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Cryopreservation of brown bear (Ursus arctos) semen requires centrifugation to increase concentration and/or remove urine contamination. However, a percentage of the spermatozoa are lost in the process. This percentage varies considerably between males and ejaculates, and we have studied the effect of sperm quality and seminal plasma characteristics on the spermatozoa recovery rate after centrifugation. One hundred and thirty one sperm samples obtained from fifteen brown bear males by electroejaculation under general anaesthesia were used. The ejaculates were centrifuged 600 × g for 6 min. Motility was assessed by CASA, and acrosomal status (PNA-FITC) and viability (SYBR-14/propidium iodide) were determined by flow cytometry. Seminal plasma characteristics (albumin, alkaline phosphatase, calcium, cholesterol, creatine, glucose, glutamic oxaloacetic transaminase (GOT), lactate, lipase, magnesium, phosphate and total protein) were determined by a biochemical and gas analysis. Total motility (r = 0.26; P = 0.005) and cell viability (r = 0.20; P = 0.033) were positively correlated with the percentage of recovered spermatozoa. Sperm recovery was correlated with the concentration of several components of seminal plasma: negatively with glucose concentration (r = −0.47; P = 0.005) and positively with the enzymes GOT (r = 0.36; P = 0.040) and lactate dehydrogenase (r = 0.36; P = 0.041). After sorting the data into classes according to sperm recovery (Low: 0–39, Medium: 40–69, High: 70–100), we observed that the samples with a lower recovery rate derived from ejaculates with lower values for TM, VAP and viability (P < 0.05). Multiple regression analysis rendered two models to define the post-centrifugation spermatozoa recovery which included total motility and damaged acrosome or glucose, GOT and lactate dehydrogenase. We discuss these relationships and their implications in the electroejaculation procedure and the handling of the sample during centrifugation.

Genome Resource Banks are keystones in the ex-situ conservation of wild species. Post-mortem (PM) collection of epididymal spermatozoa is an opportunistic and valuable source of germplasm, the time from the death of the animal limits its use. Seeking to improve germplasm preservation strategies for the chamois (Rupicapra sp.), the effect of PM time on epididymal sperm quality and freezability was studied using the Cantabrian chamois. Samples were classified according to PM collection time, up to 216 h (refrigerated), and cryopreserved (Tris-citric acid-fructose, 430 mOsm/kg, 15% egg yolk, 8% glycerol; freezing at −20 °C/min). Sperm quality was assessed after recovery and post-thawing (motility by CASA, HOS test, abnormal forms, cytoplasmic droplets, and viability and acrosomal damage by flow cytometry). The sperm mass pH and osmolality showed a positive correlation with time. Total sperm motility dropped after 2 days PM, with progressivity and sperm velocities remained similar up to 3 days PM. Sperm freezability was acceptable, with the post-thawing HOST, motility, progressivity, VAP, VCL, VSL and BCF negatively correlating with PM time. Overall, chamois epidydimal samples were not adequate for preservation after 6 days PM. Freezability capacity could make these spermatozoa suitable for specific ART even if kept refrigerated for several days PM.

The use of assisted reproductive techniques in cervids is increasing as the commercial use of these species increase. We have tested the suitability of the antioxidants Trolox and reduced glutathione (GSH) for freezing red deer epididymal spermatozoa, aiming at improving post-thawing quality. Samples from 19 stags were frozen in a TES-Tris-fructose extender (20% egg yolk, 8% glycerol), with 1 or 5 mM of antioxidant. Motility (CASA), lipoperoxidation (malondialdehyde –MDA– production), membrane status, mitochondrial activity, acrosomal status (flow cytometry) and chromatin status (SCSA: %DFI and %HDS; flow cytometry) were assessed after thawing and after 6 h at 39 °C. There were few differences between treatments after thawing, with Trolox reducing MDA production in a dose–response manner. After the incubation, sperm quality decreased and %DFI increased moderately, with no change for MDA. GSH improved motility, kinematic parameters and mitochondrial status, with a slight increase in %HDS. GSH 5 mM also increased moderately MDA production and %DFI, possibly due to enhanced metabolic activity and reducing power. Trolox maintained MDA low, but was detrimental to sperm quality. Trolox might not be appropriate for the cryopreservation of red deer epididymal spermatozoa, at least at the millimolar range. GSH results are promising, especially regarding motility improvement after the post-thawing incubation, and should be selected for future fertility trials.

The aim of this study was to assess the effect of insemination timing on pregnancy rates in red deer (Cervus elaphus) when using sex-sorted sperm samples. Semen was collected by electroejaculation from 8 mature stags and processed to obtain: Conventional samples, following standard freezing procedures for commercial purposes; Control sorted samples, diluted and handled as per sorted samples but without being submitted to the sorter passage; and Y Sex Sorted (YSS) samples. Hinds were synchronized via intravaginal CIDR (Controlled Internal Drug Release) placement and given eCG (Folligon PMSG Serum Gonadotrophin) on day 12, upon CIDR removal. They were then inseminated with one of each sperm treatment, at the following post-eCG intervals: I_1, 55:01–55:30 h; I_2, 55:31–56:00 h; I_3, 56:01–56:30 h; or, I_4, 56:31–57:00 h. Pregnancy rates were assessed at parturition. Average pregnancy rates were highest (P < 0.05) for Conventional samples (77.6%), but similar between YSS (49.8%) and Control sorted (51.3%) samples. However, when insemination interval was taken into account, pregnancy rates within the YSS group, pregnancy rates were 80 and 83.1% for I_1 and I_2, respectively were obtained. Notably, these rates were similar (P > 0.05) to the average pregnancy rates obtained with Conventional samples (77.6%). As expected, YSS sperm yielded 94% male offspring contrasting with the 57% males obtained with Conventional and Control sorted samples. Our findings support the importance of developing specific insemination timing protocols to improve pregnancy rates when using frozen-thawed sex-sorted sperm. These findings provide the foundation for further investigations in order to determine why the YSS sperm are able to fertilize the oocyte in a shorter period of time than the conventional samples.

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.

The development of a species-specific conservation protocol that involves artificial insemination with frozen semen needs to validate an effective methodology for freezing semen. Colloid centrifugation has been suggested and widely applied as an effective tool for selecting animal spermatozoa for artificial breeding. The objective of the present study was to compare different methods of centrifugation, single layer using Androcoll-Bear and Percoll and double layer using PureSperm 100 (in two different discontinuous gradients 40%-80% and 45%-90%), for the selection of fresh brown bear sperm samples. In the before freezing group, all selected samples showed a higher progressive motility and viability (except Percoll for motility 43.0 ± 5.3 [P < 0.05]); all colloids except PureSperm 45/90% rendered samples with fewer damaged acrosomes. In the after thawing group, all tested centrifugation colloids showed a good capacity to decrease the number of damaged acrosomes. Furthermore, PureSperm treatment (45/90%) resulted in an increase in apoptotic-like changes not only immediately after thawing but also after the incubation test, leading us to suggest that this gradient could induce some kind of deleterious effects on the sperm samples. On the other hand, PureSperm treatment (40/80%) yielded a quality preservation capacity similar to Androcoll-Bear in number of damaged acrosomes, different relative to the control (control, 5.3 ± 0.6; PureSperm 80, 2.0 ± 0.3; Androcoll, 2.1 ± 0.9 [P < 0.05]) but a decrease in the number of viable spermatozoa recovered after thawing relative to the control (control, 21.2 ± 3.1; PureSperm 80, 13.7 ± 2.7 [P < 0.05]). In conclusion, Androcoll-Bear constitutes a useful tool for handling of brown bear ejaculates owing to its simple handling and procedure with a reliable sperm selection and freezability. This colloid yielded an improvement in several sperm parameters in brown bear frozen-thawed semen; the selected spermatozoa of fresh samples with t

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 (HO 0 mm = H000; HO 50 mm = H050; HO 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 HO 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.

El uso de técnicas de reproducción asistida en cérvidos durante los últimos años ha experimentado un importante avance no solo con propósitos conservacionistas (Jabbour et al. 1997; Pukazhenthi y Wildt 2003), sino también con propósitos económicos (carne, velvet y trofeos) cuya importancia es alta en algunos países como España, Nueva Zelanda, Australia o China (Asher et al. 2000). Esta importancia económica ha generado un interés creciente por parte de los criadores de ciervo a la hora de buscar asesoramiento de especialistas con el fin de desarrollar técnicas que les permitan optimizar sus rendimientos y producciones. Dentro de este contexto, existe una gran variedad de técnicas de reproducción asistida como la electroeyaculación, la sincronización del estro a través del uso de implantes de progestágenos, la inseminación artificial (IA), la transferencia de embriones o la fecundación in vitro (IVF) que han sido objeto de investigación en varias especies de cérvidos (Asher et al. 1999), siendo la más importante tanto para los criadores como para los investigadores el ciervo rojo (Cervus elaphus). Uno de los principales objetivos de la presente tesis doctoral ha sido evaluar el rendimiento y efectos que tiene el proceso de sex-sorting, mediante el uso del sistema (SX MoFlo, DakoCytomation Inc., Fort Collins, CO, USA) modificado para selección espermática, en eyaculados de ciervo rojo obtenidos mediante electroeyaculación. Dicho proceso nos permite separar y clasificar los espermatozoides de eyaculados en fracciones enriquecidas de espermatozoides Y- y espermatozoides X- con una pureza superior al 90%. Estudios previos en cérvidos (Gao et al. 2011) han demostrado que es posible obtener fracciones enriquecidas de espermatozoide Y- con capacidad fecundante y obtener descendencia mediante inseminación artificial en hembras superovuladas. Esta tecnología de sexado espermático es especialmente interesante en especies como el ciervo, en las cuales el interés productivo y en

Sedimentation of spermatozoa occurs during long-term liquid storage and this may produce deleterious changes. Our aim was to apply gelatine supplementation during long-term pre-freezing storage of bear sperm, applying final dilution and 6% glycerol at room temperature and cool in straws. We tested four models of sperm storage using a 1:1 dilution in TTF-ULE-Bear extender (TesT-fructose-egg yolk-glycerol 6%): (i) second 1:1 dilution at room temperature (RT), cooling at 5°C in a tube and final dilution (100 × 10 sperm ml) (Standard); (ii) final dilution at RT and cooling in a tube (FD-Tube); (iii) final dilution at RT and cooling in 0.25 ml plastic straw (FD-Straw); and (iv) final dilution at RT in extender supplemented with 1.5% gelatine (Gelatine) and cooling in a 0.25 ml plastic straw. A Standard sample was stored at 5°C for 1 hr (Control); the rest of the samples (Standard, FD-Tube, FD-Straw, Gelatine) were stored for 24 or 48 hrs before freezing (100 × 10 sperm ml, glycerol 6%). The quality of the samples was assessed for motility by CASA, and viability (SYBR-14/propidium iodide-PI-; VIAB), acrosomal status (PNA-FITC/PI; iACR) and apoptotic status (YO-PRO-1/PI; YOPRO-) by flow cytometry. At pre-freezing, after 48 hr, Gelatine showed significantly higher viability (for VIAB and YOPRO-) and progressiveness (PM, LIN and STR). At 48 hr, Gelatine showed similar YOPRO-, iACR, LIN, STR and ALH respect to Control. At both 24 and 48 h post-thawing, Gelatine sample had similar scores for YOPRO-, iACR, LIN, STR, WOB and VIAB (only 24 hr) when compared with Control, and lower for TM, PM, rapidPM, VAP and ALH. No differences were found among others experimental groups with respect to Control. In conclusion, gelatine could be a suitable alternative to preserve the viability and progressive motility of brown bear ejaculates during long-term pre-freezing storage at 5°C.

Fertility is a highly complex biological function that depends on several properties of spermatozoa that are necessary for them to overcome various barriers in the female reproductive tract to reach the fertilisation site. This ability has been evaluated in vitro using cervical mucus migration tests. Head morphology has been widely studied, and various studies have reported correlations between head morphology and motility, fertility and DNA fragmentation. In the present study, we first evaluated the relationship between the ability of ram spermatozoa to overcome the mucus surrogate barrier in an in vitro migration test and sperm head morphology. Sperm motility (determined by computer-aided sperm analysis) and the acrosomal status, viability and mitochondrial status (determined by flow cytometry) of control and migrating spermatozoa were assessed. Principal component analysis and clustering analysis of the values for the morphometric parameters assessed defined three cell subpopulations. One of these subpopulations, namely spermatozoa with a short and wide head, was absent from samples collected after conclusion of the migration test. Second, we evaluated relationships among head morphology characteristics, the ability to penetrate the artificial mucus and fertility. We did not find any correlation between fertility and the number of spermatozoa that migrated, whereas there was a negative correlation between the proportion of spermatozoa with a short and wide head in the fresh sperm sample and fertility. In conclusion, the head morphology of spermatozoa was associated with their ability to overcome a mucus barrier in a migration test, and the relative size of the non-migrating subpopulation was negatively related to male fertility.

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% CO 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.

The use of prepubertal females (calves) to obtain oocytes for in vitro fertilization (IVF) programs, is being analyzed currently. This will increase the availability of female oocytes and will allow a reduction of the interval between generations. Differentials in the development capability of calf and cow oocytes have been assessed by different authors, establishing several ultrastructural and metabolic differences between them. This paper analyzes the morphometric and cytochemical differences between calf and cow oocytes through microscopic techniques. The oocytes morphologically classified as good are processed for electron microscopy a) in Epon 812 epoxy resin for morphometric analysis or b) in low temperature Lowycril K4M resin for cytochemical evaluation using Con A, GS, LPA, UEA, and WGA lectins marked with colloidal gold as probes. Calf oocytes show a greater density of microvilli on their surface and a greater number of endocytosic vesicles than those of the cow. On the other hand, cow oocytes show a larger superior mitochondrial population. In the cumulus cells it can be seen that calf oocytes have a greater volume of lipid droplets. Cytochemical analysis shows that calf oocytes have lectin marking restricted to the plasmic membrane, highlighting the presence of LPA. In cow oocytes, lectin marking can be seen both on the plasmic membrane and in the vacuoles, in both cases, with the LPA highlighted. In the zona pellucida of calf and cow oocytes, the same sugars appear (GS, LPA, WGA), and marking with LPA is more extensive in cow oocytes. © 2001 by Elsevier Science Inc.

The potential protective effect of reduced glutathione (GSH) and trolox (TRX), an analogue of vitamin E, supplementation during in vitro culture (2. h, 39. °C) of electroejaculated frozen/thawed red deer sperm was investigated. Cryopreserved sperm were thawed and incubated with no additive (Control) and 1. mM or 5. mM 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 2. h 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 5. mM; 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 5. mM) 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.

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 2. h 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.

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 2h, 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 2h 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 2h of incubation in SOF could be a useful procedure for predicting fertility of ram spermatozoa.

Brown bear ejaculates are usually collected in field conditions and may need to be shipped to a laboratory for the application of reproductive biotechnologies before cryopreservation. The aim of this study was to extend the prefreezing step to 48 hours (1 hour vs. long-term storage [LS] to 24 and 48 hours) to enable the sample to be transported. The effects of storage temperature (experiment 1), glycerol concentration (experiment 2), and dilution rate (experiment 3) on sperm were evaluated. Electroejaculates from brown bears were stored under different experimental conditions and cryopreserved. The sperm motility and viability, apoptotic status, and acrosomal status of sperm were assessed before freezing (prefreezing), after thawing, and after 2-hour incubation at 37 °C (thermal stress test). In all experiments, one control sample was frozen using a standard protocol (control). In experiment 1, three temperatures during LS with 6% glycerol were tested: 5 °C (T5), 15 °C (T15), and room temperature (RT). The LS-T5 sample yielded the highest postthawing results for viability (42.4%), progressive motility (15.6%), and intact acrosome (83.1%) after 24 hours in comparison with the other temperatures (P < 0.05); for 48 hours, the LS-T5 sample reached higher total and progressive motility (25.9% and 9%, respectively) and nonapoptotic values (36.5%). Recovery rates revealed susceptibility to freezing at LS-15 or LS-RT samples at 24 hours (viability) or 48 hours (viability and motility). In experiment 2, samples were stored at 5 °C up to 48 hours and three glycerol concentrations were evaluated: 0% (0Gly), 3% (3Gly), and 6% (6Gly). Postthawing viability and motility increased progressively with the percentage of glycerol for 24 hours at 5 °C; 6% glycerol during 48-hour storage had beneficial effects on sperm cryopreservation. Besides, 6% glycerol had a clearly superior freezability for viability (42.7% and 40.8% for 24 hours and 48 hours, respectively) and motility (24 hours

Antioxidants could improve sperm media, extending the viability of spermatozoa. Cinnamtannin B-1 (CNB-1) is a naturally occurring Atype proanthocyanidin found in a limited number of plants including Linderae umbellateae and L. nobilis. Epididymal sperm samples from six stags were used in this experiment. Thawed sperm samples were pooled. The protective ability of CNB-1 was tested on red deer spermatozoa incubated at 37°C. Cryopreserved spermatozoa were thawed and incubated with 0, 0.1, 1, 10 and 100 lM of CNB-1, with or without oxidative stress (100 lM Fe2+). Viability (YO-PRO-1/IP), acrosome status and lipoperoxidation (C11-BODIPY 581/591) were checked at 0, 2 and 4 h. For the statistical analysis, we used the R statistical environment. To analyze the effects of time, antioxidant and supplement (none, oxidant, and/or antioxidant) on sperm parameters, we used linear mixed-effects models. We replicated the experiment six times. High concentrations of CNB-1 (100 lM) preserved acrosome status in oxidized samples after 4 h of incubation (p < 0.05). Moreover, lower concentrations of CNB-1 showed limited protection against lipoperoxidation although high concentrations (10 and 100 lM) lowered lipoperoxiation in oxidized samples after 2 h of incubation (p = 0.03) and 4 h (p = 0.04). CNB-1 seems a promising new antioxidant, but its particular effects on sperm physiology must be further studied

In the ram, oestrous sheep serum (OSS) is considered to be necessary for in vitro capacitation and fertilization. OSS increases membrane fluidity and tyrosine phosphorylation, and produce changes in the motility pattern. We hypothesized that in addition to the already known actions of OSS, its actions could be related to apoptotic processes. Thus, thawed sperm samples from nine rams were incubated at 38.5°C and 5% CO2 in Synthetic Oviductal Fluid (SOF-HEPES), under capacitating (10% of OSS) and non-capacitating conditions. Caspase activity was evaluated using the Vybrant© FAMTM Poly caspases assay kit. Sperm samples were evaluated previously (Control) and after 30 min of incubation (capacitating and non-capacitating conditions). Initially, the percentage of viable spermatozoa with no caspase activity (%NCAS) was 22.3% +/- 3.0. After 30 min of incubation, this percentage significantly decreased in non-capacitating conditions (12.3% +/- 3.0; p = 0.03), but when sperm samples were incubated with 10% OSS, the %NCAS remained similar (23.7% +/- 3.0; p = 0.01 vs. non-capacitating). Thus, after 30 min of incubation, fewer spermatozoa showed caspase activation if serum was present, providing evidence of a possible delaying of the apoptotic process during in vitro capacitation. OSS might benefit in vitro fertilisation by different ways. Supported by Education and Science Council (JCCM; PAI09-0006-3806) and by INIA (RZ2010-00006-CO2-01).

The adaptability of cryopreservation protocols for brown bear spermatozoa collected under field conditions and frozen in a nearby laboratory (transported for a few hours) or shipped to a reference laboratory for sex sorting (transported for a few days) was evaluated. Forty-nine electroejaculates from 15 mature brown bears were extended to 100×106sperm/mL in a TES-Tris-Fructose based extender and cryopreserved (-20°C/min to -100°C and stored at -196°C). After thawing, the quality of the seminal samples was assessed for total (TM), progressive (PM) motility and kinetic parameters - by CASA -, and viability (VIAB), viable and non-apoptotic status (YOPRO-), high membrane mitochondrial potential (MIT) and intact acrosomes (iACR) - by flow cytometry - In Experiment 1, we assessed different storage times (0, 0.5, 1 - control -, 4-5, 7-8 and 11-12h) at 5°C from final dilution to freezing. After thawing, non-equilibrated samples (0h) showed lower values of iACR, TM and PM. No significant differences were found for the different periods of equilibration tested. In Experiment 2, we evaluated three long-term storage times (24, 48 and 72h) at 5°C before freezing using storage for 1h as control. The post-thawing quality of brown bear spermatozoa declined markedly after 48-72h of pre-freezing. In conclusion, our findings suggest the possibility of extending the pre-freezing cooling period up to 24h post-collection without freezing. This knowledge should enable the adaptation of the freezing protocols for when a special handling conditions are required such as the shipment of seminal samples to technological centers for the pre-freezing application of enhancer spermatic biotechnologies.

Specific protocols for the cryopreservation of endangered Cantabrian brown bear spermatozoa are critical to create a genetic resource bank. The aim of this study was to assess the effect of cooling rates and equilibration time before freezing on post-thawed brown bear spermatozoa quality. Electroejaculates from 11 mature bears were extended to 100 × 106 spermatozoa/mL in a TES-Tris-Fructose-based extender, cryopreserved following performance of the respective cooling/equilibration protocol each sample was assigned to, and stored at -196 °C for further assessment. Before freezing, after thawing, and after 1 hour's incubation post-thawing at 37 °C (thermal stress test), the quality of the samples was assessed for motility by computer-assisted semen analysis, and for viability (SYBR-14/propidium iodide), acrosomal status (peanut agglutinin-fluorescein isothiocyanate /propidium iodide), and sperm chromatin stability (SCSA) by flow cytometry. In experiment 1, three cooling rates (0.25 °C/min, 1 °C/min, and 4 °C/min) to 5 °C were assessed. After thawing, total motility (%TM) was higher and percentage of damaged acrosomes (%dACR) was lower (P < 0.05) for 0.25 °C/min than for 4 °C/min. The thermal stress test data indicated equally poor quality (P < 0.05) for the 4 °C/min cooled samples in viability (%VIAB), %dACR, %TM, and progressive motility (%PM). In experiment 2, the effect of a pre-freezing equilibration period at 5 °C for 1 hour (cooling at 0.25 °C/min) was evaluated. Samples kept at 5 °C for 1 hour showed higher (P < 0.05) values than the nonequilibrated ones for both thawing (%dACR) and thermal stress test (%VIAB, %TM, and %PM). In experiment 3, samples stored without cooling and equilibration (direct freezing) were compared with the samples cooled at 0.25 °C/min and equilibrated for 1 hour (control freezing). Using thermal stress test, we observed that direct freezing causes damage in viability, acrosomal status, and motility of spermatozoa compared with the contr

The present study evaluated modifications occurring in thawed ram spermatozoa during incubation in different media that supported in vitro capacitation and fertilisation, and examines how these changes relate to IVF. Thawed sperm sampleswere incubated under capacitating (Cap) and non-capacitating (non-Cap) conditions for 0, 1 and 2 h and used in an IVF test. During incubation, changes related to membrane status and the motility pattern of spermatozoa were assessed, the latter being used to characterise sperm subpopulations. Asignificantly greater increase (P≤0.05) in the percentage of spermatozoa with higher membrane fluidity was observed in samples incubated with Cap medium from the beginning of incubation. In addition, changes over time in the distribution of the motile subpopulation were particularly evident when spermatozoa were incubated with Cap medium, with a noted increase in spermatozoa classified as 'hyperactivated like', with major changes occurring after 1 h incubation. Both characteristics (i.e. membrane fluidity and the percentage of the hyperactivated-like subpopulation) were significantly related with in vitro fertility, and only sperm samples incubated with the Cap medium were capable of fertilising oocytes. These results support the idea that changes in sperm membrane fluidity and motility pattern (i.e. an increase in hyperactivated spermatozoa) are needed for fertilisation to take place.

Background Sperm cryopreservation has become an indispensable tool in biology. Initially, studies were aimed towards the development of efficient freezing protocols in different species that would allow for an efficient storage of semen samples for long periods of time, ensuring its viability. Nowadays, it is widely known that an important individual component exists in the cryoresistance of semen, and efforts are aimed at identifying those sperm characteristics that may allow us to predict this cryoresistance. This knowledge would lead, ultimately, to the design of optimized freezing protocols for the sperm characteristics of each male. Methodology/Principal Findings We have evaluated the changes that occur in the sperm head dimensions throughout the cryopreservation process. We have found three different patterns of response, each of one related to a different sperm quality at thawing. We have been able to characterize males based on these patterns. For each male, its pattern remained constant among different ejaculates. This latter would imply that males always respond in the same way to freezing, giving even more importance to this sperm feature. Conclusions/Significance Changes in the sperm head during cryopreservation process have resulted useful to identify the ability of semen of males for freezing. We suggest that analyses of these response patterns would represent an important tool to characterize the cryoresistance of males when implemented within breeding programs. We also propose follow-up experiments to examine the outcomes of the use of different freezing protocols depending on the pattern of response of males. © 2013 Ramón et al.

The use of sexed spermatozoa has great potential to captive population management in endangered wildlife. The problem is that the sex-sorting facility is a long distance from the semen collection place and to overcome this difficulty two freeze-thaw cycles may be necessary. In this study, effects of refreezing on brown bear electroejaculated spermatozoa were analyzed. We carried out two experiments: (1) to assess the effects of the two freezing-thawing cycles on sperm quality and to analyze three different elapsed times between freezing-thawing cycles (30, 90 and 180. min), and (2) to analyze the use of PureSperm between freezing-thawing cycles to select a more motile and viable sperm subpopulation which better survived first freezing. The motility, viability and undamaged acrosomes were significantly reduced after the second thawing respect to first thawing into each elapsed time group, but the elapsed times did not significantly affect the viability and acrosome status although motility was damaged. Our results with the PureSperm gradient showed higher values of viability in freezability of select sample (pellet) respect to the rest of the groups and it also showed a significant decrease in the number of acrosome damaged. In summary, the double freezing of bear semen selected by gradient centrifugation is qualitatively efficient, and thus could be useful to carry out a sex-sorting of frozen-thawed bear spermatozoa before to send the cryopreserved sample to a biobank. Given the low recovery of spermatozoa after applying a selection gradient, further studies will be needed to increase the recovery rate without damaging of the cell quality.

The use of assisted reproductive techniques in cervids is increasing as the commercial use of these species increase. We have tested the suitability of the antioxidants Trolox and reduced glutathione (GSH) for freezing red deer epididymal spermatozoa, aiming at improving post-thawing quality. Samples from 19 stags were frozen in a TES-Tris-fructose extender (20% egg yolk, 8% glycerol), with 1 or 5 mM of antioxidant. Motility (CASA), lipoperoxidation (malondialdehyde -MDA- production), membrane status, mitochondrial activity, acrosomal status (flow cytometry) and chromatin status (SCSA: %DFI and %HDS; flow cytometry) were assessed after thawing and after 6 h at 39°C. There were few differences between treatments after thawing, with Trolox reducing MDA production in a dose-response manner. After the incubation, sperm quality decreased and %DFI increased moderately, with no change for MDA. GSH improved motility, kinematic parameters and mitochondrial status, with a slight increase in %HDS. GSH 5 mM also increased moderately MDA production and %DFI, possibly due to enhanced metabolic activity and reducing power. Trolox maintained MDA low, but was detrimental to sperm quality. Trolox might not be appropriate for the cryopreservation of red deer epididymal spermatozoa, at least at the millimolar range. GSH results are promising, especially regarding motility improvement after the post-thawing incubation, and should be selected for future fertility trials.

The use of assisted reproductive techniques in cervids is increasing as the commercial use of these species increase. We have tested the suitability of the antioxidants Trolox and reduced glutathione (GSH) for freezing red deer epididymal spermatozoa, aiming at improving post-thawing quality. Samples from 19 stags were frozen in a TES-Tris-fructose extender (20% egg yolk, 8% glycerol), with 1 or 5. mM of antioxidant. Motility (CASA), lipoperoxidation (malondialdehyde -MDA- production), membrane status, mitochondrial activity, acrosomal status (flow cytometry) and chromatin status (SCSA: %DFI and %HDS; flow cytometry) were assessed after thawing and after 6. h at 39. °C. There were few differences between treatments after thawing, with Trolox reducing MDA production in a dose-response manner. After the incubation, sperm quality decreased and %DFI increased moderately, with no change for MDA. GSH improved motility, kinematic parameters and mitochondrial status, with a slight increase in %HDS. GSH 5. mM also increased moderately MDA production and %DFI, possibly due to enhanced metabolic activity and reducing power. Trolox maintained MDA low, but was detrimental to sperm quality. Trolox might not be appropriate for the cryopreservation of red deer epididymal spermatozoa, at least at the millimolar range. GSH results are promising, especially regarding motility improvement after the post-thawing incubation, and should be selected for future fertility trials.

Thawed ram spermatozoa were incubated at 37°C in the presence of dehydroascorbic acid (DHA), TEMPOL (TPL), N-acetyl-cysteine (NAC) and rutin (RUT), at 0.1 and 1mm, in order to test their effects on sperm physiology. Cryopreserved spermatozoa from four rams were thawed, pooled, washed and incubated in TALP-Hepes with 1mm or 0.1mm of each antioxidant, performing a replicate with induced oxidative stress (Fe 2+/ascorbate). Motility (CASA), viability and mitochondrial membrane potential (flow cytometry) were analysed at 2 and 4h. Lipoperoxidation (MDA production), intracellular reactive oxygen species (ROS) and DNA status (TUNEL) were analysed at 4h. Antioxidants, except DHA 0.1mm, decreased motility and kinematic parameters, but had little effect on viability or mitochondrial activity. Except 1mm DHA, the antioxidants reduced ROS at 4h. Moreover, NAC 1mm, rutin and TEMPOL reduced ROS and DNA damage in the presence of oxidative stress. N-acetyl-cysteine, rutin 1mm and TEMPOL reduced lipoperoxidation in the presence of oxidative stress. However, DHA did not affect lipoperoxidation. At 1mm, DHA increased DNA damage in the absence of oxidative stress. Dehydroascorbic acid effects could arise from spermatozoa having a low capacity for reducing it to ascorbic acid, and it may be tested in the presence of other antioxidants or reducing power. Future research should focus in testing whether the inhibition of motility observed for NAC, rutin and TEMPOL is reversible. These antioxidants might be useful at lower temperatures (refrigerated storage or cryopreservation) when their protective effects could be advantageous.

Computer-assisted sperm analysis (CASA) allows assessing the motility of individual spermatozoa, generating huge datasets. These datasets can be analyzed using data mining techniques such as cluster analysis, to group the spermatozoa in subpopulations with biological meaning. This review considers the use of statistical techniques for clustering CASA data, their challenges and possibilities. There are many clustering approaches potentially useful for grouping sperm motility data, but some options may be more appropriate than others. Future development should focus not only in improvements of subpopulation analysis, but also in finding consistent biological meanings for these subpopulations.

To try new acrosomal probes for assessing ram spermatozoa, we compared the LysoSensor™ probe, which labels acidic organelles, with the frequently used peanut agglutinin acrosomal probe (PNA-PE; phycoerythrin as fluorescent moiety). The previous microscopic observations showed a lack of relationship of LysoSensor™ with acrosomal status. Semen was obtained from five rams and frozen in four pools. Each pool was analysed carrying out a triple staining propidium ioide/PNA-PE/LysoSensor™ Green DND-189 to test acrosome labelling, and a double staining SYBR-14/PI, to assess sperm viability. Stained samples were analysed by flow cytometry. All measurements were replicated. Data were processed using agreement and repeatability tests. LysoSensor™ labelling did not agree with PNA (mean of differences: 30.8%; coefficient of agreement: 22.6%), confirming microscopic observations. Nevertheless, when LysoSensor™ was compared with SYBR-14/PI, the agreement was high (mean of differences: -0.05%; coefficient of agreement: 5.07%). Repeatability of both methods was high and similar. LysoSensor™ did not seem to specifically stain the acrosome, but it may accumulate in the cytoplasm and label viable spermatozoa. Therefore, LysoSensor™ might not be used as an acrosomal probe in ram spermatozoa, but it could be used in other kind of studies, taking advantage of its pH sensitivity.