scholarly journals Free-radical production after post-thaw incubation of ram spermatozoa is related to decreased in vivo fertility

2015 ◽  
Vol 27 (8) ◽  
pp. 1187 ◽  
Author(s):  
Enrique Del Olmo ◽  
Alfonso Bisbal ◽  
Olga García-Álvarez ◽  
Alejandro Maroto-Morales ◽  
Manuel Ramón ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Pregnancy Rate ◽  
Male Fertility ◽  
Sperm Quality ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Sperm Chromatin ◽  
High Fertility ◽  
Ros Production ◽  
Sperm Viability

The aim of the present study was to evaluate the effect of sperm reactive oxygen species (ROS) production and DNA changes on male fertility. For that purpose, six rams with significantly different pregnancy rates were used; these were classified as having high fertility, i.e. 59.4% average pregnancy rate, or low fertility, i.e. 23.1% average pregnancy rate. Sperm quality was assessed after a two-step process of sample thawing followed by an incubation of 2 h, either in the freezing extender (37°C) or after dilution in synthetic oviductal fluid (SOF; 38°C, 5%CO2). Sperm viability (YO-PRO-1), ROS production (5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein acetyl ester (CM-H2DCFDA)) and undamaged chromatin (sperm chromatin structure assay, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling, chromomycin A3) were evaluated by flow cytometry. Although no significant differences in sperm viability were observed, our results showed increased ROS production during incubation in the freezing extender as well as in SOF medium. Comparison between fertility groups showed significant differences in ROS production after 2 h of incubation for the two treatments. Regarding DNA integrity, our results showed no significant differences either between treatments and incubation times or fertility groups. Linear regression analysis showed that ROS production determined by CM-H2DCFDA was a good indicator parameter for in vivo male fertility of SOF-incubated samples, yielding a fair correlation between both parameters (r = –0.92). These results indicate that detection of ROS production by CM-H2DCFDA and flow cytometry after 2 h of incubation in SOF could be a useful procedure for predicting fertility of ram spermatozoa.

scholarly journals Sperm chromatin condensation as an in vivo fertility biomarker in bulls: a flow cytometry approach

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Marc Llavanera ◽  
Jordi Ribas-Maynou ◽  
Ariadna Delgado-Bermúdez ◽  
Sandra Recuero ◽  
Rodrigo Muiño ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Sperm Quality ◽  
Chromatin Condensation ◽  
Pearson Correlation ◽  
Correlation Coefficients ◽  
Quality Parameters ◽  
Sperm Chromatin ◽  
Fertility Rates ◽  
Sperm Chromatin Condensation

Abstract Background Genetic selection in cattle has been directed to increase milk production. This, coupled to the fact that the vast majority of bovine artificial inseminations (AI) are performed using cryopreserved sperm, have led to a reduction of fertility rates over the years. Thus, seeking sensitive and specific sperm biomarkers able to predict fertility rates is of vital importance to improve cattle reproductive efficiency. In humans, sperm chromatin condensation evaluated through chromomycin A3 (CMA3) has recently been purported to be a powerful biomarker for sperm functional status and male infertility. The objectives of the present study were: a) to set up a flow cytometry method for simultaneously evaluating chromatin condensation and sperm viability, and b) to test whether this parameter could be used as a predictor of in vivo fertility in bulls. The study included pools of three independent cryopreserved ejaculates per bull from 25 Holstein males. Reproductive outcomes of each sire were determined by non-return rates, which were used to classify bulls into two groups (highly fertile and subfertile). Results Chromatin condensation status of bovine sperm was evaluated through the combination of CMA3 and Yo-Pro-1 staining and flow cytometry. Sperm quality parameters (morphology, viability, total and progressive motility) were also assessed. Pearson correlation coefficients and ROC curves were calculated to assess their capacity to predict in vivo fertility. Sperm morphology, viability and total motility presented an area under the ROC curve (AUC) of 0.54, 0.64 and 0.68, respectively (P > 0.05), and thus were not able to discriminate between fertile and subfertile individuals. Alternatively, while the percentage of progressively motile sperm showed a significant predictive value, with an AUC of 0.73 (P = 0.05), CMA3/Yo-Pro-1 staining even depicted superior results for the prediction of in vivo fertility in bulls. Specifically, the percentage of viable sperm with poor chromatin condensation showed better accuracy and precision to predict in vivo fertility, with an AUC of 0.78 (P = 0.02). Conclusions Chromatin condensation evaluated through CMA3/Yo-Pro-1 and flow cytometry is defined here as a more powerful tool than conventional sperm parameters to predict bull in vivo fertility, with a potential ability to maximising the efficiency of dairy breeding industry.

54 SINGLE LAYER CENTRIFUGATION BEFORE CRYOPRESERVATION IMPROVES BULL SPERM QUALITY

2017 ◽  
Vol 29 (1) ◽  
pp. 134
Author(s):  
T. Nongbua ◽  
A. Utta ◽  
N. Am-In ◽  
J. Suwimonteerabutr ◽  
A. Johannisson ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Chromatin Structure ◽  
Mitochondrial Membrane ◽  
Linear Mixed Model ◽  
Sperm Quality ◽  
Single Layer ◽  
Sperm Chromatin ◽  
Sperm Viability ◽  
Bull Sperm

Single layer centrifugation (SLC) with Bovicoll is a technique to enhance sperm quality. The purpose of this study was to investigate the effect of SLC before cryopreservation on bull sperm quality after thawing. Semen was collected from 8 bulls (American Brahman, n = 5 and Sahiwal, n = 3) at the North Eastern Bull Centre (KhonKaen, Thailand). The ejaculate was split: one part was prepared following the standard procedure at the bull centre (n = 88) as control. The other part was used for SLC with Bovicoll-B (Johannisson et al. 2016 Theriogenology 86, 140). The SLC-selected sperm samples were frozen using the same protocol as control (n = 88). After thawing at 37°C for 12 s, motility analysis was performed using the CEROS II® (Hamilton Thorne, Beverly, MA, USA); sperm chromatin structure, mitochondrial membrane potential, and sperm viability were assessed using a FC500 flow cytometer (Beckman Coulter, Brea, CA, USA). Treatment means were compared using the linear mixed model (Proc MIXED, SAS®, 9.3, SAS Institute Inc., Cary, NC, USA). Results are reported as least-squares means ± standard error. The sperm kinematics for SLC samples were higher than controls for progressive motility (26.37 ± 1.59%, 19.56 ± 1.59%), Linearity (LIN) (52.80 ± 0.87%, 44.94 ± 0.87%), Straightness (STR) (83.06% ± 0.59, 76.20 ± 0.59%), beat cross frequency (BCF) (29.25 ± 0.50 Hz, 24.35 ± 0.50 Hz) and wobble (WOB) (61.78 ± 0.63%, 57.40 ± 0.63%) (all P < 0.0001) respectively, whereas SLC-selected samples were lower than controls for slow motility (13.61 ± 0.71%, 15.56 ± 0.71%; P < 0.05), Amplitude of lateral head displacement (ALH) (4.88 ± 0.18 μm, 6.67 ± 0.18 μm), velocity average path, (VAP) (61.17 ± 1.93μ/s, 67.88 ± 1.93μ/s), and curvilinear velocity (VCL) (99.78 ± 3.77 μ/s, 122.91 ± 3.77 μ/s) (all P < 0.0001), respectively. Other parameters of sperm quality were not different between treatments, although there was considerable variation among individual bulls in sperm chromatin structure assay, mitochondrial membrane potential, and sperm viability. These results suggest that SLC can be used before cryopreservation to improve the kinematics of thawed bull sperm samples without adversely affecting other parameters of sperm quality.

scholarly journals Sperm traits and male fertility in natural populations

Reproduction ◽  
2007 ◽  
Vol 134 (1) ◽  
pp. 19-29 ◽  
Author(s):  
Montserrat Gomendio ◽  
Aurelio F Malo ◽  
Julian Garde ◽  
Eduardo R S Roldan
Keyword(s):  
Swimming Speed ◽  
Male Fertility ◽  
Semen Quality ◽  
Sperm Quality ◽  
Natural Populations ◽  
Economic Benefits ◽  
High Fertility ◽  
Domestic Livestock ◽  
Antler Size ◽  
Sperm Swimming Speed

Male fertility has seldom been studied in natural populations because it has been assumed that strong selection would result in uniformly high values among males, and therefore mating success has been equated with fertilisation success. In contrast, male fertility has received much attention in studies of domestic livestock, where economic benefits rely on improving productivity, and in human infertility studies, where the efficiency of treatments depends on understanding which ejaculate traits explain reproductive failures and predict success at assisted conception. Despite years of efforts, no conclusive results have been obtained, probably because such studies have focused on opposite extremes of the range with little variation: domestic livestock have often been subject to strong artificial selection for high fertility, and human patients requiring treatment have compromised fertility. Recent findings from natural populations of red deer have shown that males differ markedly in their fertility, and have revealed the degree of variation found in different semen traits, both between and within males. Fertility trials have shown that male fertility is determined mainly by sperm swimming speed and the proportion of normal sperm, when sperm numbers are kept constant. Sperm design exerts a strong influence on sperm swimming speed, with faster swimming sperm having elongated heads, shorter midpieces and a longer principal plus terminal pieces in relation to total flagellum length. Thus, the large inter-male variation in sperm design found among natural populations underlies differences in sperm swimming speed which, in turn, determine differences in male fertility rates. Secondary sexual characters are honest indicators of male fertility, so males with large and elaborated antlers have larger testes and faster swimming sperm. Testosterone does not seem to mediate the relationship between antler size and semen quality, since it is associated with sperm production, but not with sperm quality or antler size. Finally, more fertile males produce a greater proportion of sons, who will inherit the semen traits which will enhance their fertility.

Loss of livestock breeding efficiency due to uncompensable sperm nuclear defects

1999 ◽  
Vol 11 (1) ◽  
pp. 1 ◽  
Author(s):  
D. P. Evenson
Keyword(s):  
Chromatin Structure ◽  
Male Fertility ◽  
Nuclear Dna ◽  
Early Embryo ◽  
Sperm Chromatin ◽  
Sperm Chromatin Structure Assay ◽  
Fertility Potential ◽  
Post Stress

An important goal of modern analyses of semen is to elucidate the molecular traits of mammalian sperm chromatin structural abnormalities, defined here as ‘uncompensable’, that lead to abnormalities in fertility, pronuclear formation, early embryo quality and pregnancy outcome. Sperm with uncompensable nuclear abnormalities are able to fertilize oocytes both in vivo and in vitro; however, due to the uncompensable trait(s), the embryo development may be abnormal. Uncompensable nuclear traits can be experimentally induced in bull sperm by a mild thermal insult to the testis. Sperm nuclear morphology abnormalities seen in ejaculates 11-days post stress are likely related to molecular changes in chromatin observed 3-days post stress by the flow cytometric sperm chromatin structure assay (SCSA). The SCSA measures the susceptibility of sperm nuclear DNA to denaturation in situ. This susceptibility has been correlated with the presence of DNA strand breaks that may be derived in part by oxidative stress and possibly by a unique, abortive apoptotic mechanism. The extent of DNA denaturation is not significantly related to the level of disulfide bonding between the chromatin protamines. The use of human sperm with uncompensable nuclear traits for artificial reproductive techniques is also discussed. The goal of this research is to remove from semen doses those sperm with uncompensable nuclear traits and thereby increase male fertility potential. Extra key words: male fertility potential, sperm chromatin structure assay (SCSA).

Sperm superoxide dismutase is associated with bull fertility

2016 ◽  
Vol 28 (9) ◽  
pp. 1405 ◽  
Author(s):  
Kamilah E. Grant ◽  
Rodrigo V. de Oliveira ◽  
Bettye Sue Hennington ◽  
Aruna Govindaraju ◽  
Andy Perkins ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Biological Networks ◽  
Sperm Quality ◽  
High Fertility ◽  
Protein Markers ◽  
Mammalian Development ◽  
Sperm Proteins ◽  
Thomson Reuters ◽  
Early Mammalian Development

Decreasing mammalian fertility and sperm quality have created an urgent need to find effective methods to distinguish non-viable from viable fertilising spermatozoa. The aims of the present study were to evaluate expression levels of β-tubulin 2C (TUBB2C), heat shock protein 10 (HSP10), hexokinase 1 (HXK1) and superoxide dismutase 1 (SOD1) in spermatozoa from Holstein bulls with varying fertility using western blotting and to analyse the biological networks of these key sperm proteins using a bioinformatics software (Metacore; Thomson-Reuters, Philadelphia, PA, USA). The rationales behind this study were that the sperm proteins play crucial roles in fertilisation and early embryonic development in mammals and ascertaining the biological networks of the proteins helps us better understand sperm physiology and early mammalian development. The results showed that expression of SOD1 was higher in spermatozoa from high fertility bulls (P < 0.05) and that SOD1 is the best protein to diagnose bulls based on the fertility index (P < 0.05). Using Metacore analysis, we identified an SOD1 network with pathways and linkages with other relevant molecules. We concluded that SOD1 sperm expression is associated with in vivo bull fertility. The findings are important because they illuminate molecular and cellular determinants of sperm viability and the identified protein markers can be used to determine bull fertility.

scholarly journals 9 DOES STRAW CONCENTRATION AFFECT POST-THAW SPERM QUALITY IN AI BULL SIRES?

2005 ◽  
Vol 17 (2) ◽  
pp. 155
Author(s):  
J. Ballester ◽  
A. Johannisson ◽  
M. Håård ◽  
H. Gustafsson ◽  
H. Rodriguez-Martinez
Keyword(s):  
Plasma Membrane ◽  
Sperm Quality ◽  
Chromatin Condensation ◽  
Membrane Integrity ◽  
Annexin V ◽  
Membrane Stability ◽  
Semen Parameters ◽  
Sperm Chromatin ◽  
Sperm Viability ◽  
Low Concentrations

There is increasing interest in decreasing the number of spermatozoa per AI dose, owing to the discrimination of threshold concentration and fertility, economical revenues and the use of sexed semen. This study evaluated the quality (as sperm viability, acrosome integrity, membrane stability and chromatin stability) post-thaw of semen collected from four elite AI sires and frozen at 15 × 106 (control) or 2 × 106 spermatozoa (spz) per dose to disclose eventual deleterious extension effects. Semen collected via a.v. from 4 élite AI sires was split-processed and frozen in 0.25 mL plastic straws under commercial conditions, at either 2 × 106 or 15 × 106 spz/straw (the latter as control). The semen parameters were within acceptable limits of normality. The post-thaw samples showed acceptable motility (above 50%). Sperm viability and stability were assessed post-thaw using flow cytometry of cells loaded with SYBR-14 and propidium iodide (PI) for sperm viability (membrane integrity), and with carboxy-SNARF-1 (SNARF; Invitrogen AB, Frolunda, Sweden), PI, and FITC-Pisum sativum agglutinin (PSA, triple stain) for acrosome status. Membrane stability status was measured with Annexin-V/PI, while sperm chromatin condensation and stability were assessed following in situ acid-induced DNA denaturation and staining with acridine orange. No significant differences were seen between the two concentrations regarding sperm motility, plasma membrane integrity (SYBR/PI, SNARF), or chromatin stability. The highly extended semen (2 × 106 spz/straw), however, showed a higher (P < 0.05) frequency of spermatozoa with translocated phosphatydil serine (as detected with Annexin-V), indicating their plasma membranes had become unstable. Also, there were more spermatozoa showing acrosomal damage (PSA). In conclusion, low concentrations of spermatozoa do not properly sustain conventional cryopreservation, although damages appear restricted to the sperm membrane. These changes in the stability of the plasma membrane apparently did not affect the viability of the spermatozoa, although they may negatively affect their lifespan. These findings may have an impact on the care that must be taken when cryopreserving low concentrations of spermatozoa with conventional freezing protocols. This work was supported by the Swedish Farmers’ Foundation for Research in Agriculture (SLF) and FORMAS, Stockholm.

scholarly journals Glycerophospholipids protect stallion spermatozoa from oxidative damage in vitro.

2021 ◽  
Author(s):  
Ashlee Jade Medica ◽  
Robert John Aitken ◽  
Garth Nicolson ◽  
Alecia Sheridan ◽  
Aleona Swegen ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Oxidative Damage ◽  
Unsaturated Fatty Acids ◽  
Sperm Quality ◽  
Membrane Lipid ◽  
Ros Generation ◽  
Sperm Viability ◽  
Commercial Preparation

Stallion sperm membranes comprise of a high proportion of poly-unsaturated fatty acids, making stallion spermatozoa especially vulnerable to peroxidative damage from reactive oxygen species generated as a by-product of cell metabolism. Membrane Lipid Replacement therapy with glycerophospholipid (GPL) mixtures has been shown to reduce oxidative damage in vitro and in vivo. The aims of this study were to test the effects of a commercial preparation of GPL, NTFactor® Lipids, on stallion spermatozoa under oxidative stress. When oxidative damage was induced by the addition of arachidonic acid to stallion spermatozoa, the subsequent addition of GPL reduced the percentage of 4-hydroxynonenal (4-HNE; a key end product of lipid peroxidation) positive cells (32.9±2.7 vs 20.9±2.3%; P≤0.05) and increased the concentration of 4-HNE within the spent media (0.026±0.003 vs 0.039±0.004 μg/mL; P≤0.001), suggesting that oxidized lipids had been replaced by exogenous GPL. Lipid replacement improved several motility parameters (total motility, 2.0±1.0 vs 68.8±2.9%; progressive motility, 0±0 vs 19.3±2.6%; straight line velocity, 9.5±2.1 vs 50.9±4.1 μm/s; curvilinear velocity, 40.8±10 vs 160.7±7.8 μm/s; average path velocity 13.4±2.9 vs 81.9±5.9 μm/s; P≤0.001), sperm viability (13.5±2.9 vs 80.2±1.6%; P≤0.001) and reduced mitochondrial ROS generation (98.2±0.6 vs 74.8±6.1%; P≤0.001). Supplementation with GPL during 17 oC in vitro sperm storage over 72 h improved sperm viability (66.4±2.6 vs 78.1±2.9%; P≤0.01) and total motility (53±5.6 vs 66.3±3.5%; P≤0.05). It is concluded that incubation of stallion spermatozoa with sub-mm-sized GPL micelles results in the incorporation of exogenous GPL into sperm membranes, diminishing lipid peroxidation and improving sperm quality in vitro.

scholarly journals Molecular morphology and function of bull spermatozoa linked to histones and associated with fertility

Reproduction ◽  
2013 ◽  
Vol 146 (3) ◽  
pp. 263-272 ◽  
Author(s):  
Rodrigo V de Oliveira ◽  
Sule Dogan ◽  
Lauren E Belser ◽  
Abdullah Kaya ◽  
Einko Topper ◽  
...  
Keyword(s):  
Chromatin Condensation ◽  
Mammalian Species ◽  
Sperm Concentration ◽  
Low Fertility ◽  
Sperm Chromatin ◽  
High Fertility ◽  
Expression Levels ◽  
Molecular Morphology ◽  
Sperm Chromatin Condensation

Sub-par fertility in bulls is influenced by alterations in sperm chromatin, and it might not be solved with increased sperm concentration in artificial insemination. Appropriate histone retention during sperm chromatin condensation plays critical roles in male fertility. The objective of this study was to determine failures of sperm chromatin condensation associated with abnormal persistence or accessibility of histones by aniline blue (ANBL) test, expression levels, and cellular localizations of one variant and two core histones (H3.3, H2B, and H4 respectively) in the spermatozoa of low-fertility (LF) vs high-fertility (HF) bulls. The expression levels and cellular localizations of histones in spermatozoa were studied using immunoblotting, immunocytochemistry, and staining methods. The bioinformatics focused on the sequence identity and evolutionary distance of these proteins among three mammalian species: bovine, mouse, and human. We demonstrated that ANBL staining was different within the LF (1.73 (0.55, 0.19)) and HF (0.67 (0.17, 0.06)) groups (P<0.0001), which was also negatively correlated within vivobull fertility (r=−0.90,P<0.0001). Although these histones were consistently detectable and specifically localized in bull sperm cells, they were not different between the two groups. Except H2B variants, H3.3 and H4 showed 100% identity and were evolutionarily conserved in bulls, mice and humans. The H2B variants were more conserved between bulls and humans, than in mice. In conclusion, we showed that H2B, H3.3, and H4 were detectable in bull spermatozoa and that sperm chromatin condensation status, changed by histone retention, is related to bull fertility.

Probiotic administration improves sperm quality in asthenozoospermic human donors

2017 ◽  
Vol 8 (2) ◽  
pp. 193-206 ◽  
Author(s):  
D.G. Valcarce ◽  
S. Genovés ◽  
M.F. Riesco ◽  
P. Martorell ◽  
M.P. Herráez ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Viability ◽  
Dna Fragmentation ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Bifidobacterium Longum ◽  
Fold Change ◽  
Sperm Chromatin ◽  
Computer Assisted ◽  
Sperm Analysis

The objective of this study is to analyse the effect of the ingestion of two selected antioxidant probiotics strains (Lactobacillus rhamnosus CECT8361 and Bifidobacterium longum CECT7347) on sperm quality parameters in asthenozoospermic males after three and six weeks of administration. Nine asthenozoospermic men without any medical treatment under similar diet conditions participated in the study. The quality of individual sperm samples was evaluated before (previous to ingestion), during (after 3 and 6 weeks of ingestion) and after probiotic administration (3 and 6 weeks after finishing the treatment). Sperm motility was evaluated by computer-assisted sperm analysis system, DNA fragmentation by sperm chromatin structure assay, cell viability by flow cytometry and measurement of intracellular H2O2 (reactive oxygen species; ROS) by flow cytometry using dichloro-dihydrofluorescein diacetate. Sperm motility was drastically improved after the treatment (approximately 6 fold change), DNA fragmentation was statistically reduced after probiotic administration from (approximately 1.2 fold change) and intracellular H2O2 level was decreased (approximately 3.5 fold change). Cell viability was not affected by the treatment. The significant improvement in sperm motility and the decrease in DNA fragmentation reported in this study provide preliminary evidence that probiotics could be administrated to improve motility and decrease DNA fragmentation and ROS levels in asthenozoospermic human males.

scholarly journals Multiparametric Study of Antioxidant Effect on Ram Sperm Cryopreservation—From Field Trials to Research Bench

Animals ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 283
Author(s):  
Marta F. Riesco ◽  
Mercedes Alvarez ◽  
Luis Anel-Lopez ◽  
Marta Neila-Montero ◽  
Cristina Palacin-Martinez ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Redox Balance ◽  
Field Trials ◽  
Low Fertility ◽  
High Fertility ◽  
Sperm Cryopreservation ◽  
Ram Sperm ◽  
Positive Effect

The optimization of sperm cryopreservation protocols in ram is a feasible tool to reinforce artificial insemination technologies considering the desirable application of sperm by vaginal/cervical or transcervical deposition. Cryopreservation provokes different types of damage on spermatozoa and many of these detrimental effects are triggered by redox deregulation. For this reason, the antioxidant supplementation in sperm cryopreservation protocols to decrease reactive oxygen species (ROS) levels and to equilibrate redox status has been widely employed in different species. Despite this, more fertility trials are necessary to provide the definitive tool to ensure the antioxidant effectiveness on sperm quality. For this reason, in this work, we performed a multiparametric analysis of some previously tested antioxidants (crocin, GSH and Trolox) on ram sperm cryopreservation from field trials to sperm quality analyses focused on new strategies to measure redox balance. Attending to fertility trial, Trolox supplementation registered an improvement concerning to fertility (when we considered high fertility males) and multiple lambing frequency and other complementary and descriptive data related to lambing performance such as prolificacy and fecundity. This positive effect was more evident in multiple lambing frequency when we considered low fertility males than in global male analysis. In vitro analyses of sperm quality confirmed in vivo trials registering a positive effect on sperm viability and redox balance. In this study, we provided the definitive evidence that the role of trolox on redox balance maintenance has a direct effect on fertility parameters, such as prolificacy. The effectiveness of antioxidant treatments was tested, for the first time in ovine species, using an integrative and multiparametric approach combining in vivo and in vitro analyses and novel approaches, such as RedoxSYS. These types of strategies should be applied to improve sperm conservation methods and optimize AI technologies upgrading the correlation between in vitro and in vivo analyses.

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