Fluorescein Isothiocyanate-peanut Agglutinin Research Articles

Sperm vitrification supports in vitro blastocyst development after ICSI in horses

In the last decade, the introduction of advanced assisted reproductive techniques has significantly impacted the horse breeding industry. Specifically, in vitro embryo production by intracytoplasmic sperm injection (ICSI) has gained relevance and begun to be used to conserve wild equids. Kinetic sperm vitrification permits cell cryopreservation without permeating cryoprotective agents, facilitating the international commercialization and transportation of samples. This study aimed to assess embryo morpho-kinetic dynamics up to the blastocyst stage of ICSI embryos produced by injection of frozen (F) or vitrified (V) spermatozoa into horse eggs. Semen collection, as well as sperm vitrification and analysis were done as described by Consuegra et al. (Animal Reproduction Sciences. 2019; 206:69-77). Computer-assisted sperm analysis (Sperm Class Analyzer® CASA) and flow cytometry with double stain PNA-FITC (peanut agglutinin-fluorescein isothiocyanate) and PI (propidium iodide) were performed to assess acrosomal integrity and sperm viability respectively. Frozen and vitrified sperm cells from a single ejaculate of a horse and a donkey were used. Oocyte collection, in vitro maturation, ICSI, time-lapse imaging andembryo culture were performed as reported by Bragulat et. al. (Theriogenology 2023; 195:199-208). Sperm analysis before cryopreservation showed: total motility (TMOT) 98.34%; progressive sperm motility (PMOT) 78.60%; intact-membrane sperm (IMS) 86.00%, and normal form (NF) 79.37%. After thawing, frozen sperm (F) analysis showed: TMOT 84.36%; PMOT 61.28%; IMS 77.50% and NF 86.21% and for vitrified sperm (V): TMOT 80.64%; PMOT 61.28%, IMS 81.00% and NF 68.97%. A total of 89 matured oocytes were injected. No significant differences were found in cleavage rates (F: 35/43, 81.39%; V: 35/46, 76.08%) or blastocyst rates at day 8 (F: 3/43, 6.97%; V: 8/46, 17.39%). Preliminary data from time-lapse imaging showed similar embryo morpho-kinetic dynamics between groups. Average hours after ICSI for time to cleavage were (mean ± SEM): F, n = 4, 27.00 ± 1.41; V, n = 3, 27.67 ± 1.20. Second cell division: F, n = 3, 38.33 ± 6.64; V, n = 3, 32.33 ± 3.38. Morula stage: F, n = 1, 85; V, n = 1, 120. Blastocyst stage: F, n = 1, 150; V, n = 1, 180. Our results demonstrate for the first time that vitrified sperm cells can be used successfully to produce blastocysts by ICSI without compromising developmental competence in horses.

Effectiveness of Anthelmintic Treatments in Small Ruminants in Germany.

Simple SummaryParasitic worms are a major threat to the health and production of sheep and goats worldwide, particularly because many worms have become resistant to commonly applied drugs. This problem is well known around the world, but the situation is currently not well studied in Germany. This study was therefore performed to evaluate the effectiveness of treatments against infection with gastrointestinal nematodes. Faecal samples from animal groups were examined before and after treatment, the worm eggs in the feaces were counted, and the reduction in egg excretion following treatment was calculated. Eggs of Haemonchus contortus (barber’s pole worm) were stained to differentiate them from other gastrointestinal nematodes. Treatments were chosen and carried out by farmers together with their local veterinarian. Additional information was collected by questionnaires to check if the treatments were performed correctly. Reduced effectiveness was observed for all available drugs, but some showed better treatment success than others. The barber’s pole worm frequently survived treatments by most products. The high percentage of treatment failures is highly concerning, and it is necessary to develop and/or apply alternative methods of worm control to prevent the deterioration of this situation.Widespread anthelmintic resistance is a concern for small ruminant health and production worldwide. The current situation regarding anthelmintic efficacy is, however, not very well studied in Germany. Thus, a nationwide field study was undertaken to assess the effectiveness of 253 treatments performed in 223 small ruminant flocks by faecal egg count reduction test (FECRT) using pooled samples and a modified McMaster method. The percentage of Haemonchus contortus and non-Haemonchus eggs was determined by fluorescence microscopy following peanut agglutinin–fluorescein isothiocyanate staining. Treatments were chosen and performed by farmers together with their local veterinarian, and potentially confounding factors for FECRT results were addressed as far as possible by rigorous inclusion criteria. Reduced effectiveness was observed for treatments with all examined anthelmintic classes, but treatments with benzimidazoles and moxidectin showed significantly poorer results than monepantel, a closantel and mebendazole combination, and levamisole. Low case numbers precluded reliable assessment of avermectins. Unsuccessful treatments were frequently associated with the survival of H. contortus, but this was also observed for non-Haemonchus genera. The results are highly concerning, and sustainable approaches to parasite control are urgently needed to prevent further deterioration of this situation.

114 GETTING THE YOLK OUT: THE USE OF A SOY LECITHIN-BASED CRYOMEDIUM FOR SEMEN BANKING IN THE PALLAS’ CAT AND FISHING CAT

Classically, semen from nondomestic felids has been frozen in an egg yolk-based cryomedium (TEY). However, the inclusion of egg yolk is problematic, resulting in possible batch to batch variation, microbial contamination, and regulatory concerns with international semen transport. Our previous research revealed that a soy lecithin-based cryopreservation medium (SOY) was superior to TEY for freezing domestic cat sperm. However, there is limited information on the efficacy of SOY in nondomestic felids. In the present study, our aim was to assess the effect of SOY v. TEY on motility, acrosome status, and fertilizing capacity of frozen-thawed sperm in 2 wild cat species: the Pallas’ cat (Otocolobus manul) and fishing cat (Prionailurus viverrinus). Semen was collected via electroejaculation from male cats (n = 3/species), split into 2 aliquots, extended in either SOY or TEY (containing 4% glycerol), slow-cooled, and frozen in straws over liquid nitrogen vapor. Sperm motility (percent progressively motile, PM; rate of progressive motility on 1 to 5 scale, RPM) was evaluated at 0, 1, 3, 6, and 24 h post-thaw and acrosome status (AS) was assessed via fluorescein isothiocyanate-peanut agglutinin staining at 0 and 6 h post-thaw. Heterologous IVF was performed using oocytes (n = 10–15/experimental unit, 2 replicates) collected laparoscopically from gonadotropin-treated domestic cats. At 48 h post-insemination, Hoechst33342 staining was used to determine oocyte stage, number of blastomeres, and number of accessory sperm bound to the zona pellucidae of embryos and mature oocytes. The PM, RPM, and AS were analysed with repeated-measures ANCOVA, using pre-freeze values as a covariate. Embryo cleavage % and accessory sperm number were analysed with ANOVA. All data are reported as mean ± s.e. In the Pallas’ cat, PM, RPM, and AS of SOY-treated sperm (35.0 ± 7.6% motile, 2.8 ± 0.4 RPM, 41.2 ± 5.4% intact; 0 h) did not differ (P > 0.05) from TEY-treated sperm (36.7 ± 4.4% motile, 2.8 ± 0.4 RPM, 45.3 ± 9.4% intact; 0 h) at any post-thaw time point. Similarly in the fishing cat, post-thaw PM, RPM, and AS of SOY-treated sperm (38.3 ± 7.3% motile, 2.6 ± 0.2 progression, 20.0 ± 2.9% intact; 0 h) did not differ (P > 0.05) from TEY-treated sperm (31.7 ± 8.7% motile, 2.4 ± 0.3 RPM, 18.3 ± 6.4% intact; 0 h) at any time point. In the Pallas’ cat, neither embryo cleavage % (42.6 ± 7.0% SOY; 61.4 ± 14.8% TEY) nor accessory sperm number (12.2 ± 2.8 SOY; 18.1 ± 4.0 TEY) differed (P > 0.05) between treatments. Fishing cat results were similar, with no difference (P > 0.05) between SOY and TEY for cleavage % (60.4 ± 9.4% SOY; 47.9 ± 5.0% TEY) or accessory sperm number (4.6 ± 1.0 SOY; 5.5 ± 1.0 TEY). Collectively, these findings demonstrate that our SOY medium is an effective alternative to TEY for sperm cryopreservation in 2 nondomestic felid species. The replacement of an egg yolk-based cryomedium with a chemically defined, animal protein-free option represents a significant advance in quality control and biosecurity for cat semen banking and may enhance the use of assisted reproductive technologies for population management of imperiled felids. Research was funded by the Institute of Museum and Library Services.

Changes of IZUMO1 in bull spermatozoa during the maturation, acrosome reaction, and cryopreservation

To obtain basic information of bull IZUMO1 (a sperm protein essential for sperm–egg fusion) and disclose possible causes for the impaired fertilizing ability in bull cryopreserved spermatozoa, we investigated this protein in bull spermatozoa collected from various regions of epididymides, freshly ejaculated spermatozoa, acrosome-reacted spermatozoa, and cryopreserved spermatozoa by Western blotting and the triple staining with the anti-IZUMO1 antibody, fluorescein isothiocyanate–peanut agglutinin, and 4′,6-diamidino-2-phenylindole. In the cauda epididymal spermatozoa and freshly ejaculated spermatozoa, bull IZUMO1 was detected mainly as a 45-kDa major form. This major form was derived probably from a 52-kDa precursor form in the epididymis. Bull IZUMO1 was immunolocalized along the border between the principal and equatorial segments of the acrosomal region (pattern P1 of IZUMO1) in the most of epididymal and freshly ejaculated spermatozoa with normal acrosomes. In the samples after the treatments to induce the acrosome reaction, the percentages of spermatozoa without acrosomes and with IZUMO1 in whole equatorial segment (pattern P2 of IZUMO1) significantly increased. These results indicate that bull IZUMO1 undergoes maturation-related changes during sperm transit through the epididymis and that it is translocated to the equatorial segment of acrosomal region during the acrosome reaction. On the other hand, severe damages were observed in the acrosomes of 60% of the cryopreserved spermatozoa. Localization of IZUMO1 in these spermatozoa was pattern P2 (IZUMO1 in whole equatorial segment), P3 (IZUMO1 in whole acrosomal region), or P4 (IZUMO was lost). Moreover, after the incubation to compare the stability of acrosomes and IZUMO1 localization between cryopreserved spermatozoa and freshly ejaculated spermatozoa, much more spermatozoa lost acrosomes and IZUMO1 in the cryopreserved samples compared with freshly ejaculated samples. These findings indicate that impaired fertilizing ability of bull cryopreserved spermatozoa with damaged acrosomes is related partially to the aberrant translocation of IZUMO1 which may be followed by the loss of intact IZUMO1.

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.

Salvaging urospermic ejaculates from brown bear (Ursus arctos)

The objective of this study was to reverse the osmotic stress of sperm in urine contaminated bear ejaculates that were obtained by electroejaculation using pre-freezing washing or density gradient centrifugation isolation. In Experiment 1, ejaculates were divided into six aliquots, five were diluted in each washing extender: 200, 300, 400, 500 and 700mOsm/kg (prepared from a Tes–Tris–Fructose base, adding water or fructose as corresponds), at a 1:2 ratio (raw semen: washing solution, v/v); and the other aliquot was handled without washing (Control group). Samples were centrifuged at 600×g for 6min prior to freezing. In Experiment 2, ejaculates were divided into two aliquots: one was diluted 1:1 with TCG (Tris–Citric acid–Glucose) and centrifuged at 600×g for 6min (Centrifugation Control; C-Control); the other was treated with PureSperm® density gradient column. After treatments, samples were cryopreserved. Sperm motility, viability (SYBR-14/propidium iodide (PI)) and acrosomal status (peanut agglutinin-fluorescein isothiocyanate (PNA-FITC)/PI) were analyzed before and after freezing. Ejaculates with an initial osmolality of less than 120mOsm/kg treated with pre-freezing washing, and the Control sample had greater pre-freezing sperm motility than the raw ejaculate, but sperm viability was not different among these groups. The samples washed with 700mOsm/kg solutions had the least pre-freezing viability. In the post-thawing evaluation, pre-freezing washing treatments did not provide any improvement in comparison with the Control sample, and treatment with 700mOsm/kg extender had deleterious effects in all urospermic samples. PureSperm® density gradient centrifugation applied to urospermic raw semen was suitable for improving sperm motility and viability of pre-freezing samples and the selected spermatozoa had greater freezing capacity.

Tolerance of brown bear spermatozoa to conditions of pre-freezing cooling rate and equilibration time

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 control group (P < 0.05). In conclusion, our results suggest that slow cooling rates to 5 °C and at least 1 hour equilibration time are necessary for the effective cryopreservation of brown bear sperm.

61 TRANSGENIC HUNTINGTON'S DISEASE MONKEY SPERM HAS A LOWER CRYOTOLERANCE

Cryopreservation is an important tool routinely used for preserving sperm for artificial reproductive technologies (ART), as well as genetic preservation of unique animal models. The cryopreservation process is harsh and detrimental to the fragile gametes, and damage to the sperm is not only known, but inevitable. This study presents new data in which sperm from 3 transgenic Huntington's disease (HD) monkeys (rhesus macaques) are compared with 3 wild-type (WT) rhesus sperm donors. Currently, there are no data comparing HD versus WT sperm viability and cryotolerance in humans. The goal of this study was to investigate differences between fresh and frozen semen by quantitative analysis on sperm viability based on (1) motility, (2) membrane integrity, and (3) acrosome integrity. Sperm motility was determined by visual evaluation. Membrane and acrosome integrity were assessed simultaneously by Hoechst 33342, propidium iodide (PI), and fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) triple staining. Sperm viability analysis was divided into 3 groups: (1) fresh HD versus fresh WT, (2) fresh versus cryopreserved-thawed WT, (3) and fresh HD versus cryopreserved-thawed HD sperm. Interestingly, fresh HD sperm had a lower percentage of membrane-damaged cells (38.57 ± 3.15) compared with WT (49.67 ± 3.56; P &lt; 0.03). However, after cryopreservation and subsequent thawing, HD sperm had a significantly higher percentage increase in damaged membranes than WT sperm (27.91 ± 2.93 v. 8.27 ± 8.28; P &lt; 0.001), respectively. No significant difference in acrosome damage between groups was identified in either fresh or cryopreserved sperm populations. Motility significantly declined in both cryopreserved populations [HD: 89.7 to 43.4% (P &lt; 0.001) and WT: 90.0 to 45.6% (P &lt; 0.001)]. There was no significant difference between either freeze-thawed group. These data illustrate that HD sperm have a lower cryotolerance than WT sperm. Our findings suggest that the optimization of the HD sperm cryopreservation method and investigation on biochemical differences (e.g. membrane lipid composition) are necessary to improve post-thaw survival. This in turn is important for the establishment of a sperm cryobank and future derivation of a unique animal model such as HD monkey. Our study also suggests that HD monkey could be a useful model for optimizing cryopreservation method for HD patients.

The addition of heat shock protein HSPA8 to cryoprotective media improves the survival of brown bear (Ursus arctos) spermatozoa during chilling and after cryopreservation

The Cantabrian brown bear survives as a small remnant population in northern Spain and semen cryopreservation for future artificial insemination is one of the measures being implemented for conservation of this species. As part of this program we investigated the value of adding heat shock protein A8 (HSPA8) to media (N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid-TRIS-fructose with 20% egg yolk) used for chilling and cryopreserving the spermatozoa. Semen samples from eight brown bears were obtained by electroejaculation during the breeding season. In experiment 1, we tested three concentrations of HSPA8 (0.5, 1, and 5 μg/mL) to determine whether sperm motility (computer assisted sperm analysis system) and sperm survival could be improved during refrigeration (5 °C) up to 48 hours. Results showed that sperm viability (test with propidium iodide) was improved by the addition of 0.5 and 5 μg/mL HSPA8. In experiment 2, HSPA8 was added to the cryopreservation media (6% final glycerol concentration) before the freezing process. Though there were no differences in sperm viability immediately after thawing (analyses to 0 hours), plasma membrane permeability (test with YO-PRO-1) was significantly lower by the presence of HSPA8 (1 μg/mL) and acrosomal damage (test with peanut agglutinin-fluorescein isothiocyanate conjugate) was reduced by higher concentrations of HSPA8 (1 and 5 μg/mL) (analyses after thermal stress test incubating over 2 hours to 37 °C). In experiment 3, results of a simple progression test carried out through artificial mucus (hyaluronic acid 4 mg/mL) showed a significant decrease in the total number of sperm able to swim a distance of 0.5 to 2 cm through a capillary tube for all HSPA8-based extenders. Nevertheless, the distance traveled by the vanguard spermatozoa, which represent a highly motile subpopulation, was restored by the inclusion of 1 and 5 μg/mL HSPA8 in the cryopreservation media. Thus, the HSPA8 addition to extender improves the quality of brown bear (Ursus arctos) sperm during chilling (viability) and after cryopreservation (number of sperm with damaged acrosomes and “apoptotic-like” changes).

Methodological factors affecting the results of staining frozen–thawed fertile and subfertile Japanese Black bull spermatozoa for acrosomal status

In the present study, some methodological factors affecting the acrosomal staining of frozen–thawed Japanese Black bull spermatozoa were investigated by examining; the effect of fixation/permeabilization procedure on intact acrosome percentage after fluorescein isothiocyanate peanut agglutinin (FITC-PNA) staining, the acrosomal staining patterns by using two types of fluorescent probes FITC-PSA (Pisum Sativum Agglutinin) and FITC-PNA and the effect of staining methods, either smear or vial, on intact acrosome percentage. Then intact acrosome percentage was compared between the samples stained by thus established method and those simply fixed with glutaraldehyde (glutaraldehyde fixation method). A possibility that FITC-PNA staining or the glutaraldehyde fixation methods could detect any difference in intact acrosome percentage or acrosomal staining patterns between fertile and subfertile bulls was also examined. The results showed that (1) 4% paraformaldehyde fixation plus 1% Triton X-100 permeabilization was better than absolute ethanol alone, (2) FITC-PNA acrosomal labeling was more specific than FITC-PSA, (3) sperm suspensions should be smeared and gently processed before acrosomal staining rather than spotted onto glass slides after staining in vial in order to avoid excessive mechanical damage of the sperm acrosome, and (4) staining spermatozoa with FITC-PNA had no major advantages over examination of simply glutaraldehyde fixed sperm samples and both failed to detect any significant difference in intact acrosome percentage between the fertile and the subfertile bulls used here. The present study demonstrates important methodological considerations which need to be taken into account in order to design a reliable and reproducible protocol for the study of the acrosome.

Effect of insulin-like growth factor I on functional parameters of ram cooled-stored spermatozoa

The aim of the study was to examine the effects of insulin-like growth factor I (IGF-I) on ram sperm traits after hypothermic storage. Sperm ejaculates from Lacaune rams were diluted in a Tris extender, pooled, divided into groups of IGF-I doses tested (0, 10, 100 or 200 ng.ml-1) and stored (0-5°C) for 96 h. IGF-I elevated whole sperm motility as measured by a Computer-assisted Sperm Analyser (CASA) system, by 24 h (10 ng.ml-1) and 48 h (200 ng.ml-1) of storage, and by progressive movement on each day of storage. After 72 h the sperm samples were analysed for plasma membrane integrity (peanut agglutinin-fluorescein isothiocyanate), membrane stability (annexin V-Fluos) and apoptosis (Yo-Pro®-1) using fluorescence microscopy. The addition of IGF-I (at 100 or 200 ng.ml-1) reduced the ratio of sperm with disrupted membranes and the ratio of annexin V-labelled sperm. The ratio of apoptotic sperm was reduced by IGF-I given at 10 or 100 ng.ml-1 compared with control. Sperm fertilizing ability, determined at 48 h by an in vitro fertilization (IVF) test on bovine oocytes, was increased by IGF-I given at 100 ng.ml-1 from 47.0 to 67.7%. In conclusion, IGF-I maintained ram sperm functions following cooling storage and its effects were reflected in sperm fertilizing ability in vitro.

A flow-cytometric study on the effect of myeloperoxidase on stallion spermatozoal motility and structure

Myeloperoxidase (MPO) is a pro-oxidant enzyme that is normally contained in neutrophils. MPO has recently been associated with keratinized cells and with decreased postthawmotility in stallion semen [1]. The aimof the studywas to determine effects of experimental addition of activeMPO on motility, mitochondrial potential, apoptosis induction, membrane and acrosome integrity in equine semen. Semen was collected four times from three stallions. Extended (INRA96 ) semen was processed for density gradient centrifugation (Equipure Bottom Layer ) [2]. Purified pellet was re-extended to 100 x1 06spermatozoa/ ml in INRA96 and divided in 3 samples. One sample was used for control, and active human MPO (Calbiochem, Merck) was added in the other two samples to a final concentration of 5 or 50ng/ml. After incubation (2 hours, 20 C), motility was analysed with Computer Assisted Semen Analysis (IVOS, Hamilton-Throne, Beverly, MA, USA) and cytometric analyzes were perfomed with EasyCyte (IMV). Mitochondrial potential and apoptosis were assayed using Guava Mitopotential JC-1 and 7-AAD kit (Millipore). Membrane and acrosome integrity were respectively assayed with PI (Propidium Iodide) (Invitrogen) and PNA (Peanut Agglutinin-Fluorescein Iso Thio Cyanate) (SigmaAldrich). Statistical differences (P < 0.05) were determined using Kruskall-Wallis test. No effect of the stallions was observed on parameters assayed in this study. Unlike total motility, progressive motility was decreased in both MPO concentrations (P < 0.001). MPO addition had no effect on membrane and acrosome integrity. No differences were detected for the percentages of spermatozoa having polarised or depolarised mitochondria. Apoptosis, assayed by 7-AAD fluorescence, was not increased by the treatments. Our results agree with previously published effects on in vitro ROS production systems with xanthine oxidase [3], showing an effect on motility but no influence on mitochondria and membrane or acrosome integrity. However, membrane lipoperoxidation was increased by ROS in this study [3], and it could be linked to the impaired motility also observed in our protocol. Further studies with increasing concentrations of added MPO should be conducted to correlate motility with lipoperoxidation. References

259 ADDITION OF CHOLESTEROL TO FLOW-SORTED BULL SPERMATOZOA TO ENHANCE CRYOSURVIVAL DOES NOT AFFECT THE ACROSOME REACTION

Treating bull sperm with cholesterol-loaded cyclodextrins (CLCs) improves post-thaw motility and viability (Purdy and Graham 2004 Biol. Reprod. 71, 522–527). Unpublished research in our laboratory demonstrated similar improvements for cryosurvival of CLC-treated sex-sorted bull sperm. Our objective was to determine the effects of CLCs on the dilauroylphosphatidylcholine (PC12)-induced acrosome reaction of flow-sorted and non-sorted (control), frozen/thawed bull sperm. Sperm concentrations (3 ejaculates, 4 bulls) were adjusted to 200 × 106 sperm mL–1 with TALP without Ca++. Sperm for sorting were stained (63 μm Hoechst 33342, 34.5°C, 45 min), and then incubated an additional 15 min with 2.5 mg CLC in TALP (+CLC) or an equivalent volume of TALP without CLCs (–CLC), adjusted to 100 × 106 sperm mL–1 with 4% egg yolk TALP containing 0.002% food dye, filtered, and ‘bulk’ sorted (Schenk et al. 1999 Theriogenology 52, 1375–1391) into 2.0 mL aliquots of 20% egg yolk Tris buffer. Controls were extended in 20% egg yolk Tris A to result in a final concentration of 21 × 106 sperm mL–1. All samples were cooled to 5°C over 90 min, and Tris buffer with (controls) or without (flow-sorted) egg yolk (12% glycerol) was added (v/v). Sorted samples were centrifuged (850g, 20 min), and pellets were suspended in 20% egg yolk/6% glycerol Tris, resulting in 21 × 106 sperm mL–1. Sperm were loaded into 0.25-mL straws and frozen over LN2 vapor. Straws were subsequently thawed (30 s, 37°C), and 25 million sperm/treatment were centrifuged through 60% Percoll® to remove egg yolk. Pellets were adjusted to 20 × 106 sperm mL–1 with TALP containing 2.0 mm Ca++. Aliquots (30 μL) were stained with propidium iodide (PI) and fluorescein isothiocyanate–peanut agglutinin (FITC-PNA); treated with 0, 25, or 35 μL PC12; and brought to a constant volume (300 μL) with TALP. TALP (30 μL) containing 42 mm Ca++ was added, resulting in final concentrations of 0, 68, and 95 μm PC12 and 6 mm Ca++. Following incubation (38°C, 35–40 min) 50 000 sperm were analyzed on an SX MoFlo® (Dako, Fort Collins, CO, USA) to determine the percentage of live acrosome-reacted sperm/live sperm(PI –ve, FITC +ve). There were no differences between CLC-treated and non-treated sorted or non-sorted sperm (Table 1) suggesting that, as previously shown for non-sorted sperm (Purdy and Graham 2004), treatment of flow-sorted sperm with CLCs does not interfere with the progression of the acrosome reaction. Table 1. Effect of treatment with CLCs on % live acrosome-reacted (mean ± SEM) non-sorted and flow-sorted cryopreserved bull spermatozoa

Early pre-pubertal exposure to low-dose oral di(2-ethylhexyl) phthalate does not affect sperm plasma membrane stability, acrosomal integrity or chromatin structure in the post-pubertal boar

This study aimed to determine whether pre-pubertal exposure in boars to di(2-ethylhexyl) phthalate (DEHP), a plasticizer reported to have toxic effects on rodent reproduction, would affect the sperm ability to undergo capacitation and acrosome reaction (AR) in vitro or give rise to a higher degree of chromatin instability associated with acid-induced denaturation. Spermatozoa were collected from 16 boars ( n = 8/group) 8–9 months of age, exposed to 300 mg/kg body weight of DEHP or placebo per os three times a week, from 3 to 7 weeks of age. The spermatozoa were cryopreserved and examined post-thaw by flow cytometry for their ability to capacitate in vitro when exposed to the effector bicarbonate and to acrosome-react when exposed to calcium ionophores, using the lipid stain Merocyanine-540 (m-540), and peanut agglutinin-fluorescein isothiocyanate, respectively, as probes. The ability of the DNA to sustain denaturation in vitro was tested using a sperm chromatin structure assay (SCSA). No significant differences between the DEHP-exposed group and controls were found for any of the sperm attributes examined. Frozen–thawed spermatozoa showed similar rates of non-capacitated cells between groups, and were capacitated at similar rates. Rates of induced ARs were also similar. Values of DNA denaturation were low and showed no differences between groups. In conclusion, pre-pubertal exposure to DEHP does not seem, under the conditions of the present experiment, to affect the ability of frozen–thawed spermatozoa collected post-puberty to capacitate or acrosome-react (the main requisites for fertilization) or to present damage in their nuclear genome.

The Effect of Oestradiol, Progesterone and Heparin on Bovine Spermatozoa Function After Thawing

The present experiment was designed to determine the effects of various biologically active substances, such as oestradiol (OE), progesterone (P4) and heparin (Hep) alone or in combination on sperm plasma membrane scrambling, capacitation and acrosome reaction (AR) of post-thaw bovine spermatozoa. Spermatozoa were incubated for 180 min in capacitation medium supplemented with (i) 1 mug/ml OE; (ii) 1 mug/ml P4; (iii) 1 mug/ml OE and 1 mug/ml P4; (iv) 1 mug/ml OE and 5 mug/ml Hep; (v) 1 mug/ml P4 and 5 mug/ml Hep; (vi) 1 mug/ml OE, 1 mug/ml P4 and 5 mug/ml Hep. At predetermined time intervals aliquots were taken to assess sperm plasma membrane scrambling, or capacitation (AR induced by lysophosphatidylcholine) in spermatozoa. The second experiment was aimed to study the effects of OE, P4 and OE/P4 as potential inducers of AR in Hep-capacitated spermatozoa. Plasma membrane scrambling was assessed by a flow cytometer, using Merocyanine staining. Acrosomal status and viability of spermatozoa were evaluated under epifluorescence microscope with Ethidium homodimer-1/peanut agglutinin fluorescein isothiocyanate staining method (EthD-1/PNA-FITC). The results show that OE, P4 and a combination of OE/P4 at concentrations used did not affect sperm viability. Heparin significantly (p < 0.001) increased sperm plasma membrane scrambling of OE and P4-treated spermatozoa. P4 significantly affected the rate of sperm capacitation (p < 0.001) and AR (p < 0.05), but OE expressed membrane-stabilizing properties (p < 0.05). It can be concluded that in frozen-thawed bovine spermatozoa OE presents plasma membrane stabilizing properties that can be abolished by Hep, but not by P4. Progesterone possesses capacitating and AR-inducing properties in frozen-thawed bovine spermatozoa that can be alleviated by OE.

Effects of hypothermic liquid storage and cryopreservation on basal and induced plasma membrane phospholipid disorder and acrosome exocytosis in boar spermatozoa

Flow cytometry was utilised to determine whether short-term (Day 1) or long-term hypothermic liquid storage (Day 5), or cryopreservation of boar spermatozoa (1) caused changes in plasma membrane phospholipid disorder (MPLD) and acrosome exocytosis (AE), indicative of an advanced stage of capacitation or acrosome status, and (2) facilitated or inhibited the induction of capacitation and the acrosome reaction. Merocyanine with Yo-Pro-1 and peanut agglutinin-fluorescein isothiocyanate with propidium iodide were used to identify MPLD and AE, respectively, in viable spermatozoa. The incidence of basal sperm MPLD and AE in fresh semen was very low (1.1 and 2.2%, respectively) and was increased (P < 0.05) only a small amount in Day 5 and cryopreserved semen (3-8%). Compared to no bicarbonate, incubation with bicarbonate increased MPLD, but the response was greatest (P < 0.05) in fresh sperm (52.3%) compared with Day 1 (36.6%), Day 5 (13.9%) and cryopreserved sperm (13.6%). Incubation with calcium ionophore A23187 increased AE in spermatozoa, but the response was less (P < 0.05) for fresh (34%) and cryopreserved (27%) semen than for Day 1 (45%) and Day 5 (57%) semen. In summary, hypothermic liquid storage and cryopreservation of boar spermatozoa did not advance capacitation or acrosome status in viable spermatozoa, but did alter their responses to induction of capacitation and the acrosome reaction.

Induction of capacitation and the acrosome reaction of boar spermatozoa by L-arginine and nitric oxide synthesis associated with the anion transport system

The aim of this study was to determine the effect of L-arginine on nitric oxide (NO) synthesis, capacitation and acrosome reaction of boar spermatozoa. Ejaculated boar spermatozoa were washed and then cultured in a bicarbonate:CO(2)-buffered medium, modified NCSU-37, for 2 h. At the end of the culture, the status of spermatozoa was determined. The presence of (0.1-2.0 mmol l(-1)) L-arginine in the culture medium induced an acrosome reaction as determined by fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and increased intracellular NO content, as quantified by a fluorescent indicator, diaminofluorescein-2 diacetate (DAF-2 DA). This stimulatory effect of L-arginine was neutralized by supplementation with an NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (1 mmol l(-1)). However, the inactive enantiomorph, N(omega)-nitro-D-arginine methyl ester, did not affect the stimulatory effect of L-arginine. These results indicate that L-arginine induces an acrosome reaction through the NO signal pathway in boar spermatozoa. Furthermore, the stimulatory effect of L-arginine was inhibited in the presence of an anion transport inhibitor, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonic acid (SITS; 0.1 mmol l(-1)), whereas any responses of spermatozoa to caffeine were not inhibited by SITS. A stimulatory effect of L-arginine on capacitation and acrosome reaction of spermatozoa was also observed in modified NCSU37 medium by using a chlortetracycline fluorescence assay, but not in supplemented bicarbonate-free Tris-buffered medium. These results indicate that the presence of L-arginine induces nitric oxide synthesis and stimulates capacitation and acrosome reaction of boar spermatozoa only when active sperm anion transport is present as a result of bicarbonate supplementation.

Induction of capacitation and the acrosome reaction of boar spermatozoa by L-arginine and nitric oxide synthesis associated with the anion transport system

The aim of this study was to determine the effect of L-arginine on nitric oxide (NO) synthesis, capacitation and acrosome reaction of boar spermatozoa. Ejaculated boar spermatozoa were washed and then cultured in a bicarbonate:CO(2)-buffered medium, modified NCSU-37, for 2 h. At the end of the culture, the status of spermatozoa was determined. The presence of (0.1-2.0 mmol l(-1)) L-arginine in the culture medium induced an acrosome reaction as determined by fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and increased intracellular NO content, as quantified by a fluorescent indicator, diaminofluorescein-2 diacetate (DAF-2 DA). This stimulatory effect of L-arginine was neutralized by supplementation with an NO synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (1 mmol l(-1)). However, the inactive enantiomorph, N(omega)-nitro-D-arginine methyl ester, did not affect the stimulatory effect of L-arginine. These results indicate that L-arginine induces an acrosome reaction through the NO signal pathway in boar spermatozoa. Furthermore, the stimulatory effect of L-arginine was inhibited in the presence of an anion transport inhibitor, 4-acetamido-4'-isothiocyano-stilbene-2,2'-disulphonic acid (SITS; 0.1 mmol l(-1)), whereas any responses of spermatozoa to caffeine were not inhibited by SITS. A stimulatory effect of L-arginine on capacitation and acrosome reaction of spermatozoa was also observed in modified NCSU37 medium by using a chlortetracycline fluorescence assay, but not in supplemented bicarbonate-free Tris-buffered medium. These results indicate that the presence of L-arginine induces nitric oxide synthesis and stimulates capacitation and acrosome reaction of boar spermatozoa only when active sperm anion transport is present as a result of bicarbonate supplementation.

Evaluation on sperm quality of freshly ejaculated boar semen during in vitro storage under different temperatures

The purpose of this study was to assess the sperm quality of fresh ejaculated boar semen stored under different temperatures for up to 48 h in order to use the fresh semen efficiently. Spermatozoa were evaluated by 4 methods: Using trypan blue staining, the viability of spermatozoa stored at 39, 20, 15 and 4 °C for 48 h were 1.6, 46.9, 42.0 and 31.0%, respectively. Employing the hypoosmotic swelling test (HOST) showed 1.7%(39 °C), 28.7%(20 °C), 24.1%(15 °C), and 20.1%(4 °C) coiled-tail spermatozoa following 48 h storage. With Coomassie blue staining, the rates of acrosome-intact spermatozoa stored for 48 h were 4.5%(39 °C), 35.3%(20 °C), 55.7%(15 °C) and 22.8%(4 °C). Using fluorescein isothiocyanate-peanut agglutinin (FITC-PNA), the percentages of acrosome-intact spermatozoa stored for 48 h were 4.3%(39 °C), 43.2%(20 °C), 17.3%(15 °C) and 14.8%(4 °C), respectively. The cytoplasmic droplets were found in 18.66% of the spermatozoa in fresh semen and were gradually shed during storage. The results of these 4 methods were highly correlated and could be used to characterized sperm-cell quality effectively. These findings indicated that both membrane integrity and viability of spermatozoa could be preserved well during in vitro storage at 20 °C and 15 °C for 24 to 48 h.

Activation of protein kinase C induces cortical granule exocytosis in a Ca(2+)-independent manner, but not the resumption of cell cycle in porcine eggs.

The effects of protein kinase C (PKC) activation on meiotic resumption and cortical granule (CG) exocytosis as well as its dependence on Ca2+ in porcine eggs matured in vitro were studied. Cortical granule release was judged by both confocal laser microscopy after the eggs were labeled with fluorescein isothiocyanate-peanut agglutinin (FITC-PNA) and electron microscopy. Meiotic resumption and pronuclear formation were observed after eggs were stained with acetic orcein. When eggs were treated with PKC activators, 1-oleyl-2-acetyl-glycerol (OAG) or phorbol 12-myristate 13-acetate (PMA), the pronuclear formation percentage was significantly lower than that of Ca2+ ionophore A23187-treated group, but not statistically different from that in negative control group (P > 0.05), and most of the eggs were still arrested at metaphase II stage, suggesting that PKC activation does not induce the resumption of meiosis and pronuclear formation. In contrast, PKC activation induced 89.1% to 100% of the eggs completely or partially released their CG in different groups, not statistically different from A23187-treated group, and this effect could be overcome by PKC inhibition. When the intracellular free Ca2+ was chelated with acetoxymethal ester form of 1,2-bis(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM), and then treated with PMA or OAG in Ca(2+)-free medium, the proportions of eggs with CG release were 90.9% and 78.1%, respectively, not statistically different from the above-treated groups, suggesting that CG exocytosis induced by PKC activation is independent of Ca2+ rise. The results indicate that different events of porcine egg activation may be uncoupled from one another.