Aging of stallion spermatozoa stored in vitro is delayed at 22°C using a 67 mm glucose-10 mm pyruvate-based media.

Background: Most commerce of equine seminal doses is carried out using commercial extenders under refrigeration at 5°C.

Objectives: To determine if 10 mm pyruvate in a 67 mm glucose extender and storage at 22°C could be the basis of an alternative storage method to cooling to 5°C.

Material And Methods: Stallion ejaculates were extendedin: INRA96 (67 mm glucose, non-pyruvate control), modified Tyrode's (67 mm glucose-10 mm pyruvate), supplemented with 0, 10, 50, and 100 μM itaconate.

Pyruvate enhances stallion sperm function in high glucose media improving overall metabolic efficiency.

If a mechanism of more efficient glycolysis depending on pyruvate is present in stallion spermatozoa, detrimental effects of higher glucose concentrations that are common in current commercial extenders could be counteracted. To test this hypothesis, spermatozoa were incubated in a 67 mM Glucose modified Tyrode's media in the presence of 1- or 10-mM pyruvate and in the Tyrode's basal media which contains 5 mM glucose. Spermatozoa incubated for 3 h at 37 °C in 67 mM Tyrode's media with 10 mM pyruvate showed increased motility in comparison with aliquots incubated in Tyrode's 5 mM glucose and Tyrode's 67 mM glucose (57.

The concentration of glucose in the media influences the susceptibility of stallion spermatozoa to ferroptosis.

In Brief: Although common in many commercial extenders, supraphysiological concentrations of glucose in the media may be detrimental to stallion spermatozoa. In this study, we present evidence that these elevated glucose levels may predispose spermatozoa to ferroptosis.

Abstract: Stallion spermatozoa depend on oxidative phosphorylation as their major source of ATP; however, the metabolism of these cells is complex and a great degree of metabolic plasticity exists.

Ovine fertility by artificial insemination in the breeding season could be affected by intraseasonal variations in ram sperm proteomic profile.

It is important to note that seasonality could affect ram reproductive parameters, and therefore, fertility results after artificial insemination. In this work, 1) we assessed fertility rates after cervical artificial insemination of 11,805 ewes at the beginning (June 21 to July 20) and at the end (November 20 to December 21) of the reproductive season in the Assaf breed for the last four years, and 2) we aimed to identify male factors influencing the different reproductive success obtained depending on the time at the mating season in which ovine artificial insemination was performed. For this purpose, we evaluated certain ram reproductive and ultrasonographical parameters as well as we performed a multiparametric and proteomic sperm analysis of 6-19 rams at two very distant points in the mating season (July as Early Breeding Season -EBS- and November as Late Breeding Season -LBS-).

Dataset of the sperm proteome of stallions with different motility.

This paper provides a detailed set of data on how the stallion sperm proteome differs among stallions with different sperm motilities, although within normal ranges. Findings distinguish proteins that may help to identify stallions of superior sperm motility. Sperm proteins were analyzed using a UHPLC/MS/MS system comprising of an Agilent 1290 infinity series UHPLC coupled to an Agilent 6550 Q-TOF mass spectrometer (Agilent Technologies, Santa Clara, CA, USA).

The seminal plasma proteins Peptidyl arginine deaminase 2, rRNA adenine N (6)-methyltransferase and KIAA0825 are linked to better motility post thaw in stallions.

Seminal plasma plays an important role in sperm physiology. Seminal plasma proteins vehiculated in microvesicles, carry RNAs and proteins with a potential role in early embryo development. Additionally, proteins present in seminal plasma participate in redox regulation and energy metabolism.

Proteins involved in mitochondrial metabolic functions and fertilization predominate in stallions with better motility.

Even in stallions with sperm quality within normal reference ranges at ejaculation, subtle differences in sperm quality exist that in many cases lead to reduced time frames for conservation of the ejaculate and/or reduced fertility. The spermatozoon is a cell highly suitable for proteomics studies, and the use of this technique is allowing rapid advances in the understanding of sperm biology. The aim of the present study was to investigate differences among stallions of variable sperm quality (based on motility and sperm velocities), although all horses had sperm characteristics within normal ranges.

Differences in the proteome of stallion spermatozoa explain stallion-to-stallion variability in sperm quality post-thaw†.

The identification of stallions and or ejaculates that will provide commercially acceptable quality post-thaw before cryopreservation is of great interest, avoiding wasting time and resources freezing ejaculates that will not achieve sufficient quality to be marketed. Our hypothesis was that after bioinformatic analysis, the study of the stallion sperm proteome can provide discriminant variables able to predict the post-thaw quality of the ejaculate. At least three ejaculates from 10 different stallions were frozen following a split sample design.

Seminal plasma AnnexinA2 protein is a relevant biomarker for stallions which require removal of seminal plasma for sperm survival upon refrigeration†.

Some stallions yield ejaculates that do not tolerate conservation by refrigeration prior to artificial insemination (AI), showing improvement after removal of most of the seminal plasma (SP) by centrifugation. In this study, the SP-proteome of 10 different stallions was defined through high-performance liquid chromatography with tandem mass spectrometry and bioinformatic analysis in relation to the ability of the ejaculates to maintain semen quality when cooled and stored at 5°C. Stallions were classified into three groups, depending on this ability: those maintaining good quality after direct extension in a commercial extender (good), stallions requiring removal of seminal plasma (RSP) to maintain seminal quality (good-RSP), and stallions, unable to maintain good semen quality even after RSP (poor).

Mass spectrometry reveals distinct proteomic profiles in high- and low-quality stallion spermatozoa.

The horse breeding industry relies upon optimal stallion fertility. Conventional sperm assessments provide limited information regarding ejaculate quality and are not individually predictive of fertilizing potential. The aim of this study was to harness mass spectrometry to compare the proteomic profiles of high- and low-quality stallion spermatozoa, with the ultimate goal of identifying fertility biomarker candidates.

Proteomic profiling of stallion spermatozoa suggests changes in sperm metabolism and compromised redox regulation after cryopreservation.

Proteomic technologies allow the detection of thousands of proteins at the same time, being a powerful technique to reveal molecular regulatory mechanisms in spermatozoa and also sperm damage linked to low fertility or specific biotechnologies. Modifications induced by the cryopreservation in the stallion sperm proteome were studied using UHPLC/MS/MS. Ejaculates from fertile stallions were collected and split in two subsamples, one was investigated as fresh (control) samples, and the other aliquot frozen and thawed using standard procedures and investigated as frozen thawed subsamples.

Depletion of thiols leads to redox deregulation, production of 4-hydroxinonenal and sperm senescence: a possible role for GSH regulation in spermatozoa†.

We hypothesized that thiols and particularly glutathione (GSH) are essential for the regulation of stallion sperm functionality. To test this hypothesis, we initially investigated the relationship between sperm function and GSH content, revealing highly significant correlations between GSH, sperm viability, motility, and velocity parameters (P < 0.001).

Second-order advantage maintenance with voltammetric data modeling for quantitation of ethiofencarb in the presence of interferences.

This work presents the development of a method based on a voltammetric determination coupled to second-order data modeling with multivariate curve resolution-alternating least-square (MCR-ALS) and unfolded partial least squares regression followed by residual bilinearization (U-PLS/RBL) for the quantitation of the pesticide ethiofencarb in the presence of interferences in tap water. The determination of ethiofencarb by voltammetry is possible on the basis that this pesticide is hydrolysed in alkaline media giving rise to a compound which shows an oxidation peak at Ep=0.80 V.

Determination of tricyclic antidepressants in human breast milk by capillary electrophoresis.

A method for the determination of several tricyclic antidepressants (imipramine, desipramine, amitriptyline, nortriptyline, clomipramine, norclomipramine, doxepine and nordoxepine) in breast milk has been developed. This assay consists of a common extraction process in an organic phase, which is evaporated until dried and finally reconstituted in the appropriate buffer for injection in a capillary electrophoresis system. The capillary electrophoresis method used is an "acetonitrile stacking" method previously reported for determining these drugs in serum samples.

Development of a non-aqueous electrophoresis method for the simultaneous determination of tricyclic antidepressants in human serum.

In the present work we report a non-aqueous electrophoresis method (NACE) for the separation and determination of eight tricyclic antidepressants (TCAs) in human serum. Separation is carried out in 1 M acetic acid, 50 mM ammonium acetate and 10 mM SDS in ACN. Standards and samples were prepared in 0.

Response surface methodology for the optimisation of flow-injection analysis with in situ solvent extraction and fluorimetric assay of tricyclic antidepressants.

A semiautomatic extraction-fluorimetric method for the determination of tricyclic antidepressant drugs (TCAs) based in the formation of ion pairs with 9,10-dimethoxyanthracene-2-sulphonate (DMAS) has been developed. The aqueous solutions of the TCAs (imipramine, desipramine, amitriptyline, nortriptyline, clomipramine or doxepine) are injected into a carrier composed by DMAS in an acid medium and the ion pair formed is extracted into dichloromethane where the fluorescence is measured. An experimental design (Central Composite Design) together with the Response Surface Methodology has been used to find the optimal instrumental FIA and chemical variables.

Comparative study of different approaches to the determination of robustness for a sensitive-stacking capillary electrophoresis method. Estimation of system suitability test limits from the robustness test.

A robustness study for a sensitive-stacking capillary electrophoresis method based on "acetonitrile-stacking" was carried out. Ten variables (pH, acetonitrile and triethanolamine in the buffer, injection time, injection pressure, acetonitrile and NaCl in the sample, capillary and tray temperature and separation voltage), whose levels were varied by 10% around the nominal level, were examined by a Plackett-Burman design (two-level design). The effects on corrected peak area and resolution (responses) were calculated and interpreted using three statistical approaches: dummy variables, distribution effects (Dong's algorithm) and calibration curve.

Response surface methodology in the development of a stacking-sensitive capillary electrophoresis method by field-amplified injection for the analysis of tricyclic antidepressants in the presence of salts.

The work presented here explores the possibilities of the electrokinetic injection (EK) to achieve sensitive methods for the determination of tricyclic antidepressants in biological samples (serum). The addition of ACN to the sample, with high content in salts, causes stacking at the tip of the capillary, in a similar way as for hydrodynamic injection. An experimental design with the response surface methodology has been used to find the optimum composition of the matrix of the sample (sodium chloride and ACN percentages) and the conditions for the EK (water-plug length, time, and voltage of injection) in few experiments.

Response surface methodology in the development of a stacking-sensitive capillary electrophoresis method for the analysis of tricyclic antidepressants in human serum.

Stacking methods are very important in overcoming the poor detection limits in capillary electrophoresis (CE). In this paper, the separation and determination of several tricyclic antidepressants by a stacking method is described. The inclusion of acetonitrile (ACN) in the sample causes stacking (transient pseudoisotachophoresis) especially in presence of sodium chloride.