Sperm parameters in the Great Dane: Influence of age on semen quality.

Not all sires have sperm suitable for chilled or frozen storage, and success in artificial insemination (AI) varies highly among individual dogs and breeds. Fertilizing potential is further complicated as sperm quality declines with the aging process. Due to the rapidity of aging and senescence in large breed dogs, associated health and fertility changes may be observed over a shorter period, though this period remains undefined for any breed. Working with a population of purebred Great Danes (GD), our aims were (1) to characterize the distribution of a series of sperm parameters, (2) to distinguish sources of variation in sperm quality within this rapidly aging breed, and (3) to identify changes in sperm quality that may accompany aging. Ejaculates collected from young, middle-aged, and senior Great Dane dogs (n = 50) were evaluated for semen volume, total sperm number and viability, and reactive oxygen species (ROS), in addition to sperm morphology and kinematic parameters. Total testicular volume was also determined using ultrasonography. Testicular volume was not a predictor of sperm production in the GD, however, significant differences between coat colors were identified. Age was negatively associated with total motility, progressive motility, and amplitude of lateral head displacement (ALH) (p < .05). We identified significant relationships between GD male age and TM, PM, and immotility with -9.9%, -9.0%, and +8.3% change per year of age, respectively, which support the anecdotal reports of decline of the fertility with the advance of age in this breed. Sperm of younger GD dogs aged 12 ≤ x < 24 months had significantly higher TM, PM, ALH, and nonlinear motility (p < .05) than older dogs (x ≥ 48 months). High ROS levels were positively associated with TM and PM, average pathway distance (DAP) and straight line distance (DSL), average pathway velocity (VAP), straight line velocity (VSL), and the presence of hairpin tails (p < .05). While age and ROS have significant influences on sperm parameters in the GD, the influence of selection for breed specific phenotypes could help explain the functional significance of the diversity among GD males.

Imaging flow cytometry to characterize the relationship between abnormal sperm morphologies and reactive oxygen species in stallion sperm.

Low levels of intracellular reactive oxygen species (ROS) are essential for normal sperm function and are produced by sperm mitochondria as a byproduct of metabolism, but in excess, ROS can cause catastrophic cellular damage and has been correlated with infertility, poor sperm motility and abnormal morphology in humans. Stallion sperm motility is fueled predominantly by oxidative phosphorylation-produced ATP, requiring high basal rates of mitochondrial function. Consequently, whether elevated ROS production by stallion sperm is an indicator of dysfunctional or highly motile cells has been debated by researchers over the last decade. The objective of this study was to evaluate the relationship between various sperm morphologies and ROS production in fresh and cooled stallion semen by employing the novel method of imaging flow cytometry for stallion semen assessment. For evaluation of fresh semen, single ejaculates (n = 5) were collected from four resident stallions at the University of California, Davis. For the evaluation of 24-h cool-stored semen, single ejaculates were collected from stallions at Texas A&M University (n = 5) and shipped to the University of California, Davis overnight for evaluation. Ejaculate volume, sperm concentration and motility parameters were recorded. Samples were co-stained for viability and ROS detection with SytoxGreen™ and dihydroethidium (DHE), respectively, and evaluated with the Amnis® ImageStream® system (Luminex Corporation). Antimycin, an electron transport chain inhibitor that triggers ROS production (1 µM), was used as a positive control for DHE, while dead cells (2× snap frozen in liquid nitrogen) served as a positive control for SytoxGreen™. Unstained samples were also evaluated as controls. Imaging flow cytometric analysis was performed with the ideas® software (Luminex Corporation). Evaluated morphologies included abnormal head (AH), abnormal midpiece (AM), abnormal tail (AT), proximal cytoplasmic droplet (PD), or distal cytoplasmic droplet (DD), and morphologically normal (MN) cells. For fresh semen, an additional abnormality, coiled tail and midpiece (CTM) was assessed; 24-h cool-stored semen did not contain enough viable CTM cells for analysis. Only cells with obvious, single abnormalities were selected for the first portion of analysis to minimize subjectivity. Mixed effects modelling was used to evaluate the relationship between each morphologic classification and the corresponding DHE fluorescence intensity. Compared to the MN population, ROS production was significantly higher in viable cells with AH, PD and AM (p < .0001) in both fresh and cooled semen. CTM cells had significantly higher levels of ROS production compared to MN cells in fresh semen (p < .0001). There was no significant difference in ROS levels between MN cells and AT and DD cells in either fresh or cooled semen (p > .05). These results suggest that ROS generation is indicative of abnormal cell morphology and function and confirm that imaging flow cytometry is a valuable tool for the assessment of stallion semen.

Good prognosis for survival to hospital discharge in a group of horses with uterine prolapse treated at a veterinary medical teaching hospital.

OBJECTIVE: To describe uterine prolapse, predispositions, and outcomes in mares treated between 1988 and 2019. ANIMALS: 24 mares with uterine prolapse. PROCEDURES: Clinical records were retrospectively reviewed, and follow-up information was gathered. The Mann-Whitney U test and Fisher exact test were used to analyze results for history and outcome variables for potential association with mare survival to hospital discharge. The χ2 test was used to compare breed distribution. RESULTS: Age was known in 23 mares (median, 11.1 years). For 15 mares with exact known parity, the median parity was 3 births (range, 1 to 13 births). For 22 mares, the maiden status was known (7 [32%] maiden; 15 [68%] multiparous). Breed distribution differed significantly between mares with uterine prolapse and all mares treated at the Veterinary Medical Teaching Hospital. Arabians were overrepresented in the uterine prolapse group (7/24 [29%]), compared with all mares treated (4,174/44,474 [9%]). Uterine prolapse occurred within 2 hours after parturition for 10 of 11 mares with known foaling times, after dystocia for 7 mares, and after abortion for 4 mares. Seventeen of 23 (74%) mares survived to hospital discharge. Acute hemorrhage was the most frequent cause of death. There were no correlations between mare survival to hospital discharge and mare parity or age, foal sex, retained fetal membranes, sepsis, or maiden status. Colts were overrepresented in foals with a known sex (12/17). CLINICAL RELEVANCE: Results indicated a breed predisposition to uterine prolapse, with Arabian mares overrepresented among affected mares. No characteristics correlated with mare survival to hospital discharge.

Effects from disruption of mitochondrial electron transport chain function on bull sperm motility.

Sperm mitochondrial function is essential for normal physiology and fertility, but the importance of mitochondrial activity to support specific sperm functions, such as motility, varies between species. It was previously believed that mitochondrial function was not necessary for bull sperm motility [1]; however, this theory is contradicted by recently reported findings that the upper fraction of bull sperm swim-up preparations had both high motility and elevated mitochondrial oxygen consumption rates [2]. The objective of this study was to investigate the relationship between mitochondrial function and motility in bull sperm. We hypothesized that sperm motility would be positively correlated with mitochondrial oxygen consumption (MITOX) but unaffected by pharmacological inhibition of electron transport chain (ETC) activity. This was accomplished by monitoring both mitochondrial oxygen consumption and motility parameters in the presence of mitochondrial effector drug treatments. Duplicate ejaculates were collected by electroejaculation from Black Angus bulls (n = 4). Oxygen consumption, as % air saturation (pO2; oxygen partial pressure), over time was monitored in the presence of 5 drug treatments: vehicle control, FCCP, Antimycin (ANTI), Oligomycin (Oligo), and FCCP + Oligomycin (FCCP + OLIGO). Duplicate aliquots were prepared for concurrent motility assessment by computer-assisted sperm analysis (CASA) at 6 and 30 min post-treatment (t6 and t30). The impact of treatments on pO2 (in % air saturation) over time were assessed by generalized linear mixed effects modeling (GLMM) which was also used to test for differences in average motility across treatment conditions and time points (t6 and t30). Pearson product-moment correlation was used to investigate relationships between oxygen consumption and motility parameters. Overall, pO2 differed over time between treatment conditions (p < 0.0001). When compared to the vehicle treatment, ANTI and OLIGO significantly inhibited oxygen consumption (p < 0.05, adjusted), and FCCP stimulated a marked increase in oxygen consumption. No significant differences in motility over time were observed between treatments, so comparison of motility parameters between treatment conditions was performed with pooled timepoints. Motility parameters were only observed to differ significantly from the vehicle with ANTI Treatment, for which significant decreases in numerous parameters, including total motility (p = 0.007), progressive motility (p = 0.01), DAP (p = 0.01), VAP (p = 0.01) and VSL (p = 0.02) were identified. For the vehicle treatment, correlational analysis did not reveal any significant correlations between pO2 and any motility parameters at t6; however, several significant correlations were identified at t30. Mean pO2 was negatively correlated with local motility (p < 0.01) and positively correlated with DCL, DAP, and VCL (p < 0.05). Results from this study suggest that bovine sperm motility is impacted by mitochondrial functionality, with ETC inhibition by ANTI causing significant reduction in motility parameters. This study also demonstrates the use of a new technology for the assessment of bovine sperm mitochondrial function. This modality for evaluation of bull sperm mitochondrial function will inform future efforts to understand bull sperm function and fertility and aid investigations into toxicological agents.

Assessment of an iPad-based sperm motility analyzer for determination of canine sperm motility.

The objective of this study was to evaluate the repeatability and accuracy of canine sperm motility (total and progressive) assessment with a tablet-based Canine iSperm instrument compared to computer-assisted sperm analysis (CASA). The experiment used fresh and frozen/thawed canine semen samples for comparisons of semen analysis parameters (concentration, total motility, and progressive motility) between a CASA system, iSperm, and NucleoCounter SP-100 (concentration) instruments. Spearman's Rho correlational analysis was used to identify significant associations between motility assessment methods. Significant positive correlations were found between CASA assessment and iSperm for both progressive and total motility measurements. We also determined the coefficient of variation (CV) for repeatability of sample analysis for iSperm and CASA for fresh sperm, wherein each sample was assessed 10 times on both devices. For fresh and frozen-thawed samples, concentration assessment by iSperm showed high variability (CV= 19.9 ± 1.5%). For iSperm assessment of total and progressive motility, the CVs were 6.3 ± 0.5% and 10.7 ± 0.8%, respectively. The results indicate that the iSperm application offers an accurate and alternative measurement of motility to traditional CASA analysis, though caution should be taken when assessing concentration due to the high CV observed in this study.

Histological, Immunological, and Genetic Analysis of Feline Chronic Gingivostomatitis.

Feline chronic gingivostomatitis (FCGS) is an immune-mediated inflammatory condition affecting the oral mucosa that results in substantial pain and suffering. The goal of this study was to complete an in-depth immunohistochemistry analysis of affected FCGS mucosa, to perform and compare immune cell phenotypes in the blood of FCGS and healthy controls cats, and to determine a transcriptomic profile of the affected and normal oral mucosa of FCGS cats. We hypothesized that cats with FCGS would have circulating activated CD8+ T cells and that tissues would be infiltrated with activated B and T cells with a highly proinflammatory transcriptome. We found that oral mucosal tissues from cats with FCGS have high tissue infiltration of B cells and that T cells include both CD4+ and CD8+ lymphocytes. Cells positive for CD25 (IL2 receptor, indicative of lymphocyte activation) and FOXP3 (indicative of regulatory T cells) were scattered throughout the mucosa. Compared to healthy individuals, cats with FCGS had high circulating CD8+ effector memory cells with a concurrent decrease in central memory cells and evidence of circulating activated CD8+ T cells (CD25+, CD62L-). Gene expression in the affected tissues was enriched for genes associated with T-cell signaling, cell adhesion molecules, leukocyte migration, inflammatory signaling pathways, extracellular matrix-receptor interactions, cytokine-cytokine receptor interactions, and natural killer cell-mediated cytotoxicity, among others. These data are essential to understand disease pathogenesis, to inform mechanism of action studies for future and current therapies, and to help select prognostic biomarkers and potency assays for stem cell treatment of FCGS.

Sperm mitochondrial regulation in motility and fertility in horses.

The biological nature of age-related declines in fertility in males of any species, including stallions, has been elusive. In horses, the economic costs to the breeding industry are frequently extensive. Mitochondrial function in ejaculated sperm, which is essential for sperm motility, is reflected by adenosine triphosphate production, mitochondrial oxidative efficiency and production of reactive oxygen species, and that this balance may become compromised in ageing stallions and during the process of cryopreservation. This presentation will focus on mitochondrial integrity and function as an avenue for understanding the pathophysiology of sperm when undergoing cryopreservation and male ageing. We discuss the importance of understanding the differences and similarities of sperm mitochondria to that of somatic cells regarding structure and mitochondrial biochemistry relating to sperm function. The roles of oxidative phosphorylation and glycolysis in sperm mitochondria are outlined as is the method of determining oxygen consumption and calcium homoeostasis in sperm mitochondria. Further, we outline the role of oxidative stress and reactive oxygen species.