Effects of substrate availability and mitochondrial disruption on oxidative metabolism and sperm motility in fertile dogs.

In several mammalian species, the measurement of mitochondrial oxygen consumption (MITOX) under different metabolic conditions has demonstrated a positive correlation with sperm motility and may be a sensitive indicator of mitochondrial health. In general, the maintenance of sperm motility and many key sperm functions and fertilizing events are heavily energy-dependent processes, and some species-specific substrate preferences exist. Although canine sperm have been known to undergo capacitation and maintain motility with supplementation of a wide range of energy substrates, the relationship between mitochondrial function, and the maintenance of oxidative metabolism and sperm motility remain unclear. The objective of this study was to explore the metabolic flexibility of canine sperm, and to investigate the relationship between mitochondrial function, and maintenance of motility under differing nutrient conditions. We explored substrate preferences and the bioenergetics underlying maintenance of canine sperm motility by monitoring mitochondrial oxidative function and sperm kinematics in the presence of mitochondrial effector drug treatments: FCCP, antimycin (ANTI), and oligomycin (OLIGO). We hypothesized that canine sperm possess the ability to use compensatory pathways and utilize diverse nutrient sources in the maintenance of motility. Oxygen consumption (change in pO2, oxygen partial pressure) and sperm kinematics (CASA) were measured concurrently (t0-t30) to assess the relationship between oxidative metabolism and maintenance of sperm motility in dogs. Four media were tested: containing glucose, lactate, and pyruvate (GLP), containing glucose (G), fructose (F), or lactate and pyruvate (LP). In the absence of pharmacological inhibition of the electron transport chain, energetic substrate had no effect on sperm kinematics in fertile dogs. Following mitochondrial disruption by ANTI and OLIGO, mitochondrial oxygen consumption was negatively correlated with several sperm motility parameters in GLP, G, F, and LP media. In every media, FCCP treatment quickly induced significantly higher oxygen consumption than in untreated sperm, and spare respiratory capacity, the maximal inducible oxidative metabolism, was high. With respiratory control ratios RCR >1 there was no indication of bioenergetic dysfunction in any media type, indicating that sperm mitochondria of fertile dogs have a high capacity for substrate oxidation and ATP turnover regardless of substrate. Our results suggest MITOX assessment is a valuable tool for assessing mitochondrial functionality, and that canine sperm employ flexible energy management systems which may be exploited to improve sperm handling and storage.

Embryo-endometrial interaction associated with the location of the embryo during the mobility phase in mares.

Embryo-maternal crosstalk is essential to establish pregnancy, with the equine embryo moving throughout the uterus on days 9-15 (ovulation = day 0) as part of this interaction. We hypothesized that the presence of a mobile embryo induces local changes in the gene expression of the endometrium. On Day 12, the endometrial transcripts were compared among three groups: uterine horn with an embryo (P+, n = 7), without an embryo (P-, n = 7) in pregnant mares, and both uterine horns of nonbred mares (NB, n = 6). We identified 1,101 differentially expressed genes (DEGs) between P+ vs. NB and 1,229 DEGs between P- vs. NB. The genes upregulated in both P+ and P- relative to NB were involved in growth factor pathway and fatty acid activation, while downregulated genes were associated with oxytocin signaling pathway and estrogen receptor signaling. Comparing the transcriptome of P+ to that of P-, we found 59 DEGs, of which 30 genes had a higher expression in P+. These genes are associated with regulating vascular growth factors and the immune system, all known to be essential in early pregnancy. Overall, this study suggests that the mobile embryo influences the endometrial gene expression locally.

Germ cell recovery, cryopreservation and transplantation in the California white sturgeon, Acipenser transmontanus.

The white sturgeon (Acipenser transmontanus) is the largest freshwater fish in North America. Because of the unique life history characteristics of sturgeon, including longevity, late maturation and long spawning intervals, their aquaculture can be a significant investment of resources. As a result of habitat loss and overharvesting, natural populations of white sturgeon are threatened and there is a growing effort to improve conservation aquaculture programs. Germ cell transplantation is an innovative technology previously demonstrated in a variety of fish species to be able to produce a surrogate broodstock. The technique relies upon optimal donor germ cell recovery and transplantation into a recipient fish. In this study, we developed and optimized the harvest of donor cells for germline transplantation and evaluated methods for ovary cryopreservation for the first time in the white sturgeon. We found that harvesting gonads from juveniles between the ages of 1.5 and 2.5-years resulted in reliably high proportions of pre-meiotic cells regardless of sex, a critical feature for using white sturgeon for transplantation studies since the species shows no distinguishing external sex characteristics. From the viable cells, we identified germline cells using immunolabeling with the antibody DDX4, a marker specific to the germline. For in vivo tracking of donor cells during transplantations, gonadal cells were stained with a long half-life non-toxic cell membrane dye, PKH26, and microinjected into the peritoneal cavity of newly hatched white sturgeon larvae. Larvae were reared until 3 months post-transplantation to monitor for colonization and proliferation of PKH26-labeled cells within the recipient larval gonads. Furthermore, viable cell detection, assessment of germline-specificity, and transplantation was determined for cells recovered from cryopreserved ovarian tissue from sexually immature females. Transplantations using cells cryopreserved with media supplemented with dimethyl sulfoxide (DMSO) rather than ethylene glycol (EG) demonstrated the highest number of PKH26-labeled cells distributed along the gonadal ridges of the larval recipient. Determining optimal methods of tissue cryopreservation, and germ cell recovery and transplantation are foundational to the future development of germ cell transplantation as a strategy to improve the aquaculture and conservation of this species. Our study demonstrates that conservation actions, such as surrogate breeding, could be utilized by hatcheries to retain or improve natural gamete production without genetic modification, and provide an encouraging approach to the management of threatened sturgeon species.

Transcriptome Signature of Immature and In Vitro-Matured Equine Cumulus-Oocytes Complex.

Maturation is a critical step in the development of an oocyte, and it is during this time that the oocyte advances to metaphase II (MII) of the meiotic cycle and acquires developmental competence to be fertilized and become an embryo. However, in vitro maturation (IVM) remains one of the limiting steps in the in vitro production of embryos (IVP), with a variable percentage of oocytes reaching the MII stage and unpredictable levels of developmental competence. Understanding the dynamics of oocyte maturation is essential for the optimization of IVM culture conditions and subsequent IVP outcomes. Thus, the aim of this study was to elucidate the transcriptome dynamics of oocyte maturation by comparing transcriptomic changes during in vitro maturation in both oocytes and their surrounding cumulus cells. Cumulus-oocyte complexes were obtained from antral follicles and divided into two groups: immature and in vitro-matured (MII). RNA was extracted separately from oocytes (OC) and cumulus cells (CC), followed by library preparation and RNA sequencing. A total of 13,918 gene transcripts were identified in OC, with 538 differentially expressed genes (DEG) between immature OC and in vitro-matured OC. In CC, 13,104 genes were expressed with 871 DEG. Gene ontology (GO) analysis showed an association between the DEGs and pathways relating to nuclear maturation in OC and GTPase activity, extracellular matrix organization, and collagen trimers in CC. Additionally, the follicle-stimulating hormone receptor gene (FSHR) and luteinizing hormone/choriogonadotropin receptor gene (LHCGR) showed differential expressions between CC-MII and immature CC samples. Overall, these results serve as a foundation to further investigate the biological pathways relevant to oocyte maturation in horses and pave the road to improve the IVP outcomes and the overall clinical management of equine assisted reproductive technologies (ART).

Embryo Pulsing: Repeated Expansion and Contraction of In Vivo and In Vitro Equine Blastocysts.

Morphokinetic evaluation of embryo development has allowed the discovery of events occurring during blastulation. Here, we describe equine embryo pulsing, determined as continued expansion and contraction of both in vivo and in vitro produced blastocysts. Using time-lapse imaging, we demonstrated that pulsing starts during early blastocyst development of in vitro-produced embryos in horses. The median time for a complete contraction was 0.22h (0.08h-2h; min-max) where embryos reduced their sizes around 12.0% (median; 2.3%-27.0%) and the median time for an expansion was 3.3h (0.75-9.0h) where embryo re-expanded around 16.9% (3.2%-42.8%). We also found that pulsing can be observed in in vivo-produced embryos obtained from mares 6.5 days after ovulation and continues during the expansion of the blastocysts. Even though its exact mechanism remains unknown, studies in human IVF suggest that the pulsing of embryos is associated with embryo quality and implantation rates. Thus, further investigations regarding this event in equine in vitro production procedures are warranted. Additionally, the pulsing in the in vivo-produced embryos could explain the diverse morphology occasionally observed in the collected or shipped embryos. Future studies are necessary to understand the underlying mechanism of pulsing and its association with embryo quality and embryo transfer outcome.

Canine Semen Evaluation and Processing.

Advances in canine semen evaluation have progressed over time in fits and spurts, interspersed with long periods of relative inactivity. Despite exciting advances in the semen analysis, clinical canine theriogenology has been in a period of relative inactivity for a number of decades since initial advances in canine semen freezing in the mid 20th century. This review describes ways that the clinical practice of canine semen evaluation should improve, given the state of current knowledge.

Effect of pentobarbital as a euthanasia agent on equine in vitro embryo production.

Postmortem and pre-euthanasia oocyte retrieval provides the last opportunity to preserve the genetic material in mares. Pentobarbital (PB) is the most common euthanasia agent; however, its effect on the developmental competence of oocytes has not been determined. Here, we evaluated the concentration of PB in equine follicular fluid (FF) and investigated its effect on the developmental competence of oocytes using a bovine IVF model to overcome the low availability of equine oocytes. The concentration of PB was measured by gas-chromatography/mass-spectrometry in FF collected from mare ovaries immediately after euthanasia (n = 10), 24 h post-euthanasia (n = 10), and from the ovaries collected by ovariectomy (negative control; n = 10). The serum concentration of PB was also evaluated as a positive control. PB was detected in all FF samples with an average concentration of 56.5 µg/ml. Next, bovine cumulus-oocyte complexes (COC) were held in holding media with PB for 6 h at 60 µg/ml (H60, n = 196), 164 µg/ml (H164, n = 215) or without PB (control; n = 212). After holding, the oocytes were matured and fertilized in vitro, followed by in vitro culture to the blastocyst stage. The cumulus expansion grade, cleavage rate, blastocyst rate, embryo kinetic rate and the blastocyst cell numbers were compared among the experimental groups of bovine COC. Higher rates of Grade 1 cumulus expansion were found in controls (54%, 32-76%; median, min-max) in comparison to H60 and H164 (24%,11-33% and 13%, 8-44%; P < 0.001). The cleavage rate was higher in the controls than in H164 (64% vs. 44%; P < 0.01). Blastocyst rates (blastocyst/cleaved oocytes) and total cell number were not different among the groups (control 29%, H60 25%, and H164 24%). In a preliminary study, equine oocytes (n = 28) were exposed to PB in vitro for 6 h followed by intracytoplasmic sperm injection (ICSI) and in vitro embryo production. Exposed oocytes showed a numerically lower maturation rate (43% Vs 52%; P > 0.05) in comparison to the laboratory-established rate during the same timepoints. Overall, we showed that PB reaches the FF immediately after euthanasia, exposing oocytes to this drug. This exposure affected cumulus expansion and cleavage rates in a bovine model, suggesting initial damage caused by PB that may not completely impede the formation of embryos, although lower overall embryo numbers might be obtained.

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.

Comparative oxidative metabolism in mammalian sperm.

In this review, we discuss the physiology of mitochondrial function in sperm using a comparative species approach. Mitochondria impart the ability for sperm from internal fertilizing species to attain individual motility and the ability to navigate the female reproductive tract to the site of fertilization. The presence of reactive oxygen species (ROS) is a normal physiological event of the mitochondrial electron transport chain (ETC); however, when excessive leakage of ROS occurs, sperm damage may follow. ROS production is associated with high levels of sperm motility but must be delicately balanced to prevent cellular damage during post-ejaculatory transport events. We discuss the differences in fundamental oxygen and ATP substrate balance in three mammalian species of veterinary importance, with an emphasis on ETC function, ROS production, and the balance of glycolytic and oxidative phosphorylation production of ATP in sperm.

Characterization of sperm cell membrane charge and selection of high-quality sperm using microfluidics in stallions.

Intracytoplasmic sperm injection (ICSI) is the only method for in vitro embryo production (IVP) in horses. Besides oocyte developmental competence, the outcome of IVP is also highly dependent on sperm quality. Therefore, it is not only essential to employ superior methods of selecting high quality sperm, but also to be able to characterize which quantifiable properties of sperm quality are most indicative of its fertility. In men, a net negative surface charge, estimated by zeta potential (ZP) is highly correlated with sperm quality and in vitro embryo developmental outcomes. However, there is no information available about approximate charges or ZP in equine sperm. Therefore, in this study we aimed to characterize equine sperm ZP and identify its associations with known measures of sperm quality. Additionally, we aimed to complete a comprehensive comparison of conventional sperm selection techniques as compared to the novel method of microfluidic sorting. Ejaculates (n = 22) were partitioned into fresh (∼23 °C, 0 h; n = 12) and cooled (∼4 °C, 24 h; n = 10) groups, and processed by swim up (SU), density gradient centrifugation (DGC), density gradient-swim up combination (DG-SU), and microfluidic chip (MF) sorting. Motility, progressive motility, cell viability, normal morphology, and ZP were evaluated for both unprocessed fractions and post-selected fractions. The ZP of both fresh and cooled samples was net negative and also correlated with motility and progressive motility for both fresh and cooled samples (P < 0.05). The ZP of cooled samples was also correlated with viability (P < 0.05). Among the compared methods of sperm selection, MF was highly effective in selecting high quality sperm as determined by the measured parameters. Percent motility, progressive motility, normal morphology, and viability of MF selected sperm were of higher quality than sperm selected by SU, and of similar to DG-SU and DGC without the use of potentially harmful centrifugation steps. Correlations between ZP, motility, and viability parameters may indicate a role of external charge on the motility and survival of sperm within the female reproductive tract. In conclusion, we identified an average net negative ZP on equine sperm and correlations between ZP and other measures of sperm quality, as well as having identified MF as a novel effective method of equine sperm selection for IVP.

Effect of Transvaginal Aspiration of Oocytes on Blood And Peritoneal Fluid Parameters in Mares.

Transvaginal aspiration of oocytes (TVA) is a commonly used clinical procedure to obtain oocytes for in vitro production of embryos in horses. The goal of this study was to evaluate the effect of the TVA procedure on blood and peritoneal parameters, and to investigate the association of these findings with variables such as use of antibiotics, number of ovarian punctures, and length of the procedure. Physical examination was performed and blood and peritoneal fluid were obtained from 14 mares before they underwent TVA and the same parameters were assessed 24 hours after the procedure. On examination, 13/14 mares remained clinically healthy after the procedure. One mare developed low-grade fever, transient anorexia and mild colic that resolved within 12 hours post-TVA. The use of antibiotics, length of procedure and number of ovarian punctures did not have an effect on the measured parameters. All the mares presented significant changes in the leukogram, but these mostly remained within normal reference range values. The peritoneal parameters were also consistently affected after TVA. A significant post-TVA increase in lactate, total protein, and peritoneal neutrophil count was observed in peritoneal fluid. Peritoneal lactate level was elevated above baseline physiological levels in more than 50% of the mares. Results from this study indicate that there is an expected degree of inflammation after TVA procedures and peritoneal fluid parameters could be successfully used to monitor inflammation in the early stages.

An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section I.

As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of semen physiology as it applies to overall fertility. This segment of our two-section review will focus on normal sperm parameters, beginning with development and extending through the basic morphology of mature spermatozoa, as well as common issues with male factor infertility in IVP. Ultimately, the relevance of sperm parameters to overall male factor fertility in equine IVP will be assessed.

An Update on Semen Physiology, Technologies, and Selection Techniques for the Advancement of In Vitro Equine Embryo Production: Section II.

As the use of assisted reproductive technologies (ART) and in vitro embryo production (IVP) expand in the equine industry, it has become necessary to further our understanding of available semen selection techniques. This segment of our two-section review will focus on the selection of spermatozoa based on quality and sex for equine intracytoplasmic sperm injection (ICSI), as well as current and future developments in sperm sorting technologies. Ultimately, novel methods of semen selection will be assessed based on their efficacy in other species and their relevance and future application towards ARTs in the horse.

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.

Genetic Manipulation of the Equine Oocyte and Embryo.

As standard in vitro fertilization is not a viable technique in horses yet, many different techniques have been used to create equine embryos for research purposes. One such method is parthenogenesis in which an oocyte is induced to mature into an embryo-like state without the introduction of a spermatozoon, and thus they are not considered true embryos. Another method is somatic cell nuclear transfer (SCNT), in which a somatic cell nucleus from an extant horse is inserted into an enucleated oocyte, creating a genetic clone of the donor horse. Due to limited availability of equine oocytes in the United States, researchers have investigated the potential for combining equine somatic cell nuclei with oocytes from other species to make embryos for research purposes, which has not been successful to date. There has also been a rising interest in producing transgenic animals using sperm exposed to exogenous DNA. The successful creation of transgenic equine blastocysts shows the promise of sperm mediated gene transfer (SMGT), but this method is not ideal for other applications, like gene therapy, because it cannot be used to induce targeted mutations. That is why technologies like CRISPR/Cas9 are vital. In this review, we argue that parthenogenesis, SCNT, and interspecies SCNT can be considered genetic manipulation strategies as they create embryos that are genetically identical to their parent cell. Here, we describe how these methods are performed and their applications and we also describe the few methods that have been used to directly modify equine embryos: SMGT and CRISPR/Cas9.

A Review of OCT4 Functions and Applications to Equine Embryos.

OCT4 is a core transcription factor involved in pluripotency maintenance in the early mammalian embryo. The POU5F1 gene that encodes the OCT4 protein is highly conserved across species, suggesting conserved function. However, studies in several species including mice, cattle, and pigs, suggest that there are differences in where and when OCT4 is expressed. Specifically, in the horse, several studies have shown that exposure to the uterine environment may be necessary to induce OCT4 expression restriction to the inner cell mass (ICM) of the developing embryo, suggesting that there may be equine-specific extrinsic regulators of OCT4 expression that have not yet been investigated. However, an alternative hypothesis is that this restriction may not be evident in equine embryos because of our inability to culture them to the epiblast stage, preventing the observation of this restriction. In vitro studies have identified that OCT4 is expressed in the immature equine oocyte and in the early equine embryo, but OCT4 expression has not been studied after the formation of the ICM in the equine embryo. Despite the gaps in knowledge about equine-specific functions of OCT4, this factor has been used in studies assessing equine embryonic stem cells and to induce pluripotency in equine somatic cells. This review describes the role of OCT4 in the equine embryo and its applications in equine stem cell research.

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.

Effects from aging on semen quality of fresh and cryopreserved semen in Labrador Retrievers.

Characteristics of frozen sperm associated with fertility and aging have not been fully determined in dogs. The aim of this study was to determine the relationship between fresh and post-thaw sperm quality, age, among the factors of motility, viability, morphology and oxidative stress in a group of fertile stud dogs with extensive breeding records and in dogs with reported subfertility problems. Sperm parameters from 39 fertile dogs were measured in fresh semen and frozen thawed semen. Additionally, frozen semen from 9 subfertile dogs was thawed and analyzed. Subfertile dogs were defined by referring veterinarians solely on the basis of owner history; breeding records were not available to this study. Evaluation included total motility (TM) and progressive motility (PM), average path velocity (VAP), viability, morphology and presence of sperm reactive oxygen species (ROS). Fertile males' ages ranged from 1 to 10 years, subfertile males' ages ranged from 4 to 14 years. All dogs were assigned to age groups according to age as young (1-3 years) middle (4-6 years) and senior (>7 years). The effect of sperm state (fresh vs. frozen-thawed), age, and fertility history (fertile vs. subfertile) on the measured endpoints were determined using a mixed effects model. TM showed a negative correlation with age in the frozen-thawed semen. Additionally, TM in fresh semen was higher in all age groups compared to post thaw semen (P < 0.05). PM was higher in all age dogs of fresh semen compared to frozen-thawed semen (P < 0.05). As such, TM and PM in post thaw semen statistically decreased compared to fresh semen regardless of age of stud male (P < 0.05). Differences in VAP were only observed between young and senior age groups in fresh semen (P < 0.05) while young and middle age dogs from fresh semen were different in all age groups for frozen-thawed semen (P < 0.05). ROS was higher in the young frozen-thawed semen compared to the young fresh semen (P < 0.05). When comparing TM, PM and VAP of fertile vs. subfertile dogs, middle aged fertile dogs are different from middle aged and senior subfertile dogs (P < 0.05). No differences were found in measures of ROS between fertile and subfertile. For sperm viability and morphology, differences were observed between all age groups of fertile dogs in comparison to all ages of subfertile dogs (P < 0.05). Sperm motility appeared to be the most affected parameter by freezing damage than any other parameter measured in this study, while we were not able to determine a significant association between ROS production and fertility status.