Acrosomal Status Research Articles

Pre- and Post-Thaw Addition of L-Carnitine and Pyruvate: Effect on Stallion Sperm Parameters.

The addition of antioxidants to cryopreservation media reportedly improves sperm post-thaw quality and reproductive performance after artificial insemination. Therefore, the objectives of this study were to evaluate if the addition of L-carnitine and pyruvate to freezing media, or their addition to samples after thawing, improves the post-thaw quality of equine spermatozoa. Thus, in Experiment 1, stallion semen samples were cryopreserved in: (1) EDTA-glucose-based extender with 20% egg yolk and 5% dimethylformamide (EDTA control); (2) skim milk-based extender with 20% egg yolk and 5% dimethylformamide (milk control); (3) Extender1 supplemented with 50 mM L-carnitine and 10 mM pyruvate (EDTA-carnitine-pyruvate); and (4) Extender 2 supplemented with 50 mM L-carnitine and 10 mM pyruvate (milk-carnitine-pyruvate). In Experiment 2, 50 mM L-carnitine and 10 mM pyruvate were added post-thaw to samples cryopreserved with extenders 1 and 2 (EDTA control and milk control). Sperm kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability were evaluated after thawing. No significant differences (p > 0.05) were observed for most of the kinematic parameters, DNA fragmentation, membrane lipid peroxidation, acrosome status and viability of spermatozoa, between the samples frozen in the presence or absence of L-carnitine and pyruvate, nor between the samples after the post-thaw addition of these components. A higher (p < 0.05) mean velocity and higher (p < 0.05) amplitude of lateral head displacement were observed in the samples frozen in the milk-based extender with the addition of L-carnitine and pyruvate after thawing. The addition of 50 mM L-carnitine and 10 mM pyruvate, either to the freezing extenders or after thawing, was not deleterious for sperm; however, it did not improve equine sperm motility, viability, acrosome and DNA integrity, nor decrease membrane lipid peroxidation after thawing.

In vitro effects of antidepressants on human sperm function.

Depression currently affects about 280 million people worldwide and its prevalence has been increasing dramatically, especially among the young and people of reproductive age, which consequently leads to an increase in antidepressant consumption. Antidepressants are associated with sexual dysfunction in both men and women; however, their role in male fertility has been scarcely studied. Fluoxetine and sertraline, two serotonin reuptake inhibitors (SSRIs), are among the most prescribed antidepressants worldwide. To determine their possible effects, human sperm cells were exposed to either sertraline or fluoxetine at concentrations previously found in blood and seminal fluid of patients undergoing treatment. Spermatozoa were incubated for up to 24 h at 37°C and 5% CO2, and important functional parameters such as sperm motility, viability, mitochondrial membrane potential, cellular reactive oxygen species (ROS) production, chromatin/DNA integrity, acrosome status, and tyrosine phosphorylation were assessed. At low levels, fluoxetine consistently decreased progressive motility throughout time while promoting fluctuations in ROS levels and sperm capacitation. Nevertheless, it did not affect viability, mitochondrial membrane potential, acrosome reaction nor chromatin/DNA integrity. Sertraline, on the other hand, had little to nonsignificant impact at low doses, but affected almost all tested parameters at supratherapeutic concentrations. Altogether, our results suggest that both antidepressants may impair sperm function, possibly through different mechanisms of action, but fluoxetine is the only exhibiting mild negative effects at doses found in vivo.

Comparison of Nanoparticles and Single-Layer Centrifugation for Separation of Dead from Live Stallion Spermatozoa.

The goal of this study was to compare the efficacy of coated iron-core nanoparticles and single-layer centrifugation for separation of dead from live stallion spermatozoa. Our hypothesis was that nanoparticles would bind to dead sperm and allow for separation from live sperm using a magnet, resulting in a population of spermatozoa with a high percentage of total and progressive motility. Treatment Group 1 was an untreated control. Treatment Group 2 (nanoparticles, NP) utilized sperm incubated with nanoparticles followed by application of a magnet to remove dead sperm adhered to the coated nanoparticles. Treatment Group 3 (single-layer centrifugation, SLC) layered sperm above EquiPure™ followed by centrifugation. Semen samples were subsequently evaluated for sperm motility parameters, plasma membrane integrity, acrosome status, and morphology. The SLC technique yielded higher (p < 0.05) progressive motility (76 ± 9.2%) than the NP separation technique (59 ± 12.2%) or the untreated control (47.3 ± 5.1%). However, the total number of sperm recovered was higher (p < 0.05) in the NP technique (526.2 ± 96.6 × 106) than the SLC procedure (211.7 ± 70 × 106), yielding a higher total number of progressively motile sperm (317.6 ± 109 × 106) recovered using the NP technique than the SLC technique (157.8 ± 43.6 × 106). The percentage of live, acrosome intact sperm recovered was higher for SLC than NP. In summary, the SLC technique yielded a higher percentage of sperm motility, intact plasma membranes, and acrosome integrity, but yielded lower total sperm than with the nanoparticle separation technique.

P-354 Advanced Paternal Age (APA) amplifies cryopreservation-induced stress in human spermatozoa

Abstract Study question Does paternal age intensify the negative effects of cryopreservation on sperm quality? Summary answer Paternal age exacerbates the adverse impact of cryopreservation on sperm quality. What is known already Despite mounting evidence indicating age-related declines in sperm quality, the impact of APA on male fertility remains unclear. APA raises questions about DNA fragmentation and mitochondrial activity, a robust indicator of sperm functionality. While sperm cryopreservation is extensively employed in fertility preservation and assisted reproductive technology (ART), its potentially detrimental effects on cells compartments, including mitochondria, persist. The mechanisms of APA on sperm susceptibility to cryopreservation-induced cellular damage still need to be addressed. Study design, size, duration This prospective multicentric study, conducted between January and October 2023, comprised 200 men. Only normozoospermic samples were included in the research (n = 81). Then, based on their age, the participants were categorised into two groups: ages ≤35 (non-APA, n = 40) and ages ≥42 (APA, n = 41). The cohorts were stratified into distinct subgroups, encompassing both fresh and frozen/thawed specimens of APA and non-APA sperm, and subjected to evaluations encompassing mitochondrial functionality, acrosomal reaction, DNA fragmentation and Tyrosine phosphorylation. Participants/materials, setting, methods Semen samples were collected after 2-7 days abstinence and analysed according to WHO 2021 guidelines. Acrosomal status was determined using FITC-conjugate PSA. DNA fragmentation was examined with the Halosperm kit. Mitochondrial functionality was gauged using MitoTracker Red CMXRos through the percentage of stained cells and fluorescence intensity and PCR for mitochondrial DNA copy number (mtDNAcn). Tyrosine phosphorylation was assessed through immunofluorescence and Western Blotting. Main results and the role of chance Non-APA group exhibited significantly superior fresh semen characteristics. DNA fragmentation was notably elevated in fresh semen from APA group, reaching 21.7%, compared to 15.4% non-APA individuals. After cryopreservation, the DNA fragmentation index further increased in men over 42 years to 26.7%. Mitochondrial functionality analysis indicated a significant decrease in staining intensity in cryopreserved sperm, notably in the APA group, with mean fluorescence intensity decreasing from 3.35±1.5 to 1.8±0.5. In contrast, the non-APA group experienced a drop from 3.33±1.6 to 2.53±1.6. Additionally, in the APA group, a significant increase in mtDNAcn was observed in both fresh and frozen/thawed sperm compared to the younger counterparts, indicative of poor mitochondrial quality. Cryopreservation negatively impacted acrosome integrity in both age groups, with a significant decrease observed in unreacted acrosomes from 71.5% to 57.7% in the non-APA group and from 75% to 63% in the APA group. Furthermore, exploration of capacitation-like changes, like tyrosine phosphorylation, demonstrated a significant decrease with age and a negative impact of cryopreservation on tyrosine phosphorylation in sperm from young males. Limitations, reasons for caution Main limitations are the small number of examined samples and the inclusion of only normozoospermic patients. Therefore, future investigations are needed to provide a more comprehensive understanding of the impact of paternal age on cryopreservation-induced stress in human spermatozoa. Wider implications of the findings Our research sheds light on the influence of paternal age on sperm cryopreservation, revealing higher vulnerability of mitochondria and elevated DNA fragmentation in APA. These findings should be considered when advising older men on choosing cryopreservation techniques in assisted reproductive treatments. Trial registration number Not applicable

Cryopreservation alters buffalo sperm kinematics and mitochondrial parameters, acrosome and intra-cellular calcium status

BACKGROUND: Little is known about the effects of different seasons on the cryopreservation success of buffalo sperm in terms of kinematics and sperm functional parameters. OBJECTIVE: To study the effect of three seasons (winter, comfort and summer) and cryopreservation on sperm kinematics and functional properties in buffalo bulls. MATERIALS AND METHODS: Semen ejaculates (n = 90) collected during three seasons i.e. winter (n = 30), comfort (n = 30), summer (n = 30) were evaluated for sperm kinematics and functional properties. RESULTS: Sperm kinematics with respect to total (TM), progressive (PM) and rapid motility (RM) was higher (P&lt;0.05) in fresh sperm compared to sperm that had been frozen-thawed. Similarly, all kinematic parameters [viz. average path velocity (VAP), straight linear velocity (VSL), curvilinear velocity (VCL), beats cross frequency (BCF), lateral head displacement (ALH), linearity (LIN) and straightness (STR)] were higher (P&lt;0.01) at the fresh stage. With respect to season, frozen-thawed semen TM (57.67 ± 115%), PM (50.2 ± 1.15%) and RM (51.6 ± 1.19%) were higher (P&lt;0.01) when using sperm collected during winter. The stage of cryopreservation (i.e., equilibration and freeze-thawing) also showed significant effects (P&lt;0.01) on mitochondrial superoxide positive status (MSPS), mitochondrial membrane potential (MMP), acrosome status and intra-cellular calcium status. CONCLUSION: The season of sperm collection and cryopreservation have significant effects on buffalo bull sperm kinematics and functional properties.

Characterization of expression patterns and dynamic relocation of Notch proteins during acrosome reaction of bull spermatozoa

Notch is a conserved cell-signaling pathway involved in spermatogenesis regulation. This study firstly evaluated the presence, localization patterns, acquisition origin and relation to acrosome reaction of Notch proteins in bull sperm. Western Blot analysis detected all Notch proteins in ejaculated bull sperm, and immunostaining described their specific sperm localization. Recovery of sperm from different segments showed that Notch proteins have testicular origin (NOTCH1, NOTCH2, DLL4), are sequentially acquired during sperm maturation along epididymal transit (NOTCH3, DLL3, JAGGED1-2), or post-ejaculation (DLL1, NOTCH4). Testis NOTCH2 is ubiquitously expressed in all germ-cell lines, whereas DLL4 is expressed in round and elongated spermatids during the Golgi, Cap, Acrosome and Maturation phases. In vitro spontaneous and induced sperm acrosome reaction induce consistent sperm regional relocation of NOTCH2, DLL4 and JAGGED1, and these relocation patterns are significantly associated to sperm acrosome status. NOTCH2 and JAGGED1 are relocated from the head apical to the post-equatorial regions, whereas DLL4 is lost along with the acrosome, evidencing that sperm spatial redistribution of NOTCH2 and JAGGED1 is linked to acrosome reaction onset, whereas DLL4 loss is linked to AR completion. Overall, results prompt for a relevant Notch role in bull sperm acrosome testicular development, epididymal maturation and acrosome reaction.

Sperm functionality is differentially regulated by porcine oviductal extracellular vesicles from the distinct phases of the estrous cycle.

Context Extracellular vesicles (EVs) derived from the oviductal fluid (oEVs) play a critical role in various reproductive processes, including sperm capacitation, fertilisation, and early embryo development. Aims To characterise porcine oEVs (poEVs) from different stages of the estrous cycle (late follicular, LF; early luteal, EL; mid luteal, ML; late luteal, LL) and investigate their impact on sperm functionality. Methods poEVs were isolated, characterised, and labelled to assess their binding to boar spermatozoa. The effects of poEVs on sperm motility, viability, acrosomal status, protein kinase A phosphorylation (pPKAs), tyrosine phosphorylation (Tyr-P), and in in vitro fertility were analysed. Key results poEVs were observed as round or cup-shaped membrane-surrounded vesicles. Statistical analysis showed that poEVs did not significantly differ in size, quantity, or protein concentration among phases of the estrous cycle. However, LF poEVs demonstrated a higher affinity for binding to sperm. Treatment with EL, ML, and LL poEVs resulted in a decrease in sperm progressive motility and total motility. Moreover, pPKA levels were reduced in presence of LF, EL, and ML poEVs, while Tyr-P levels did not differ between groups. LF poEVs also reduced sperm penetration rate and the number of spermatozoa per penetrated oocyte (P Conclusions poEVs from different stages of the estrous cycle play a modulatory role in sperm functionality by interacting with spermatozoa, affecting motility and capacitation, and participating in sperm-oocyte interaction. Implications The differential effects of LF and LL poEVs suggest the potential use of poEVs as additives in IVF systems to regulate sperm-oocyte interaction.

Evaluation of equine semen frozen in extenders free of egg yolk using two different freezing curves

A chemically defined cryopreservation extender that maintains seminal parameters is relevant. Fifteen ejaculates from 5 stallions (n= 5; r=3) were diluted in 5 extenders: 1) EDTA-glucose based extender with egg-yolk and dimethylformamide (EY); 2) commercial equine extender (CE); 3) CE with dimethylformamide (CE-3); 4) bovine commercial extender with liposomes (OP); 5) bovine commercial extender with soybean lecithin (BIO), and frozen using a slow and a rapid temperature descent curve. Post-thaw evaluations were: sperm kinematic parameters, viability and acrosome status, membrane lipoperoxidation and DNA fragmentation. Sperm data were analysed using an ANOVA or Friedman test (results mean ± SD). Paired comparison between the two freezing curves was analysed using the Wilcoxon test. Total and progressive motility were significantly higher (P<0.05) in the EY and CE-3 samples using the slow curve, whereas for the fast curve, total and progressive motility were significantly higher (P<0.05) in the EY samples compared to all the extenders and the samples frozen in CE-3 were significantly higher than the remaining extenders (P<0.05). The percentages of live acrosome intact sperm and of live non-peroxidized sperm were significantly higher (P<0.05) in the EY extender when using either of the freezing curves and in turn, were significantly higher (P<0.05) in samples frozen in CE-3 compared to the remaining extenders. Intact DNA was significantly lower (P<0.05) in the BIO extender, using the rapid curve. To conclude, the commercial equine extender with 3% dimethylformamide, without egg-yolk, could be a suitable alternative for extenders with egg-yolk.

Evaluation of Different Thawing Protocols on Iberian Boar Sperm Preserved for 10 Years at Different Liquid Nitrogen Levels.

The conservation of genetic resources in pig breeds, notably the Iberian pig, is crucial for genetic improvement and sustainable production. Prolonged storage in liquid nitrogen (LN2) is recognized for preserving genetic diversity, but potential adverse effects on seminal quality remain debated. This study aims to assess the impact of ten years of storage at different LN2 levels and to optimize thawing protocols for Iberian pig sperm. Sperm samples from 53 boars were cryopreserved and stored at varying LN2 levels and, a decade later, the samples were thawed at 37 °C for 20 s or at 70 °C for 8 s. Sperm motility, membrane integrity, acrosome status, and DNA fragmentation were evaluated in year 0 and year 10. Overall, no significant differences were observed in post-thaw sperm quality between storage levels in year 0 or year 10. But thawing at 70 °C 8 s showed significant improvements, particularly in samples that were always stored in LN2, in all analyzed parameters except fragmentation, which was not affected by cryostorage. This study suggests that the long-term preservation of Iberian pig sperm does not affect quality over time, regardless of whether the samples were fully submerged in LN2. Furthermore, it is determined that thawing at 70 °C for 8 s maximizes post-thaw sperm quality, especially in those samples stored constantly submerged in LN2.

Cold-induced damage in boar sperm cooled to 5°C in Lactose Egg Yolk (LEY) medium with different Glycerol concentrations

Boar sperm is highly susceptible to cold damage. When temperature drops to 5°C, the plasmatic membrane is destabilized. The freezing process causes a reduction of the fertility window because frozen/thawed boar sperm has less survivability. The aim of this work was to analyze the effect on sperm characteristics and response to capacitation stimuli of cooling to 5°C using a controlled protocol. Also, we evaluated if the addition of Glycerol 2% or 3% at 5°C was able to modify these parameters. For this purpose, we assessed motility, plasmatic membrane integrity and acrosomal membrane status. Capacitation was induced using Tyrode´s capacitating medium (TCM) and assessed by chlortetracycline stain and induction of acrosomal reaction with Progesterone. Motility patterns were analyzed using a CASA system. These tests were performed at three different points of the freezing curve: 37°C; 17°C and 5°C. Response to TCM vs TBM was only significant at 37°C. While at 37°C and 17°C capacitated sperm was below 20%, at 5°C reached 50% both in the TBM and TCM. CASA analysis showed that spermatozoa exposed to TCM had higher LIN and WOB than those in TBM. All parameters were similar in the Glycerol concentrations studied. These results suggest that the chilling process may be causing an effect similar to cryocapacitation along the cooling curve, starting subtle at 17°C and reaching 50% of the sperm population at 5°C, being independent of Glycerol concentration.

Oxidative Stress and Acrosomal Status of Human Spermatozoa Subjected to Hydrophobic Carbon Soot Treatments.

The fourth industrial revolution extensively reshapes the reality we are living in by blurring the boundaries of physical, digital and biological worlds. A good example is the previously unthinkable incursion of nanoscale waste materials, such as soot, into the technologies for assisted reproduction. Although the rapeseed oil soot may efficiently enhance the progressive motility of human spermatozoa, it is yet unknown whether this material induces undesirable oxidative stress and premature acrosome reaction, endangering the sperm-oocyte fusion and blastocyst formation. In an attempt to clarify this issue, we reveal that the three-hour incubation of human semen mixed with three main types of soot does not cause oxidative stress and spontaneous acrosome reaction of the sperm. These unique findings are attributed to synchronous elimination and stabilization of the oxidants via hydrogen bonding to the acidic groups of the soot (i.e., C=O and/or C-O-C) and electron donation by its basic chemical sites (i.e., C-OH and/or COOH). Moreover, the soot nanoparticles are electrostatically attracted by discrete positively charged areas on the sperm head, increasing its negative charge and in some cases interfering the acrosome reaction. Such novel mechanistic insights emphasize the credibility of rapeseed oil soot to confidently shift from the purely diagnostic and therapeutic phases in reproductive medicine to research dealing with the effect of carbon nanomaterials on the embryo development and implantation.

IZUMO1 Receptor Localization during Hyaluronic Acid Selection in Human Spermatozoa.

IZUMO1 is an acrosome transmembrane protein implicated in the adhesion and fusion of gametes. This study aims to describe the distribution of IZUMO1 in human sperm under different physiological conditions: before capacitation (NCS), at one-hour capacitation (CS1), after a hyaluronic acid (HA) selection test (mature, MS1 and immature, IS1), and induced acrosome reaction from one-hour-capacitated sperm (ARS1). The data obtained in NCS, CS1, and MS1 significantly highlight dotted fluorescence in the acrosomal region (P1) as the major staining pattern (~70%). Moreover, we describe a new distribution pattern (P2) with a dotted acrosomal region and a labelled equatorial region that significantly increases in HA-bound spermatozoa, suggesting the onset of the migration of IZUMO1. In contrast, unbound spermatozoa presented an increase in P3 (equatorial region labelled) and P4 (not labelled). Finally, costaining to observe IZUMO1 distribution and acrosome status was performed in ARS1. Interestingly, we reported a variety of combinations between the IZUMO1 staining patterns and the acrosomal stages. In conclusion, these data show as a novelty the diffusion of the IZUMO1 protein during different physiological conditions that could contribute to the improvement in sperm selection techniques.

The Beneficial Effects of Pterostilbene on Post-Thawed Bovine Spermatozoa.

Reactive Oxygen Species (ROS), primarily produced by cellular metabolism, are highly reactive molecules that modify cellular compounds. During sperm preparation in Assisted Reproductive Techniques (ARTs), intrinsic and extrinsic sources of ROS can impact spermatozoa's oxidative status. The modification of the media with compounds that enhance sperm quality characteristics is of great significance. The current study investigated the effect of pterostilbene, a phenolic compound, on bovine sperm quality. Cryopreserved spermatozoa from six bulls were thawed, supplemented with pterostilbene (0, 10 μΜ, 25 μΜ) and incubated for 60 min and 240 min. Spermatozoa were analyzed in terms of motility, viability, acrosomal status and intracellular concentration of superoxide anion in each time point. The incubation of spermatozoa with 25 μΜ pterostilbene resulted in the preservation of quality parameters through superoxide anion mitigation, while its presence in capacitating conditions resulted in higher percentage of acrosome-reacted spermatozoa. The results of the present study indicate that the addition of pterostilbene prevents oxidative insult to spermatozoa and preserves the sperm quality parameters.

Exogenous progesterone during in vitro fertilization improves developmental competence of partially cumulus-denuded bovine oocytes

The progesterone (P4) secreted by cumulus cells has gained attention for its role as a possible physiological inducer of sperm acrosome exocytosis. In mammals, it is generally accepted that fertilization rates of oocytes without cumulus are markedly low. This study assessed the integrity of capacitated bovine sperm acrosome when exposed to increasing concentrations of P4, and evaluated whether exogenous P4 during in vitro fertilization (IVF) increases the developmental competence of partially cumulus-denuded oocytes in serum-free conditions. After a 4-h capacitation induction, sperm were incubated with increasing concentrations of P4 (0, 0.1, 10 and 100 μM) and evaluated for viability, caspase activation and acrosome status at three different times (4, 5, and 22 h), including the capacitation induction period. Progesterone induced sperm acrosomal exocytosis without compromising sperm viability or activating sperm caspases. Sperm undergoing acrosome reaction exhibited three differential Concanavalin A patterns, corresponding to early, intermediate and late acrosomal exocytosis. The percentage of these patterns significantly increased over time, regardless of P4 concentration, except for those spermatozoa with late acrosomal exocytosis, which only showed an increase at 22 h of incubation. After incubation for 1 h with 100 μM P4, spermatozoa showing intermediate acrosomal exocytosis significantly increased. At 22 h of incubation, the pattern corresponding to early acrosomal exocytosis evidenced a dose-dependent increase. However, prematurely high levels of acrosome reaction induced by 100 μM P4 led to inefficient IVF outcomes (P < 0.05). Therefore, IVF trials with partially cumulus-denuded oocytes were carried out with lower P4 concentrations (0, 0.1, 1, 5, 10 μM). Cleavage rate significantly increased at 1 μM P4, which translated to increased total embryo production after 7 days of in vitro culture (P < 0.05). Significantly higher percentages of expanded blastocysts were observed at both 1 μM and 10 μM P4 as compared to the other experimental conditions. In conclusion, the different patterns of acrosomal exocytosis identified over time by incubation of live sperm with a fluorescent lectin revealed the existence of sperm subpopulations heterogeneous in their physiological states. Moreover, exogenous P4 at 1 μM during IVF improved the developmental competence of partially cumulus-denuded oocytes in serum-free conditions.

Evaluation of sperm and hormonal assessments in Wagyu, Nellore, and Angus bulls.

Wagyu bulls are known to have a highly exacerbated libido, as shown by the intense sexual interest of young calves. Therefore we believe that Wagyu male animals have specialized Sertoli and Leydig cells that are directly involved with the sexual precocity in this breed as mature bulls have a small scrotal circumference. This study aimed to evaluate whether there were differences in the hormone and sperm characteristics of Wagyu bulls compared with the same characteristics of subspecies Bos indicus and Bos taurus sires. Frozen-thawed semen from Wagyu, Nellore, and Angus sires were analyzed for sperm kinetics (computer-assisted sperm analysis), plasma membrane integrity, chromatin integrity, acrosome status, mitochondrial activity, lipid peroxidation and hormone [luteinizing hormone (LH) and testosterone] serum concentration. The results showed that Wagyu had lower total motility and an increased number of sperm with no motility when compared with Nellore and Angus bulls. Wagyu breed did not differ from those breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation or hormone (LH and testosterone) concentrations. We concluded that Wagyu sires had lower total motility when compared with Nellore and Angus bulls. Wagyu breed did not differ from these breeds when considering plasma and acrosome membranes integrity, mitochondrial potential, chromatin resistance, sperm lipid peroxidation, or hormone (LH and testosterone) concentrations.

Boar sperm motility is modulated by CCK at a low concentration of bicarbonate under capacitation conditions.

In a previous study, our group detected the cholecystokinin (CCK) protein in the porcine oviduct. This fact, together with the involvement of CCK in the regulation of sperm protein tyrosine phosphorylation by the modulation of HCO3 - uptake (in mice and humans) suggests a role for CCK during sperm capacitation. Therefore, on the one hand, the expression of CCK receptors (CCK1R and CCK2R) on boar testes has been investigated and probed; on the other hand, boar spermatozoa (from seminal doses of 1-day and 5-day storage) were exposed to different concentrations of CCK (0-control, 25 or 50 μM) in a medium supporting capacitation supplemented with 0, 5 or 25 mmol/L of HCO3 - for 1 h at 38.5°C. Sperm motion (total and progressive motility), kinetic parameters, viability, acrosome status, and mitochondrial activity were determined. No differences between groups (0, 25 or 50 μM of CCK) were observed when HCO3 - was absent in the media (p > .05). However, the results showed that when the media was supplemented with 5 mmol/L HCO3 - in 1-day seminal dose storage, the linearity index (LIN, %), straightness index (STR, %) and oscillation index (WOB, %) (sperm kinetics parameters) increased in the presence of CCK regardless the concentration (p < .05). Nevertheless, CCK in sperm from 5-day storage only increased the WOB parameter in comparison to the control (p < .05). Furthermore, the average amplitude of the lateral displacement of the sperm head (ALH, μm) and curvilinear velocity (VCL, μm/s) decreased when CCK was present, depending on its concentration and sperm aging (1-day vs. 5-days) (p < .05). In the case of the media supporting capacitation supplemented with 25 mmol/L HCO3 - , any differences were observed except for sperm viability in the 5-day seminal doses, which increased in the 50 μM-CCK group compared to the control (p < .05). In conclusion, these data suggest an implication of CCK protein during sperm capacitation under low bicarbonate concentration increasing the sperm linear trajectory.

Lactate as the sole energy substrate induces spontaneous acrosome reaction in viable stallion spermatozoa.

Equine spermatozoa appear to differ from spermatozoa of other species in using oxidative phosphorylation preferentially over glycolysis. However, there is little information regarding effects of different energy sources on measured parameters in equine spermatozoa. To determine the effect of three individual energy substrates, glucose, pyruvate, and lactate, on motion characteristics, membrane integrity, and acrosomal status of stallion spermatozoa. Freshly ejaculated stallion spermatozoa were incubated with combinations of glucose (5mm), pyruvate (10mm), and lactate (10mm) for 0.5 to 4h. Response to calcium ionophore A23187 (5μm) was used to evaluate capacitation status. Motility was evaluated using computer-assisted sperm analysis, and plasma membrane and acrosomal integrity were evaluated by flow cytometry. Incubation with lactate alone for 2h increased acrosomal sensitivity to A23187. Notably, incubation with lactate alone for 4h induced a significant spontaneous increase in acrosome-reacted, membrane-intact (viable) spermatozoa, to approximately 50% of the live population, whereas no increase was seen with incubation in glucose or pyruvate alone. This acrosomal effect was observed in spermatozoa incubated at physiological pH as well as under alkaline conditions (medium pH approximately 8.5). Sperm motility declined concomitantly with the increase in acrosome-reacted spermatozoa. Sperm motility was significantly higher in pyruvate-only medium than in glucose or lactate. The addition of pyruvate to lactate-containing medium increased sperm motility but reduced the proportion of live acrosome-reacted spermatozoa in a dose-dependent fashion. This is the first study to demonstrate that incubation with a specific energy substrate, lactate, is associated with spontaneous acrosome reaction in spermatozoa. The proportion of live, acrosome-reacted spermatozoa obtained is among the highest reported for equine spermatozoa. These findings highlight the delicate control of key sperm functions, and may serve as a basis to increase our understanding of stallion sperm physiology.

Comparison of different cryoprotectants for freezing donkey jack (Equus asinus) semen

The objective of this study was to extrapolate a rapid sperm freezing technique developed in horses to Remonta Argentino donkey semen, evaluating, in vitro and in vivo, the effect of freezing media that included two cryoprotectants, dimethylformamide (DMF) and methylformamide (MF) at two different concentrations (5 and 7%). Twenty-four ejaculates from 8 fertile jacks (n=8; r=3) were processed with 8 extenders: BotuSemen Gold with 5% or 7% MF and 5% or 7% DMF and EDTA-glucose with 5% or 7% MF and 5% or 7% DMF, all containing 11% lactose, 20% egg-yolk and Equex and frozen: to -15°C (10-12°C/min) then to -120°C (25-40°C/min.), then plunged in N2. Post-thaw evaluations included sperm motility (Computer Assisted Semen Analysis; AndroVision, Minitube, Tiefenbach, Germany), morphology (Diff Quick stain), membrane function and acrosome status (combined hypoosmotic test and Coomassie blue stain). Sperm data were analyzed using Kruskal Wallis and pregnancy data using Chi-Square test. Values are mean ± SD. Differences were observed in the post-thaw progressive sperm motility between individuals (p<0.05) resulting in three subgroups (low: 9.23 ± 0.16; intermediate: 22.92 ± 9.81 and high: 41.33 ± 5.41). Within each subgroup, there were no in vitro differences among the 8media used for any of the sperm characteristics evaluated. Samples in BotuSemen Gold with 5% DMF, however, tended to show highest percentages (P>0.05) of sperm with acrosomes and functional membranes (DMF: 5%: 53.67 ± 22.01; 7%: 33.92 ± 23.4; MF: 5%: 44.5 ± 20.46; 7%: 38.75 ± 27.4). In vivo, 300 × 106 PM sperm were deeply inseminated post-ovulation in 30 mares: 15 with BotuSemen Gold 5% DMF and 15 with BotuSemen Gold 7% DMF. A 46% (7/15) pregnancy rate was obtained using the extender with 5% DMF and no pregnancies (0%; 0/15) were obtained using the extender with 7% DMF (P=0.003). Perhaps uterine inflammatory response post-insemination was too high when using 7% DMF or sperm chromatin was damaged, affecting in vivo results. In conclusion, the rapid semen freezing technique was successfully applied in jacks. Regarding the extenders, no significant in vitro differences were observed using either BotuSemen Gold or EDTA-glucose media, whether adding MF or DMF at either 5% or 7%. However, when inseminating mares, pregnancies were only obtained using 5% DMF. It would be interesting to analyze sperm DNA and peroxidation characteristics as well as the uterine response to insemination to determine possible reasons for the failure in fertility and the variability observed among individuals.

Effect of post-thaw addition of seminal plasma to cryopreserved epididymal equine spermatozoa

Seminal plasma (SP) has been reported to increase the immediate motility of frozen-thawed epididymal sperm and appears to be important for plasma membrane remodeling, capacitation, acrosomal reaction and fertilization. The objective of this study was to evaluate the effect of incubating equine epididymal frozen-thawed sperm with different concentrations of seminal plasma. Sperm samples were obtained from testes after castration of 40 young Silla Argentino stallions (24 to 36 months of age). Testicles were cooled (5°C) and after 24 h, the cauda epididymis were dissected and flushed (retrograde) with Kenney extender. Epididymal sperm were cryopreserved in EDTA-glucose with 5% DMF, 11% lactose, 20% egg-yolk and Equex, using a rapid temperature-descent curve. The samples were thawed at 37°C for 1 min and then incubated 10 minutes with different concentrations of SP (10%, 50%, 100%) vs. a control (0% SP). To create a pool of SP, Silla Argentino stallions of proven fertility were used. Seminal plasma was separated and stored at -18 °C until use. Semen parameters evaluated were sperm motility (Computer Assisted Semen Analysis; ISAS v1, Proiser), morphology (Diff Quick stain), viability and membrane function (combined hypoosmotic test and Coomassie blue stain). Data were analyzed using Kruskal Wallis test with P<0.05considered significant. Post-thaw incubation with increasing concentrations of seminal plasma significantly decreased sperm total and progressive motility (0%: 33.06 ± 2.02; 26.71 ± 1.90; 10%: 24.06 ± 1.74; 18.85 ± 1.59; 50%: 19.44 ± 1.71; 14.92 ± 1.53; 100%: 14.97 ± 1.57; 11.66 ± 1.48 respectively. Values are mean +/- SEM). However, the values of Beat-Cross Frequency, Linearity and Straightness were significantly higher when the spermatozoa were incubated with SP (BCF: 0%: 10.24 ± 1.47a; 10%: 10.67 ± 3.13a,b; 50%: 10.63 ± 2.31b; 100%: 11.45 ± 2.73b; STR: 0%: 0.59 ± 0.07a; 10%: 0.62 ± 0.09a,b; 50%: 0.64 ± 0.09b; 100%: 0.64 ± 0.12b; LIN: 0%: 0.23 ± 0.05a; 10%: 0.25 ± 0.06b; 50%: 0.26 ± 0.07b; 100%: 0.26 ± 0.07b). There was no effect on membrane function, acrosome status and sperm morphology, regardless of the concentration of SP used (P>0.05). The results indicate that there is no evidence of a beneficial effect of post-thaw supplementation with SP at the concentrations used in this study. It has to be considered that the main role of SP is not associated with sperm physiology but as a modulator in the female reproductive tract. Hence we are continuing in vivo studies to assess whether SP addition can improve pregnancy rates after artificial insemination with frozen-thawed epididymal sperm.

Evaluation of equine semen frozen in extenders free of animal proteins

Various countries are reticent regarding importation of semen that contains egg-yolk, limiting its commercialization. Egg-yolk composition is complex, varying with the diet of the chicken and egg-yolk granules are similar in size to sperm, interfering with analysis. Therefore, development of a chemically defined cryopreservation extender that maintains seminal parameters is relevant. Twelve ejaculates from 4 stallions of proven fertility (n= 4; r=3) were diluted in one of 5 extenders: 1) EDTA-glucose with 11% lactose, Equex, 20% egg-yolk and 5% dimethylformamide (EY); 2) casein salt and cyclodextrin cholesterol extender (CE); 3) CE with 3% dimethylformamide (CE-3); 4) OptiXcell (OP); 5) BioXcell (BIO). Cryopreservation was carried out in a Cryogen, Neovet automatic freezing machine: -0.5°C/minute to 5°C, 180 minutes equilibration, then -5°C/minute to -20°C and -20°C/minute to -100°C, then plunging in N2. Thawing was carried out at 37°C for 1 minute and sperm kinematic parameters (CASA, AndroVision, Minitube), viability and acrosome status (FITC-PNA-PI stain), membrane lipoperoxidation (BODIPY stain) and DNA fragmentation (SCD) were evaluated. Data were analyzed using ANOVA or Friedman test and results are reported as mean ± SD. Higher (P<0.05) percentages of total and progressively motile sperm were observed in samplescryopreserved in EY (29.25 ± 11 and 21.17 ± 10), CE-3 (30.4 ± 15 and 22 ± 12) and OP (23.1 ± 14 and 15.5 ± 10) compared to those in CE (4.75 ± 3 and 1.58 ± 2) and BIO (8 ± 4 and 3.25 ± 3). Straight-line, curvilinear and average-path velocity parameters, amplitude of lateral head and beat-cross frequency were significantly higher in samples frozen with EY, CE-3 and OP compared to CE and BIO media. Straight-line and average-path velocities were also significantly higher in OP compared to EY medium. Live acrosome-intact sperm were significantly higher in EY (45 ± 14) compared to the rest of the extenders; with CE-3 (25 ± 10) being significantly higher than CE (8 ± 5), OP (9 ± 5) and BIO (13 ± 5). Live, non-peroxidized sperm were significantly higher in EY (47 ± 15) compared to the other extenders; with CE-3 (29 ± 15) being significantly higher than CE (14±10), OP (21 ± 15) and BIO (23 ± 14). No significant differences were observed in the percentages of sperm with intact DNA in any of the extenders: EY (95 ± 4); CE (95 ± 2); CE-3 (93 ± 8); OP (94 ± 7) and BIO (94 ± 5). To conclude, the casein salt and cyclodextrin cholesterol extender with 3% dimethylformamide, without egg-yolk could be a suitable alternative for extenders with animal proteins. Further studies are ongoing to evaluate in vivo fertility of ejaculates cryopreserved in this extender.