The relationship between mitochondrial DNA copy number and stallion sperm function.

Mitochondrial DNA (mtDNA) copy number has been utilized as a measure of sperm quality in several species including mice, dogs, and humans, and has been suggested as a potential biomarker of fertility in stallion sperm. The results of the present study extend this recent discovery using sperm samples from American Quarter Horse stallions of varying age. By determining copy number of three mitochondrial genes, cytochrome b (CYTB), NADH dehydrogenase 1 (ND1) and NADH dehydrogenase 4 (ND4), instead of a single gene, we demonstrate an improved understanding of mtDNA fate in stallion sperm mitochondria following spermatogenesis. Sperm samples from 37 stallions ranging from 3 to 24 years old were collected at four breeding ranches in north and central Texas during the 2015 breeding season. Samples were analyzed for sperm motion characteristics, nuclear DNA denaturability and mtDNA copy number. Mitochondrial DNA content in individual sperm was determined by real-time qPCR and normalized with a single copy nuclear gene, Beta actin. Exploratory correlation analysis revealed that total motility was negatively correlated with CYTB copy number and sperm chromatin structure. Stallion age did not have a significant effect on copy number for any of the genes. Copy number differences existed between the three genes with CYTB having the greatest number of copies (20.6 ± 1.2 copies, range: 6.0 to 41.1) followed by ND4 (15.5 ± 0.8 copies, range: 6.7 to 27.8) and finally ND1 (12.0 ± 1.0 copies, range: 0.4 to 26.6) (P < 0.05). Varying copy number across mitochondrial genes is likely to be a result of mtDNA fragmentation and degradation since downregulation of sperm mtDNA occurs during spermatogenesis and may be important for normal sperm function. Beta regression analysis suggested that for every unit increase in mtDNA copy number of CYTB, there was a 4% decrease in the odds of sperm movement (P = 0.001). Influential analysis suggested that results are robust and not highly influenced by data from individual stallions despite the low number of stallions sampled with low sperm motility. Further genome sequencing is necessary to investigate if mutations or deletions are the underlying causes of inconsistent copy numbers across mitochondrial genes. In conclusion, we show, for the first time, that increased mtDNA copy number is associated with decreased total sperm motility in stallions. We therefore suggest that mtDNA copy number may be an indicator of defective spermatogenesis in stallions.

Semen evaluation and fertility assessment in a purebred dog breeding facility.

Semen quality in dogs has not been assessed in a longitudinal study that includes endpoints of female fertility and pregnancy. Although use of artificial insemination with chilled semen is increasingly used in canine reproduction, the resultant level of predictability and odds of fertile matings for dogs is still not fully understood. This research provides, for the first time, comprehensive semen evaluation in a large population of dogs in which fertility has been tracked. Duplicate ejaculates were obtained from 39 Labrador retriever males of the Guide Dogs for the Blind (San Rafael, CA, USA) breeding program. Sperm endpoints were determined in fresh semen and extended chilled semen at 48 hour after collection. Evaluation included total and progressive motility, average path velocity, morphology, membrane lipid peroxidation, presence of sperm reactive oxygen species, sperm chromatin structure, and mitochondrial DNA copy number. Male age ranged from 1 to 10 years and were grouped as young (Y; 1-3 years, n = 21), middle aged (M; 4-6 years, n = 13), and senior (S; 7 years or greater, n = 5) for analysis. The effects of age and sperm state (fresh vs. chilled) on the above sperm endpoints were determined using a linear mixed effects model. Semen endpoint values for all parameters were established for this group of fertile males. Progressive motility was only lower in the senior male chilled samples compared to all other groups, fresh and chilled (P < 0.05). Velocity decreased with increasing age and was lower overall in chilled samples (P < 0.05). Percent morphologically normal sperm was lower in senior dogs compared with the other age groups (P < 0.05). The presence of reactive oxygen species was lower in chilled samples compared with fresh (P < 0.05). For sperm chromatin structure, the senior-aged group had a higher %COMPαt than the middle-aged group (P < 0.05). Bayesian analysis determined that no differences were seen in total motility, membrane lipid peroxidation, and mitochondrial DNA copy number, with regard to conception rate or average litter size between age groups or between fresh and chilled samples. We observed no effects from semen quality on fertility or fecundity regardless of age, despite the differences found in semen quality. The use of advanced laboratory tests to evaluate sperm parameters beyond the standard motility, morphology, and concentration will open investigation to more specific and sensitive fertility tests in canine reproduction.

Mitochondrial oxygen consumption is a unique indicator of stallion spermatozoal health and varies with cryopreservation media.

Mitochondrial oxygen consumption is a sensitive indicator of spermatozoal health in the context of cryopreservation. We investigated oxygen consumption of equine sperm mitochondria during incubation in four commercially available sperm cryopreservation extenders: modified INRA 96, BotuCrio, EZ Freezin-"LE" and "MFR5", in addition to several other parameters including motility, reactive oxygen species (ROS) production and viability. All experimental endpoints, with the exception of average path velocity, were affected significantly by freezing extender type after freezing and thawing. Sperm in INRA 96 had the lowest average progressive motility after thawing (24 ± 4.8%, P < 0.05). Sperm in EZ Freezin-"LE" had the highest post thaw viability (79 ± 3.1%, P < 0.05) and lowest post thaw ROS production (13 ± 2.4%), but sperm in BotuCrio had the highest maximal oxygen consumption levels, while also demonstrating similar ROS production and viability. This difference would not have been detected using conventional sperm analytical methods. In addition, sperm in BotuCrio had the highest average total motility (49 ± 7.4%), progressive motility (41 ± 6.4%), and velocity (VAP, 90 ± 3.6 µm/s) indicating that this medium preserved mitochondrial function optimally after cryopreservation. Mitochondrial oxygen consumption was positively correlated with traditional measures of sperm function including motility and viability (r = 0.62 and r = 0.49, respectively, P < 0.05), thus making it a sensitive method for determining cryopreservation success and mitochondrial function in stallion sperm.

Lactate and Pyruvate Are Major Sources of Energy for Stallion Sperm with Dose Effects on Mitochondrial Function, Motility, and ROS Production.

Stallion sperm rely primarily on oxidative phosphorylation for production of ATP used in sperm motility and metabolism. The objective of the study was to identify which substrates included in Biggers, Whitten, and Whittingham (BWW) media are key to optimal mitochondrial function through measurements of sperm motility parameters, mitochondrial oxygen consumption, and cellular reactive oxygen species (ROS) production. It was expected that mitochondrial substrates, pyruvate and lactate, would support sperm motility and mitochondrial function better than the glycolytic substrate, glucose, due to direct utilization within the mitochondria. Measurements were performed after incubation in modified BWW media with varying concentrations of lactate, pyruvate, and glucose. The effects of media and duration of incubation on sperm motility, ROS production, and oxygen consumption were determined using a linear mixed-effects model. Duplicate ejaculates from four stallions were used in three separate experiments to determine the effects of substrate availability and concentration on sperm motility and mitochondrial function and the relationship of oxygen consumption with cellular ROS production. The present results indicate that lactate and pyruvate are the most important sources of energy for stallion sperm motility and velocity, and elicit a dose-dependent response. Additionally, lactate and pyruvate are ideal for maximal mitochondrial function, as sperm in these media operate at a very high level of their bioenergetic capability due to the high rate of energy metabolism. Moreover, we found that addition of glucose to the media is not necessary for short-term storage of equine sperm, and may even result in reduction of mitochondrial function. Finally, we have confirmed that ROS production can be the result of mitochondrial dysfunction as well as intense mitochondrial activity.