using computer-assisted sperm analysis (CASA) and flow cytometry before freezing and after thawing. Motile SPs were determined utilizing two acknowledged clustering methods (TwoStep and K-Means) and one customized method. With the customized method, there was utilization of mean values of sperm velocity and linearity as thresholds for direct assignment of motile spermatozoa into four SPs. Regardless of approach for identifying SPs, sperm quality, as determined using flow cytometry, was correlated particularly with the subpopulation (SP) of fast and linear spermatozoa immediately after thawing and with the SP of fast and nonlinear spermatozoa before freezing and 3 h after thawing. Furthermore, there was a positive correlation between proportion of spermatozoa with fast and nonlinear movements before freezing and bull non-return to estrous rates. These results indicate that with different sperm SPs, there are different biological implications which can be evaluated to gain useful information concerning semen quality as determined using flow cytometry and fertility. Furthermore, determining SPs by assigning motile spermatozoa into clusters based on a combination of "below and "above" threshold values for sperm velocity and linearity might be considered a practical alternative to otherwise intricate clustering procedures.

Effects of Different Freezing Protocols on Motility, Viability, Mitochondrial Membrane Potential, Intracellular Calcium Level, and DNA Integrity of Cryopreserved Equine Epididymal Sperm.

The aim of the present study was to evaluate the effect of different freezing procedures on sperm motion, viability, the acrosome status, mitochondrial membrane potential (MMP), intracellular calcium content, and DNA integrity on epididymal stallion sperm. Therefore, the sperm of 10 healthy stallions was harvested by retrograde flushing after testectomy, diluted with a semen extender containing defined milk proteins and a freezing extender containing egg yolk and glycerol and frozen according to 4 different protocols, using a programmable freezer and a floating rack performing a slow (processes 1 and 2) or a fast cooling rate (processes 3 and 4, respectively). Post-thaw total motility and slow sperm values were lower when using process 4 compared with processes 1 and 2 (P < .05) after 1 hour of incubation. Progressive motility was lower in process 4 compared with process 1 immediately after thawing and after 1 hour of incubation (P < .05). The amount of rapid sperm was lower when using process 4 compared with process 1 immediately after thawing (P < .05). After 1 hour of incubation, the amount of rapid sperm was lower when using process 4 compared with processes 1 and 2 (P < .05). Higher values for viable sperm were seen in processes 1 and 2 compared with process 4 (P < .05) after 1 hour of incubation. Immediately after thawing, more viable sperm with high MMP (hMMP) were observed when using process 3 compared with process 2 (P < .05). After 1 hour of incubation, a significantly higher amount of viable hMMP sperm were detected when using processes 1 and 2 compared with process 4 (P < .05). Process 2 yielded a lower percentage of sperm containing low calcium (lCa) than process 3 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of lCa sperm was observed using process 4 (P < .05). The subpopulation of viable/hMMP/lCa sperm was higher when using process 3 compared with process 2 immediately after thawing (P < .05). After 1 hour of incubation, the lowest amount of this subpopulation was detected in process 4 (P < .05). The DNA integrity was similar in all groups. In conclusion, a slow cooling rate with a controlled rate freezer resulted in best sperm quality after thawing. Using a floating rack in nitrogen vapor as an alternative to a programmable freezer, equilibration in a cooled environment is advantageous.

"Effect of the addition of different catalase concentrations to a TRIS-egg yolk extender on quality and in vitro fertilization rate of frozen-thawed bull sperm''.

The aim of this study was to investigate the effects of different concentrations of catalase in a TRIS-egg yolk extender on sperm quality and embryonic development after in vitro fertilization of frozen-thawed bull sperm. For this purpose, from each of 7 bulls 2 ejaculates were collected and diluted with a TRIS-egg yolk extender containing 0, 5, 10, 15 or 20 IU catalase/mL. Sperm quality was evaluated 0, 3, 6, 12 and 24 h after thawing by using computer assisted analysis of motility and by flow cytometric assays. Embryonic development was determined after in vitro fertilization of bovine oocytes. Semen diluted with TRIS-egg yolk extender containing different concentrations of catalase showed more motile sperm, more sperm with intact plasma membranes, acrosomes and DNA, a high mitochondrial membrane potential, a high esterase activity, a low calcium level, a lower amount of synthesis of reactive oxygen species and lower degree of lipid peroxidation of sperm compared to semen frozen without catalase (P < 0.05), but not before 3 h after thawing. There was a dose-response relationship with the most prominent effect of 20 IU catalase/mL. However, the improvement of sperm quality had no effect (P ≥ 0.05) on embryonic development after in vitro fertilization with 20 IU catalase/mL. In conclusion, the addition of catalase to the sperm extender improved sperm quality with no obvious effect on in vitro fertility.