Nanotechnology in reproduction: Vitamin E nanoemulsions for reducing oxidative stress in sperm cells.

Vitamin E is considered a powerful biological antioxidant; however, its characteristics such as high hydrophobicity and low stability limit its application. We propose to use nanotechnology as an innovative tool in spermatology, formulating nanoemulsions (NE) that accommodate vitamin E, protecting it from oxidation and promoting its release into the medium. The protective effect of the NE against oxidative stress was assessed in red deer epididymal sperm incubated at 37 °C. Cryopreserved sperm from eleven stags were thawed and extended to 400 × 106 sperm/ml in Bovine Gamete Medium (BGM). Once aliquoted, the samples were supplemented with the NE at different concentrations (0, 6 and 12 mM), with or without induced oxidative stress (100 µM Fe2+/ascorbate). The samples were evaluated after 0, 2 and 4 h of incubation at 37 °C. Motility (CASA), viability, mitochondrial membrane potential, acrosomal status, lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (SCSA®) were assessed. After 2 and 4 h of incubation, the NE were able to prevent the deleterious effects of oxidative stress, thus improving total and progression motility (P ˂0.05). Moreover, the highest concentration tested (12 mM) improved almost every sperm kinematic variable (P ˂0.05) and preserved sperm viability in samples subjected to oxidative stress. In addition, 12 mM of NE protected the acrosomes integrity, maintained and protected mitochondrial activity, prevented sperm lipoperoxidation and reduced ROS production (P ˂0.05) in samples subjected to oxidative stress. This work indicates for the first time that vitamin E formulated in NE could be a new approach against sperm oxidative damage. This could be highly relevant for sperm physiology preservation in the context of assisted reproduction techniques.

Cinnamtannin B-1, a novel antioxidant for sperm in red deer.

Cinnamtannin B-1 (CNB-1) is a naturally occurring trimeric A-type proanthocyanidin contained in several plants such as cinnamon (Cinnamomum zeylanicum). It is considered to be a potent antioxidant. The protective effect of CNB-1 against oxidative stress was assessed in red deer epididymal sperm incubated at 37 °C. Cryopreserved sperm from six stags were thawed, pooled and extended to 400 × 106 sperm/ml in BGM (bovine gamete medium). After being aliquoted, the samples were supplemented with different concentrations of CNB-1 (0, 0.1, 1, 10 and 100 µg/mL), with or without induced oxidative stress (100 µM Fe2+/ascorbate). The samples were evaluated after 0, 2 and 4 h of incubation at 37 °C. This experiment was replicated six times. Spermmotility (CASA), viability, mitochondrial membrane potential, acrosomal status, lipoperoxidation (C11 BODIPY 581/591), intracellular reactive oxygen species (ROS) production and DNA status (TUNEL) were assessed. After 4 h of incubation, CNB-1 prevented the deleterious effects of oxidative stress, thus improved sperm progressivity and velocity (P<0.05). Furthermore, 1 and 10 µM CNB-1 improved sperm linearity, even when compared to those samples that had not been subjected to oxidative stress (P<0.05). The greatest concentration, 100 µM, prevented sperm lipoperoxidation and reduced ROS production in samples subjected to oxidative stress.

Objective evaluation of ram and buck sperm motility by using a novel sperm tracker software.

This work offers researchers the first version of an open-source sperm tracker software (Sperm Motility Tracker, V1.0) containing a novel suit of algorithms to analyze sperm motility using ram and buck sperm as models. The computer-assisted semen analysis is used in several publications with increasing trend worldwide in the last years, showing the importance of objective methodologies to evaluate semen quality. However, commercial systems are costly and versatility is constrained. In the proposed method, segmentation is applied and the tracking stage is performed by using individual Kalman filters and a simplified occlusion handling method. The tracking performance in terms of precision (number of true tracks), the percentage of fragmented paths and percentage of correctly detected particles were manually validated by three experts and compared with the performance of a commercial motility analyzer (Microptic's SCA). The precision obtained with our sperm motility tracker was higher than the one obtained with a commercial software at the current acquisition frame rate of 25 fps (P < 0.0001), concomitantly with a similar percentage of fragmentized tracks (P = 0.0709) at sperm concentrations ranging 25-37 × 106 cells/mL. Moreover, our tracker was able to detect trajectories that were unseen by SCA. Kinetic values obtained by using both methods were contrasted. The higher values found were explained based on the better performance of our sperm tracker to report speed parameters for very fast motile sperm. To standardize results, acquisition conditions are suggested. This open-source sperm tracker software has a good plasticity allowing researchers to upgrade according requirements and to apply the tool for sperm from a variety of species.

Effect of sex-sorting and cryopreservation on the post-thaw sperm quality of Iberian red deer spermatozoa.

This study investigated the effect of sex-sorting and cryopreservation on post-thaw characteristics and fertility of red deer (Cervus elaphus) sperm for the first time. Semen was collected by electroejaculation from 10 mature stags during the breeding season, and each ejaculate split into four experimental groups: Bulk sorted spermatozoa, sorted but not sexed (BSS); sorted high purity X-spermatozoa (XSS); sorted high purity Y-spermatozoa (YSS); and, control non-sorted spermatozoa (NS). Following, all samples were frozen over liquid nitrogen. Two straws per stag and sample type were analyzed immediately post-thaw and following a 2-h incubation period at 37 °C. Post-thaw total motility (TM) as assessed by CASA was not different (P < 0.05) among NS, BSS and YSS sperm. For XSS, post-thaw TM was lower (39%, P < 0.05) than that for NS (54%) or BSS (50%), but similar (P > 0.05) to that of YSS (47%) sperm. The percentage of apoptotic spermatozoa as assessed by PI/YO-PRO-1 and flow cytometry analysis, was higher (17%, P ≤ 0.05) for XSS sperm than NS (12%), BSS (13%) and YSS (14%) sperm. Following incubation there were no differences (P > 0.05) in TM or percent apoptosis among treatments. Post-thaw chromatin stability calculated as the DNA fragmentation index (%DFI) was similar among treatments; following incubation %DFI increased in all except YSS, which displayed the lowest value (P < 0.05). Artificial insemination of synchronized hinds yielded 44, 52 and 62% delivery rates for YSS, NS and standard frozen-thawed sperm, respectively (P < 0.05). Notably, 93 and 55% of fawns born were males for the YSS and NS spermatozoa, respectively (P < 0.05). In summary, Y-sorted sperm displayed acceptable post-thaw sperm evaluation parameters and the expected offspring sex ratio. More studies are needed to understand the source of sperm damage that may compromise the fertility of Y-sorted red deer sperm.

Selection of red deer spermatozoa with different cryoresistance using density gradients.

The objective of sperm selection media is selecting the best spermatozoa and to remove seminal plasma and diluent for using them in assisted reproductive techniques. It is known that individuals show different cryoresistance in response to the same freezing procedure. Our hypothesis was that the efficacy of selection media could be dissimilar for samples with different sperm quality after thawing. Epididymal sperm samples from mature Iberian red deer were collected and frozen. Males were classified as with high post-thaw sperm quality when sperm motility (SM) ≥ 70%, or as with low post-thaw sperm quality when SM ≤ 69%. Samples were centrifuged using the following density gradients (DG): Percoll® , Puresperm® and Bovipure™ , and several functional sperm parameters were assessed after sperm selecting and washing. Males classified with high sperm quality had higher post-thawing values (p > .05) for all parameters evaluated, except for linearity index, than those categorized as low sperm quality. After selection, some sperm characteristics improved (viability, apoptosis and mitochondrial activity) for both groups, showing the males with high sperm quality higher values in all sperm parameters except for kinematic traits and DNA fragmentation index (%DFI), regardless of DG. Bovipure™ yield lower values of sperm motility, viability, apoptosis and mitochondrial activity in relation to Percoll® and Puresperm® considering both quality groups. There was an interaction between the type of DG and sperm quality group for sperm viability (p = .040) and apoptosis (p = .003). Thus, Percoll® selected less live and more apoptotic spermatozoa than Puresperm® and Bovipure™ for males with low sperm quality. In conclusion, the DG are more efficient selecting spermatozoa from samples with high sperm quality, acting differently depending on initial sperm quality.

Oestrous sheep serum balances ROS levels to supply in vitro capacitation of ram spermatozoa.

Reactive oxygen species (ROS) are fundamental for intracellular signalling. In spermatozoa, they are involved both to apoptosis and to capacitation, and changes in ROS levels can alter the balance between these two processes. Oestrous sheep serum (OSS) is considered an efficient agent for in vitro capacitation of ram spermatozoa. We have explored the effects of OSS on ram sperm physiology, especially on ROS production, during in vitro capacitation. Semen samples from 15 rams were cryopreserved. After thawing, samples were submitted to four treatments: control (CTL), 10% OSS supplementation for in vitro sperm capacitation, caspase inhibitor (INH, Z-VAD-FMK 100 µM) and OSS (10%) plus caspase inhibitor (I + E). Sperm samples were incubated for 30 min at 38.5°C and 5% CO2 and evaluated motility and kinetic parameters by computer-assisted semen analysis (CASA) and viability (propidium iodide), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), intracellular calcium (FLUO-3), membrane fluidity (M540) and ROS production (CM-H2 DCFDA) by flow cytometry. OSS induced changes in kinetic parameters compatible with capacitation, with a decrease in the percentage of progressive motility and linearity, and an increase in the amplitude of the lateral displacement of the sperm head (p < .05). Moreover, OSS increased the proportion of M540+ viable spermatozoa, YO-PRO-1+ and acrosome-reacted spermatozoa (p < .05). After incubation, OSS and I+E achieved lower ROS levels (p < .05). Ca(2+) levels did not change with the incubation, but were slightly higher (p < .05) when both OSS and the inhibitor were present. We suggest that OSS may modulate ROS levels, allowing intracellular signalling for capacitation to occur while preventing higher levels that could trigger apoptosis.

An In Vitro Evaluation of Biochemical Processes Involved in Lead-Induced Changes on Ram Spermatozoa.

Lead (Pb(2+) ) is a toxic heavy metal which interferes with several physiological processes regulated by Ca(2+) , including those characterized by changes of the membrane stability and the motility of spermatozoa necessary for the fertilization of the oocyte. In this study, ejaculated sperm from six rams (Ovis aries) have been incubated in vitro with or without 50 ng Pb(2+) /ml during 30 min and in the presence or absence of three different potential modulators of the effects of Pb(2+) on changes in the sperm membrane before fertilization: charybdotoxin, quinacrine and staurosporine. Sperm samples incubated with Pb(2+) have shown significant reductions in acrosome integrity and sperm viability and an increase in progressive movement. None of the studied potential modulators had a protective effect against Pb(2+) action. On the contrary, Pb(2+) -incubated sperm in the presence of staurosporine had lower acrosome integrity, and lower sperm viability was observed when spermatozoa were incubated with Pb(2+) + charybdotoxin. Quinacrine was the only tested substance capable of increasing the concentration of Pb(2+) in spermatozoa; thus, the enhancement of Pb(2+) effects produced by staurosporine and charybdotoxin was not produced by an increased uptake of Pb(2+) by spermatozoa. However, the increase of intracellular Pb(2+) in those spermatozoa incubated with quinacrine did not result in an adverse effect on sperm motility or viability although the acrosome integrity was negatively affected.

The Effect of Oxidative Stress on Thawed Bulk-Sorted Red Deer Sperm.

The aims of this study were to assess the effects of the sex-sorting process on post-thaw sperm quality as well as on induced oxidative stress damage (H2 O2 0 mm = H000; H2 O2 50 mm = H050; H2 O2 100 mm = H100) and the protective action of reduced glutathione (GSH) and Trolox, when comparing sorted (BSS) and non-sorted (NS) red deer spermatozoa incubated at 37°C. Sperm samples from three stags were collected by electroejaculation and frozen. Immediately after thawing, sperm motility was higher (p < 0.05) for NS (59% ± 3.3) than BSS (36.9% ± 5.8) sperm. Furthermore, the percentage of apoptotic sperm was higher (p < 0.05) for BSS (21.6% ± 5.0) than NS sperm (14.6% ± 1.2). The presence of H2 O2 increased DNA damage in NS (H000 = 4.1% ± 0.9; H050 = 9.3% ± 0.7; and H100 = 10.9% ± 2.3), but not in BSS sperm. However, in the presence of oxidant, GSH addition improved (p < 0.05) sperm motility in both groups of sperm samples as compared to their controls (NS: 44.5 ± 4.8 vs 21.1 ± 3.9 and BSS: 33.3 ± 8.1 vs 8.9 ± 1.8). These results demonstrate that the sperm-sorting process induces sublethal effects, albeit selecting a sperm population with a chromatin more resistant to oxidative stress than that in non-sorted sperm. Moreover, addition of GSH at 1 mm may be a good choice for maintaining the quality of stressed sperm samples, unlike Trolox, which inhibited sperm motility.

Influence of semen collection method on sperm cryoresistance in small ruminants.

Semen collection for cryopreservation is a key step for small ruminant conservation programs. While in these species semen is mainly collected via artificial vagina (AV), electroejaculation (EE) provides a viable alternative for untrained males. Herein we investigated the effect of semen collection method on post-thaw sperm quality by comparing two small ruminant species, sheep and goats. Semen from Blanca-Celtibérica bucks and Manchega rams was collected by AV and EE on the same day and cryopreserved using a standard protocol. At thawing, sperm motion parameters were evaluated by CASA, whereas membrane stability (YO-PRO-1), sperm viability (propidium iodide, PI) and mitochondrial activity (Mitotracker Deep Red) were analyzed using flow cytometry. The semen collection method negatively affected post-thaw sperm quality in bucks but not in rams. Thus, in bucks, post-thaw sperm motility was higher for samples collected by AV as compared to those obtained via EE. Similarly, post-thaw sperm parameters evaluated by flow cytometry were worse for buck samples collected by EE than those collected by AV in the same species, or than ram samples regardless of collection method. These results suggest that ovine and caprine spermatozoa have a different response to the cryopreservation process depending upon the semen collection method used. We hypothesize that the EE procedure may lead to changes in the composition of the ejaculate in bucks that would make spermatozoa more susceptible to the cryopreservation process, whereas this procedure would have had no effect on ram spermatozoa. This assumption requires further investigation.

Estrous sheep serum enables in vitro capacitation of ram spermatozoa while preventing caspase activation.

Estrous sheep serum (ESS) is considered the most efficient agent for in vitro capacitation of ram spermatozoa. We have explored the relationship between caspase activation and capacitation in ram. Semen samples from 17 rams were cryopreserved. In vivo fertility was evaluated after intrauterine artificial insemination. Samples were submitted to four treatments: control, ESS (10%), caspase inhibitor (Z-VAD-FMK), and estrous ewe serum plus caspase inhibitor (I + E). Sperm samples were incubated for 30 minutes at 38.5 °C and 5% CO2 and analyzed with flow cytometry for mitochondrial membrane potential (MitoTracker deep red), sperm viability and apoptosis-like changes (YO-PRO-1/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate), membrane fluidity (merocyanine 540), and caspase activity (Vybrant FAM kits for polycaspases, caspase-8, and caspases 3-7). Estrous sheep serum induced changes compatible with capacitation, doubling the proportion of viable spermatozoa with increased merocyanine 540 and increasing YO-PRO-1(+) and acrosome-reacted spermatozoa (P < 0.05). Incubation increased the proportion of spermatozoa with activated caspases (P < 0.05), which was abolished by the treatments. We detected a simultaneous decrease in the proportion of the viable and caspase(-) spermatozoa after the incubation, which was prevented by the presence of estrous ewe serum (P < 0.05). The analysis of caspases 3/7 and 8 resulted in less marked differences. Fertility was positively related to viability and inactivated caspases and negatively to viable-capacitated spermatozoa and active caspases. In vitro induction of capacitation in thawed ram spermatozoa by using ESS suggests a downregulation in apoptotic pathways. However, males with the lowest fertility showed parameters similar to high-fertility males, suggesting that other factors were involved apart from capacitation and/or caspase activation.

Reduced glutathione addition improves both the kinematics and physiological quality of post-thawed red deer sperm.

The potential protective effect of reduced glutathione (GSH) and trolox (TRX), an analogue of vitamin E, supplementation during in vitro culture (2h, 39°C) of electroejaculated frozen/thawed red deer sperm was investigated. Cryopreserved sperm were thawed and incubated with no additive (Control) and 1mM or 5mM of each antioxidant to find out whether these supplementations can maintain the sperm quality, considering the use of thawed samples for in vitro techniques such as in vitro fertilisation (IVF), sperm sex sorting or refreezing. The effect of GSH on sperm motility was positive compared to TRX which was negative (P<0.001). After 2h of incubation at 39°C, use of GSH improved motility while TRX supplementation reduced sperm motility compared with Control samples without antioxidant. Use of TRX at both concentrations (1 and 5mM; TRX1 and TRX5) resulted in lesser percentages of apoptotic sperm (12.4±1.1% and 11.7±0.9%) than GSH1, GSH5 (15.2±1% and 14.6±1.1%) and Control samples (16.9±1.2%) (P<0.001). Use of GSH at both concentrations (1 and 5mM) resulted in greater mitochondrial activity as compared with findings for the Control, TRX1 and TRX5 groups. Results of this study indicate that GSH is a suitable supplement for electroejaculated red deer sperm. It would be necessary to conduct fertility trials (in vivo and in vitro), to assess whether GSH supplementation of thawed red deer sperm could improve fertility rates.

Sperm head phenotype and male fertility in ram semen.

Although there is ample evidence for the effects of sperm head shape on sperm function, its impact on fertility has not been explored in detail at the intraspecific level in mammals. Here, we assess the relationship between sperm head shape and male fertility in a large-scale study in Manchega sheep (Ovis aries), which have not undergone any selection for fertility. Semen was collected from 83 mature rams, and before insemination, head shapes were measured for five parameters: area, perimeter, length, width, and p2a (perimeter(2)/2×π×area) using a computer-assisted sperm morphometric analysis. In addition, a cluster analysis using sperm head length and p2a factor was performed to determine sperm subpopulations (SPs) structure. Our results show the existence of four sperm SPs, which present different sperm head phenotype: SP1 (large and round), SP2 (short and elongated), SP3 (shortest and round), and SP4 (large and the most elongated). No relationships were found between males' fertility rates and average values of sperm head dimensions. However, differences in fertility rates between rams were strongly associated to the proportion of spermatozoa in an ejaculate SP with short and elongated heads (P < 0.001). These findings show how the heterogeneity in sperm head shape of the ejaculate has an effect on reproductive success, and highlight the important role of modulation of the ejaculate at the intraspecific level.

Effect of different media additives on capacitation of frozen-thawed ram spermatozoa as a potential replacement for estrous sheep serum.

Capacitation is a key process through which spermatozoa acquire their fertilizing ability. This event is required for the successful application of assisted reproductive technologies such as IVF. The aim of the present study was to investigate the effect of using a synthetic oviductal fluid medium supplemented with either heparin-hypotaurine alone, in combination with progesterone (P4), 17ß-estradiol (E2), or BSA, or just ß-cyclodextrin, in replacement for estrous sheep serum (ESS) for ram sperm capacitation. After incubation in the corresponding media for 15 (time 0) or 60 minutes, sperm function was evaluated by computerized sperm motility analysis and flow cytometry (plasma membrane status and fluidity). Treatments rendering the best results in regards to sperm function parameters related to capacitation were used for an IVF test. Herein, neither heparin-hypotaurine (alone), or in combination with P4, or E2, nor ß-cyclodextrin induced capacitation-related changes in frozen-thawed ram spermatozoa. Only the medium supplemented with heparin-hypotaurine-BSA was able to induce changes compatible with in vitro capacitation relating to sperm motility pattern and plasma membrane fluidity, comparable to those in ESS-containing medium. Both media yielded sperm parameter values that differed (P < 0.05) from those obtained in the rest of the media tested. However, after the IVF trial, BSA was unable to support cleavage rates (21.80%) comparable to those obtained with ESS (52.60%; P < 0.05). We conclude that heparin-hypotaurine, P4, E2, ß-cyclodextrin, or BSA is not suitable for replacing ESS in capacitation and fertilization media for ram spermatozoa.

Use of Androcoll-S after thawing improves the quality of electroejaculated and epididymal sperm samples from red deer.

Single Layer Centrifugation is a useful technique to select sperm with good quality. The use of selection methods such as Androcoll could become an important tool to improve the quality of sperm samples and therefore to improve other artificial reproductive techniques such as sperm sex sorting, in vitro fertilization or AI. The aim of this study was to evaluate the effect of a Single Layer Centrifugation with Androcoll-S on the sperm quality of red deer sperm samples of two different origins, electroejaculated samples and epididymal samples obtained post-mortem, after thawing and after an incubation for 2h at 37°C. Sperm motility, viability, membrane permeability, mitochondrial activity, acrosomal status and DNA fragmentation were determined for all samples. The samples selected by Androcoll-S showed an improvement in sperm kinematics compared to unselected samples after thawing and after incubation. The same effect was observed in parameters such as viability, mitochondrial activity or acrosomal status which were improved after the selection. In contrast, no difference was found in DNA fragmentation between selected and unselected samples within the same sperm type. We conclude that sperm selection by SLC with Androcoll-S after thawing for red deer sperm of both types is a suitable technique that allows sperm quality in both types of sperm samples to be improved, thereby improving other assisted reproductive techniques. Further studies (IVF and in vivo fertilization) are required to determine whether this improvement can increase fertility, as has been shown for other species.

Understanding sperm heterogeneity: biological and practical implications.

Sperm are the most diverse cell type known. This diversity is thought to reflect adaptation to conditions under which sperm function as a way to ensure the survival of sperm in fertilization environments and to maximize fertilizing capacity thereof. The existence of morphological diversity among species is widely assumed, although this diversity seems less clear as we go deeper (between males, between ejaculates from the same male and even within the same ejaculate), with different theories addressing this heterogeneity. Moreover, the development of assisted reproductive techniques (ART) has led to changes in the physiological conditions in which sperm fertilize, which could lead, ultimately, to a selection towards more favourable sperm design. Regardless of the origin of this diversity, when studying the relationship between shape and function of sperm, it is advisable to assess the degree of heterogeneity of sperm and takes into account to be more likely to identify those morphological characteristics determining the fertile ability of sperm. Otherwise, these relationships could be hidden as a result of considering an average shape not representative of morphological characteristics of sperm. In addition, the knowledge of this morphological diversity in terms of changes arising from modifications in the sperm environment and mechanisms that generate these changes could be useful for understanding the reproductive capacity of males but also in enhancing their fertile ability.

Sperm characterization and identification of sperm sub-populations in ejaculates from pampas deer (Ozotoceros bezoarticus).

Pampas deer (Ozotoceros bezoarticus) is a native endangered species. Knowledge of the basic spermiogram characteristics and the morphometric descriptors is necessary to effectively develop sperm cryopreservation. In other species, sperm sub-population is related to sperm cryo-resistance. The objective was to provide a general description of the sperm, including sperm head morphometric descriptors, its repeatability, and the existence of sperm sub-populations. Sperm were obtained from adult males by electroejaculation during the breeding season. The motility score was 3.4 ± 0.2 (mean ± SEM) and progressive motility was 59.4 ± 3.7%. Ejaculated volume was 413.9 ± 51.0 µl, the total number of sperm ejaculated was 321.2 ± 55.4 × 10(6). Also, 63.3 ± 3.1% of the sperm were morphologically abnormal and 23.7 ± 2.3% had acrosome damage. The sperm head length was 7.6 ± 0.01 µm, width 4.4 ± 0.01 µm, area 28.1 ± 0.07 µm(2) and the perimeter was 21.9 ± 0.04 µm. There was a positive relationship among morphometric descriptors and the motility score, overall motility and progressive motility. Also length (P=0.011), width (P=0.003), area (P=0.006) and perimeter (P=0.009) of sperm head obtained in two different collections were positively related. Overall, the low concentration, volume, overall quality and abnormal morphology, and wide variation of these variables may be a limitation for the development of sperm cryopreserved banks. There were three sperm sub-populations with different morphometric characteristics. The morphometric descriptors are maintained similarly among different collections.

Fertility of cryopreserved ovine semen is determined by sperm velocity.

The present study aims to examine the predictive value of some sperm parameters on male fertility. Semen samples from six Manchega rams were collected and cryopreserved. Sperm quality was assessed after thawing and after 2h of incubation, either in the freezing extender (37°C) or after dilution in Synthetic Oviductal Fluid (SOF) (38°C, 5% CO2), attempting to mimic the physiological conditions of the female reproductive tract. The following sperm parameters were evaluated: motility and kinetic parameters by computer-assisted semen analyzer (CASA), and sperm viability (propidium iodide), mitochondrial membrane potential (JC-1), apoptotic-like membrane changes (YO-PRO-1), acrosomal status (PNA-FITC), and intracellular calcium (fluo-3) by flow cytometry. Results showed no significant differences between incubation media neither after thawing nor after incubation. There were no significant correlations between fertility and sperm parameters assessed by flow cytometry. However, after incubation in the freezing extender, sperm samples from males with poor fertility yielded less linearity and velocity (P<0.05) as indicated by motility parameters analyzed by CASA. These results indicate that kinematic sperm motility parameters evaluation by CASA might be useful to identify samples with poor fertility.

Single layer centrifugation (SLC) improves sperm quality of cryopreserved Blanca-Celtibérica buck semen.

The aim of the present study was to evaluate the effect of sperm selection by means of single layer centrifugation (SLC) on sperm quality after cryopreservation, either when SLC is used before freezing or after thawing, using Blanca-Celtibérica buck semen collected by electroejaculation (EE). Ejaculates from six bucks were collected by EE and divided into two aliquots. One of them (unselected) was diluted with Biladyl(®) by the two-step method and frozen over nitrogen vapor. The other aliquot was selected by the SLC technique and subsequently frozen in the same way as the unselected samples (SLC before freezing). In a further treatment, two unselected straws were thawed and SLC was carried out (SLC after thawing). At thawing, sperm motility of all samples ((i) unselected; (ii) selected before freezing and (iii) selected after thawing) was evaluated by CASA. In addition, integrity of the plasma membrane, mitochondrial membrane potential, ROS production and DNA fragmentation index were assessed by flow cytometry. Most of the sperm parameters were improved (P≤0.001) in samples selected by SLC after thawing in relation to unselected or selected by SLC before freezing. The percentage of progressive motile spermatozoa was greater (86%) for sperm samples selected after thawing compared with unselected (58%) or selected before freezing (54%). Moreover, percentages of spermatozoa with intact plasma membrane and spermatozoa with high mitochondrial membrane potential (hMMP) were also greater for sperm samples selected after thawing compared to sperm samples unselected or selected before freezing (spermatozoa with intact plasma membrane: 80% vs. 32% vs. 12%; spermatozoa with hMMP: 54% vs. 1% vs. 15%; respectively). Therefore, sperm quality after cryopreservation is improved in Blanca-Celtibérica buck ejaculates collected by EE when a sperm selection technique such as SLC is carried out after thawing.

Quality, oxidative markers and DNA damage (DNA) fragmentation of red deer thawed spermatozoa after incubation at 37 °C in presence of several antioxidants.

Antioxidants may be useful for supplementing sperm extenders. We have tested dehydroascorbic acid (DHA), TEMPOL, N-acetyl-cysteine (NAC) and rutin on epididymal spermatozoa from red deer, during incubation at 37 °C. Cryopreserved spermatozoa were thawed, washed and incubated with 1 mM or 0.1 mM of each antioxidant, including oxidative stress (Fe(2+)/ascorbate). Motility (CASA and clustering of subpopulations), viability, mitochondrial membrane potential, and acrosomal status were assessed at 2 and 4 h. Lipoperoxidation, intracellular reactive oxygen species (ROS) and DNA damage (DNA) status (TUNEL) were checked at 4 h. Oxidative stress increased ROS, lipoperoxidation and DNA damage. Overall, antioxidants negatively affected motility and physiological parameters. Only DHA 1 mm protected motility, increasing the fast and progressive subpopulation. However, it had a detrimental effect on acrosomal and DNA status, in absence of oxidative stress. Tempol and rutin efficiently reduced lipoperoxidation, ROS, and DNA damage in presence of oxidative stress. NAC was not as efficient as TEMPOL or rutin reducing lipoperoxidation or protecting DNA, and did not reduce ROS, but its negative effects were lower than the other antioxidants when used at 1 mm, increasing the subpopulation of hyperactivated-like spermatozoa at 2 h. Our results show that these antioxidants have mixed effects when spermatozoa are incubated at physiological temperatures. DHA may not be suitable because of prooxidant effects, but TEMPOL, NAC and rutin may be considered for cryopreservation trials. In general, exposure of red deer spermatozoa to these antioxidants should be limited to low temperatures, when only protective effects may develop.

Effect of semen collection method (artificial vagina vs. electroejaculation), extender and centrifugation on post-thaw sperm quality of Blanca-Celtibérica buck ejaculates.

The aim of this study was to evaluate the effect of semen collection method (artificial vagina compared to electroejaculation), season in which the semen was collected (breeding season compared to non-breeding season), freezing extender (Biladyl(®), Andromed(®) and skim milk based extender) and pre-treatment procedure (washing compared to non-washing) on post-thaw semen quality in buck. Ejaculates from seven bucks of the Blanca-Celtibérica breed were collected by artificial vagina and electroejaculation during the breeding (July to December) and non-breeding season (January to June). Samples were split in two aliquots and one of them was washed. Three freezing extenders were evaluated on washing and non-washing sperm samples. Ejaculates collected by artificial vagina had a greater sperm quality after thawing, with greater values (P≤0.05) for SM (sperm motility), NAR (acrosome intact), YO-PRO-1-/PI- (intact spermatozoa), and Mitotracker+/YO-PRO-1- (spermatozoa with active mitochondria) and lower % DFI (DNA fragmentation index). Thawed sperm samples which were collected during the breeding season had greater values (P≤0.05) for NAR, intact spermatozoa and spermatozoa with active mitochondria, than those semen samples obtained during the non-breeding season. Semen freezing with Biladyl(®) and Andromed(®) resulted in a greater sperm quality (P≤0.05) after thawing in relation to milk-based extender. Washing procedure had no effect on sperm parameters assessed at thawing. Results from the present study suggest that the success of semen cryopreservation in Blanca-Celtibérica goat depends on semen collection method and season, as well as on the extender used. Thus, the post-thaw sperm quality will be greater (P≤0.05) when samples are collected by artificial vagina during the breeding season and when Biladyl(®) or Andromed(®) are used as freezing extenders.