F.J. Peña

Antioxidant supplementation in vitro improves boar sperm motility and mitochondrial membrane potential after cryopreservation of different fractions of the ejaculate

Antioxidant supplementation during cooling was assayed to improve the motility of frozen-thawed (FT) boar spermatozoa from two different fractions of the ejaculate, the first component of the sperm-rich fraction (Fraction I) and the rest of the bulk ejaculate (Fraction II). Using a split-sample design, addition of two different concentrations (100 and 200 microMl(-1)) of the water-soluble Vitamin E analogue Trolox (6-hydroxy -2,5,7,8-tetramethylchroman -2-carboxylic acid) was evaluated for an effect on sperm motility (measured both subjectively and by means of a computer assisted motility assessment (CASA)), and on mitochondrial membrane potential using flow cytometry after cell-loading with JC-1. The effect of the Vitamin E analogue was clearly dose-dependent and varied with the fraction of the ejaculate considered. Motility was significantly higher in Trolox-treated spermatozoa (200 microm), from either ejaculate fraction, albeit the effect was more evident in spermatozoa from Fraction II (P<0.05) for any Trolox-concentration. Antioxidant supplementation resulted, also dose-dependent, in a higher number of spermatozoa showing high mitochondrial activity as assessed by the JC-1 staining, in both ejaculate fractions. In the present trial, exogenous Trolox positively affected post-thaw sperm viability (as motility and mitochondrial membrane potential) in both fractions of the ejaculate. The magnitude of the effect appeared, however, to be dependent of the fraction of the ejaculate considered.

Assessment of fresh and frozen-thawed boar semen using an Annexin-V assay: a new method of evaluating sperm membrane integrity

Detection of early changes in the sperm plasma membrane during cryopreservation is of utmost importance when designing freezing protocols. The present study evaluated the ability of an Annexin-V binding assay to detect early changes in sperm membrane integrity using flow cytometry (FC) in two different portions of the boar ejaculate, in cryopreserved semen. Using a split sample design, sperm motility was evaluated in fresh (controls) and frozen-thawed (FT) samples, both subjectively and by means of a computer-assisted motility assessment (CASA) system, while membrane integrity was assessed using Annexin-V (A) and propidium iodide (PI) staining in spermatozoa derived from the first sperm-rich fraction (Portion I) or the remaining ejaculate (Portion II). The A/PI technique revealed four sperm subpopulations, two PI negative (either A- (alive) or A+ (apoptotic)); and two PI positive (dead cells), either A+ (dead, late apoptotic or early necrotic cells) or A- (dead, late necrotic cells). Significant differences were found between the two portions of the ejaculate in the fresh (control) and FT samples. In the fresh controls, significantly more live, nonapoptotic spermatozoa (A-/PI-) were present in Portion I than in Portion II (P<0.001). Although apoptotic spermatozoa were detected in both semen portions, the frequency of live, early apoptotic (A+/PI-) cells was significantly lower in Portion I than in Portion II (P<0.001). Irrespective of the ejaculate portion considered, freezing and thawing significantly decreased the mean percentages of live spermatozoa (P<0.01), and dramatically increased the percentages of apoptotic or early necrotic cells (P<0.01), but not of early apoptotic cells (N.S.). The latter finding might suggest that apoptotic changes due to cryopreservation using the procedures applied in this trial are transient and lead to cell death. In conclusion, the Annexin-V binding assay was able to detect deleterious changes in the sperm plasma membrane at an earlier point than PI staining, thus representing a novel approach to investigating membrane integrity in this species. The finding that fewer spermatozoa in Portion I of the ejaculate showed early apoptosis post-freezing, suggests boar spermatozoa in this portion of the seminal plasma are less sensitive to the stress induced by cryopreservation.

Mitochondria in Mammalian Sperm Physiology and Pathology: A Review

While, for a long time, the role of mitochondria in sperm physiology and pathology has been largely ignored, recent research points out the mitochondria as a major organelle with key roles in sperm function both under physiological and biotechnological conditions. This paper briefly reviews these novel findings regarding the role of mitochondria in sperm, paying special attention to the most practical, readily applicable, aspects of the topic such as their role as a major source of the sublethal damage that sperm experiments after cryopreservation.

Antioxidant supplementation of boar spermatozoa from different fractions of the ejaculate improves cryopreservation: changes in sperm membrane lipid architecture

Previous studies have shown sperm quality after cryopreservation differs depending on the fraction of seminal plasma the boar spermatozoa are contained in. Thus, spermatozoa contained in the first 10 ml of the sperm-rich fraction (portion I) withstand handling procedures (extension, handling and freezing/thawing) better than those contained in the latter part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction; portion II). The present study evaluated whether an exogenous antioxidant, the water-soluble vitamin E analogue Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), could, when added to the freezing extender in a split-sample design trial, improve the post-thaw viability and membrane quality of this particular portion of the ejaculate, with particular attention to the status of the plasma membrane. Using a split-sample design, the initial changes in the fluidity status of the sperm plasmalemma after thawing were measured by flow cytometry (FC) after loading with Merocyanine-540 and YO-PRO-1. The FC-derived data revealed a clear ejaculate portion-dependent effect of the antioxidant supplementation. While no beneficial effect of the antioxidant supplementation was visible in spermatozoa from portion I, more spermatozoa with intact membranes were observed in the supplemented samples of portion II, suggesting the protective effect of vitamin E is dependent of the portion of the boar ejaculate considered.

Melatonin reduces lipid peroxidation and apoptotic‐like changes in stallion spermatozoa

Abstract:  Lipid peroxidation (LPO) has been claimed as a major factor involved in stallion damage during storage or cryopreservation. Because melatonin is a well‐known potent antioxidant, the aim of the present study was to investigate the effect of melatonin during in vitro incubation. Furthermore, we investigated the presence of specific melatonin receptors (MT1 and MT2) using specific polyclonal antibodies and western blotting. Stallion spermatozoa were incubated up to 3 hr at 37°C in the presence of different concentrations of melatonin (0, 50 pm, 100 pm, 200 pm, or 1 μm). At the beginning and at the end of the incubation period, sperm motility (using computer‐assisted sperm analysis), membrane integrity and permeability, fluidity of the sperm membrane, LPO, and mitochondrial membrane potential (Δψm) were flow cytometrically evaluated. Melatonin reduced changes in the spermatozoa related to apoptosis (increased sperm membrane permeability and lowered Δψm) (P < 0.05). Furthermore, LPO was dramatically reduced (P < 0.01) while no effect was observed on sperm motility or kinematics. Interestingly, melatonin helped maintain a more fluid sperm plasmalemma (P < 0.05). Our results clearly show the absence of MT1 and MT2 receptors in the stallion spermatozoa. It is concluded that melatonin is a useful tool to improve the quality of stored stallion sperm, increasing their life span and reducing premature aging, this likely relates to melatonin’s antioxidant properties.

Dissecting the molecular damage to stallion spermatozoa: the way to improve current cryopreservation protocols?

We review recent developments in the technology of freezing stallion sperm, paying special attention to the molecular lesions that spermatozoa suffer during freezing and thawing, such as osmotic stress, oxidative damage, and apoptotic changes. We also discuss the applicability of colloidal centrifugation in stallion sperm cryobiology. Increased knowledge about the molecular injuries that occur during cryopreservation may lead to improved protective techniques and thus to further improvements in fertility in the current decade.

Apoptotic markers can be used to forecast the freezeability of stallion spermatozoa

In an attempt to identify valuable markers for potential freezeability of the equine spermatozoa, three ejaculates were collected from five Andalusian stallions and frozen using a standard protocol. Before freezing, three apoptotic cell markers were studied by flow cytometry (early changes in sperm membranes, mitochondrial membrane potential and caspase activity). Post-thaw, spermatozoa were again evaluated for these parameters. Sperm kinematics using CASA were also studied before and after freezing and thawing. Receiving operating system curves were used to evaluate the relative value of the apoptotic markers herein studied, as forecast for potential freezeability. From all parameters studied, the outcome of JC-1 (as proportion of spermatozoa showing simultaneously orange and green fluorescence) had the highest diagnostic power. For potentially bad freezers (less than 25% of intact spermatozoa post-thaw), the significant area under the ROC-curve was 0.985, with a 100% sensitivity and 99.8% specificity for a cut off value of 55.7.

Inhibition of the mitochondrial permeability transition pore reduces "apoptosis like" changes during cryopreservation of stallion spermatozoa.

In order to evaluate to what extent the changes that occur during cryopreservation involve the mitochondrial permeability transition pore (PT-pore), a specific inhibitor was used during the cryopreservation process of stallion spermatozoa. Four ejaculates from each of 7 stallions were frozen using a standard protocol. Before freezing, each ejaculate was split into three subsamples. The first was supplemented with 2.5 microM bongkrekic acid (BA) and the second with 5 microM BA. The third subsample served as control. The BA significantly reduced the percentage of spermatozoa depicting active caspases after thawing, reduced the percentage of spermatozoa with increased membrane permeability, and increased the mitochondrial membrane potential of thawed sperm. Sperm motility was reduced as a result of the treatment. It is concluded that the mitochondrial pathway of apoptosis seems to be an important factor involved in the sublethal damage that equine spermatozoa experience after freezing and thawing, and that sperm motility in the equine species is largely dependent on mitochondrial ATP produced by oxidative phosphorylation.

Autophagy and Apoptosis Have a Role in the Survival or Death of Stallion Spermatozoa during Conservation in Refrigeration

Apoptosis has been recognized as a cause of sperm death during cryopreservation and a cause of infertility in humans, however there is no data on its role in sperm death during conservation in refrigeration; autophagy has not been described to date in mature sperm. We investigated the role of apoptosis and autophagy during cooled storage of stallion spermatozoa. Samples from seven stallions were split; half of the ejaculate was processed by single layer centrifugation, while the other half was extended unprocessed, and stored at 5°C for five days. During the time of storage, sperm motility (CASA, daily) and membrane integrity (flow cytometry, daily) were evaluated. Apoptosis was evaluated on days 1, 3 and 5 (active caspase 3, increase in membrane permeability, phosphatidylserine translocation and mitochondrial membrane potential) using flow cytometry. Furthermore, LC3B processing was investigated by western blotting at the beginning and at the end of the period of storage. The decrease in sperm quality over the period of storage was to a large extent due to apoptosis; single layer centrifugation selected non-apoptotic spermatozoa, but there were no differences in sperm motility between selected and unselected sperm. A high percentage of spermatozoa showed active caspase 3 upon ejaculation, and during the period of storage there was an increase of apoptotic spermatozoa but no changes in the percentage of live sperm, revealed by the SYBR-14/PI assay, were observed. LC3B was differentially processed in sperm after single layer centrifugation compared with native sperm. In processed sperm more LC3B-II was present than in non-processed samples; furthermore, in non-processed sperm there was an increase in LC3B-II after five days of cooled storage. These results indicate that apoptosis plays a major role in the sperm death during storage in refrigeration and that autophagy plays a role in the survival of spermatozoa representing a new pro-survival mechanism in spermatozoa not previously described.

Influence of seminal plasma on the kinematics of boar spermatozoa during freezing

Sperm motility is, for its relation to cell viability and fertility, a central component of the spermiogram, where consideration of motion patterns allows discrimination of sub-populations among boar spermatozoa. Extension and cryo-preservation imposes changes in these patterns in connection to handling, additives, temperature changes and the removal of boar seminal plasma (BSP) which apparently makes spermatozoa susceptible to oxidative stress, thus affecting survival and motility post-thaw. Detailed kinematic analyses during sperm cooling are sparse, particularly when considering the instrumentation and settings used for analyses, the effect of extenders, and of the BSP the processed spermatozoa are exposed to. Spermatozoa present in the first collectable 10mL of the sperm-rich fraction of the ejaculate (portion 1, P1-BSP), have shown an increased ability to sustain motility during and after cryo-preservation than spermatozoa immersed in the rest of the ejaculate (portion 2, P2). When P2-spermatozoa were cleansed from their BSP and exposed for 60min to pooled P1-BSP, their motility post-thaw increased to similar levels as P1-spermatozoa. This BSP-influence is sire-dependent, presumably related to the protein concentration in the different ejaculate portions, and apparently unrelated to changes in membrane integrity or membrane stability through conventional, controlled cooling.