Redox regulation and oxidative stress have become areas of major interest in spermatology. Alteration of redox homeostasis is recognized as a significant cause of male factor infertility and is behind the damage that spermatozoa experience after freezing and thawing or conservation in a liquid state. While for a long time, oxidative stress was just considered an overproduction of reactive oxygen species, nowadays it is considered as a consequence of redox deregulation. Many essential aspects of spermatozoa functionality are redox regulated, with reversible oxidation of thiols in cysteine residues of key proteins acting as an “on–off” switch controlling sperm function. However, if deregulation occurs, these residues may experience irreversible oxidation and oxidative stress, leading to malfunction and ultimately death of the spermatozoa. Stallion spermatozoa are “professional producers” of reactive oxygen species due to their intense mitochondrial activity, and thus sophisticated systems to control redox homeostasis are also characteristic of the spermatozoa in the horse. As a result, and combined with the fact that embryos can easily be collected in this species, horses are a good model for the study of redox biology in the spermatozoa and its impact on the embryo.

An overview of the sperm metabolism is presented; using the stallion as a model we review glycolysis, Krebs Cycle and oxidative phosphorylation, paying special attention to the interactions among them. In addition, metabolism implies a series of coordinated oxidation-reduction reactions and in the course of these reactions reactive oxygen species (ROS) and reactive oxoaldehydes are produced ; the electron transport chain (ETC) in the mitochondria is the main source of the anion superoxide and hydrogen peroxide, while glycolysis produces 2-oxoaldehydes such as methylglyoxal as byproducts; due to the adjacent carbonyl groups are strong electrophiles (steal electrons oxidizing other compounds). Sophisticated mechanisms exist to maintain redox homeostasis, because ROS under controlled production also have important regulatory functions in the spermatozoa. The interactions between metabolism and production of reactive oxygen species are essential for proper sperm function, and deregulation of these processes rapidly leads to sperm malfunction and finally death. Lastly, we briefly describe two techniques that will expand our knowledge on sperm metabolism in the coming decades, metabolic flow cytometry and the use of the "omics" technologies, proteomics and metabolomics, specifically the micro and nano proteomics/metabolomics. A better understanding of the metabolism of the spermatozoa will lead to big improvements in sperm technologies and the diagnosis and treatment of male factor infertility.

Testicular function is particularly susceptible to vascular insult, resulting in a negative impact on sperm production and quality of the ejaculate. A prompt diagnosis of testicular dysfunction enables implementation of appropriate treatment, hence improving fertility forecasts for stallions. The present research aims to: (1) assess if Doppler ultrasonography is a good tool to diagnose stallions with testicular dysfunction; (2) to study the relationship between Doppler parameters of the testicular artery and those of sperm quality assessed by flow cytometry and (3) to establish cut off values to differentiate fertile stallions from those with pathologies causing testicular dysfunction. A total of 10 stallions (n: 7 healthy stallions and n: 3 sub-fertile stallions) were used in this study. Two ejaculates per stallion were collected and preserved at 5°C in a commercial extender. The semen was evaluated at T0, T24 and T48h by flow cytometry. Integrity and viability of sperm (YoPro^®-1/EthD-1), mitochondrial activity (MitoTracker^® Deep Red FM) and the DNA fragmentation index (Sperm Chromatin Structure Assay) were assessed. Doppler parameters were measured at three different locations on the testicular artery (Supratesticular artery (SA); Capsular artery (CA) and Intratesticular artery (IA)). The Doppler parameters calculated were: Resistive Index (RI), Pulsatility Index (PI), Peak Systolic Velocity (PSV), End Diastolic Velocity (EDV), Time Average Maximum Velocity (TAMV), Total Arterial Blood Flow (TABF) and TABF rate. The capsular artery was the most reliable location to carry out spectral Doppler assessment, since blood flow parameters of this artery were most closely correlated with sperm quality parameters. Significant differences in all the Doppler parameters studied were observed between fertile and subfertile stallions (p ≤ 0.05). The principal components analysis assay determined that fertile stallions are characterized by high EDV, TAMV, TABF and TABF rate values (high vascular perfusion). In contrast, subfertile stallions tend to present high values of PI and RI (high vascular resistance). The ROC curves revealed that the best Doppler parameters to predict sperm quality in stallions were: Doppler velocities (PSV, EDV and TAMV), the diameter of the capsular artery and TABF parameters (tissue perfusion parameters). Cut off values were established using a Youden´s Index to identify fertile stallions from stallions with testicular dysfunction. Spectral Doppler ultrasound is a good predictive tool for sperm quality since correlations were determined among Doppler parameters and markers of sperm quality. Doppler ultrasonography could be a valuable diagnostic tool for use by clinical practitioners for the diagnosis of stallions with testicular dysfunction and could be a viable alternative to invasive procedures traditionally used for diagnosis of sub-fertility disorders.

Artificial insemination with cryopreserved spermatozoa is a major assisted reproductive technology in many species. In horses, as in humans, insemination with cryopreserved sperm is associated with lower pregnancy rates than those for fresh sperm, however, direct effects of sperm cryopreservation on the development of resulting embryos are largely unexplored. The aim of this study was to investigate differences in gene expression between embryos resulting from fertilization with fresh or cryopreserved sperm. Embryos were obtained at 8, 10 or 12 days after ovulation from mares inseminated post-ovulation on successive cycles with either fresh sperm or frozen-thawed sperm from the same stallion, providing matched embryo pairs at each day. RNA was isolated from two matched pairs (4 embryos) for each day, and cDNA libraries were built and sequenced. Significant differences in transcripts per kilobase million (TPM) were determined using (i) genes for which the expression difference between treatments was higher than 99% of that in the random case (P < 0.01), and (ii) genes for which the fold change was ≥ 2, to avoid expression bias in selection of the candidate genes. Molecular pathways were explored using the DAVID webserver, followed by network analyses using STRING, with a threshold of 0.700 for positive interactions. The transcriptional profile of embryos obtained with frozen-thawed sperm differed significantly from that for embryos derived from fresh sperm on all days, showing significant down-regulation of genes involved in biological pathways related to oxidative phosphorylation, DNA binding, DNA replication, and immune response. Many genes with reduced expression were orthologs of genes known to be embryonic lethal in mice. This study, for the first time, provides evidence of altered transcription in embryos resulting from fertilization with cryopreserved spermatozoa in any species. As sperm cryopreservation is commonly used in many species, including human, the effect of this intervention on expression of developmentally important genes in resulting embryos warrants attention.

Spermatozoa are redox-regulated cells, and stallion spermatozoa, in particular, present an intense mitochondrial activity in which large amounts of reactive oxygen species (ROS) are produced. To maintain the redox potential under physiological conditions, sophisticated mechanisms ought to be present, particularly in the mitochondria. In the present study, we investigated the role of the SLC7A11 antiporter. This antiporter exchanges intracellular glutamate for extracellular cystine. In the spermatozoa, cystine is reduced to cysteine and used for GSH synthesis. The importance of the antiporter for mitochondrial functionality was studied using flow cytometry and UHPLC/MS/MS approaches. Intracellular GSH increased in the presence of cystine, but was reduced in the presence of Buthionine sulphoximine (BSO), a γ-glutamylcysteine synthetase inhibitor (P < 0.001). Inhibition of the SLC7A11 antiporter with sulfasalazine caused a dramatic drop in intracellular GSH (P < 0.001) and in the percentage of spermatozoa showing active mitochondria (P < 0.001). These findings suggest that proper functionality of this antiporter is required for the mitochondrial function of spermatozoa. We also describe that under some conditions, glutamate may be metabolized following non-conventional pathways, also contributing to sperm functionality. We provide evidences, that the stallion spermatozoa have important metabolic plasticity, and also of the relation between redox regulation and metabolic regulation. These findings may have important implications for the understanding of sperm biology and the development of new strategies for sperm conservation and treatment of male factor infertility.