Reprod Dom Anim 49, 254–262 (2014); doi: 10.1111/rda.12264 ISSN 0936–6768

Identification of Apoptotic Bodies in Equine Semen AB Caselles1, A Miro-Moran1, A Morillo Rodriguez2, JM Gallardo Bolan˜os2, C Ortega-Ferrusola2, GM Salido1, FJ Pe~ na2, 1 1 JA Tapia and IM Aparicio 1 Cell Physiology Research Group, Department of Physiology, University of Extremadura, Caceres, Spain; 2Veterinary Teaching Hospital, Laboratory of Spermatology, University of Extremadura, Caceres, Spain

Contents Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37°C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin-V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer-assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process.

Introduction Apoptosis or ‘programmed cell death’ is a highly specialized process that controls cell death. In somatic cells, it has been characterized by an initial shrinkage of the cells resulting in the breakage of cell-to-cell contacts. The cells acquire a round shape and their internal membranes, and organelles become more concentrated in the cytoplasm. In the nucleus, there is chromatin condensation, and specific endonucleases accurately cleave the DNA between nucleosomes, giving fragments of ~180 base pairs. At the end of this process, while the process of DNA cleavage is taking place, the nucleus undergoes fragmentation, and the cell splits into a number of small intact pieces or apoptotic bodies. Cytoplasmic budding and fragmentation is followed by phagocytic removal, a process in which migrating macrophages or healthy neighbouring epithelial cells engulf cell fragments without an inflammatory response. An additional change during apoptosis is the translocation of phosphatidylserine (PS) from the inner to the outer surface of the plasma membrane, which constitutes one of the principal targets of phagocyte

recognition (reviewed by Schultz and Harrington 2003; Munoz-Pinedo 2012). During recent years, an increasing number of studies have demonstrated that cell death by apoptosis takes place during spermatogenesis and that it is a physiological process involved in the maintenance of homeostasis in testes by removing surplus and abnormal germ cells (Guo et al. 2000; Johnson et al. 2008). However, sometimes, defective germ cells that are marked for elimination during spermatogenesis escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa, in a process that has been termed abortive apoptosis (Sakkas et al. 1999a,b, 2003, 2004). Spermatozoa showing typical features associated with apoptosis have been identified in numerous mammalian species (Anzar et al. 2002; Aziz et al. 2007; OrtegaFerrusola et al. 2009; Sokolowska et al. 2009; Garcia Vazquez et al. 2012), and a correlation between markers of apoptosis, such as high levels of caspases activation, PS exposure or DNA fragmentation, with morphologically abnormal and immature forms of spermatozoa (Sakkas et al. 2004; Aziz et al. 2007), low motility (Paasch et al. 2004; Taylor et al. 2004), low viability (Anzar et al. 2002) and low in vitro fertilization and pregnancy success rates has been found (Sun et al. 1997; Host et al. 2000a,b; Fatehi et al. 2006). Yet, it is not only features characteristic of abortive apoptosis that have been found in spermatozoa in fresh semen. Numerous large spheroidal elements corresponding to apoptotic bodies have been found in the seminal plasma of infertile individuals and patients affected by neoplastic disease (Gandini et al. 2000). In 2004, Muratori et al. (2004) observed spheroidal elements devoid of nuclei in seminal plasma in humans, which they named ‘M540 bodies’ due to their bright staining with merocyanine 540 (M540). Subsequently, they showed that those elements that they first called M540 bodies were, in fact, apoptotic bodies; the percentage of these apoptotic bodies was higher in oligoasthenoteratozoospermic and asthenoteratozoospermic compared to normozoospermic subjects. Interestingly, semen from oligoasthenoteratozoospermic and asthenoteratozoospermic subjects showed the highest levels of apoptotic signs (Marchiani et al. 2007a,b). Cryopreservation (including freezing and thawing) induces cell death and subtle damage in most cells of the surviving population, leading to reduced life span while in the female reproductive tract or in vitro. The two major sources of damage are the toxicity of unequal distribution of cryoprotectants (such as glycerol) and

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Apoptotic Bodies in Stallion Sperm

the osmotic stress caused by dehydration of the extender and the cells during freezing and thawing (Morris et al. 2006, 2007). Together with these major insults, phase transitions in the sperm plasmalemma, oxidative stress, premature ageing and ‘apoptotic like phenomena’ have been recognized as factors explaining the reduced life span of cryopreserved semen (Martin et al. 2004, 2007; Ortega-Ferrusola et al. 2008). Research in mammalian species has allowed the identification of a number of indicators for differences in freezability among species and individuals from the same species (Giraud et al. 2000; Nunez-Martinez et al. 2007). Since artificial insemination (AI) in the equine species is being permitted by most stud books, this technology is becoming increasingly popular in Europe, prompting the development of intense research in the quality of semen and its tolerance for cryopreservation and cooled storage. However, due to the great individual variation in sperm survival during cold storage and cryopreservation in stallions (Metcalf 2007; Loomis and Graham 2008; Sieme et al. 2008; Ortega-Ferrusola et al. 2009; Gutierrez-Cepeda et al. 2012), the standardization of cryopreservation protocols in the equine species is a difficult task, requiring more intensive research. Therefore, taking into account that markers of apoptosis have been related to semen quality and fertility, the main objective of the present research was to investigate whether fresh equine semen contains apoptotic bodies and to establish a possible relationship between the amount of apoptotic bodies and the quality of semen parameters such as cell concentration, volume of ejaculate, viability and motility. Moreover, we also studied whether the presence of apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa of being incubated at 37°C or being frozen and thawed.

Materials and Methods Ethics Semen collection was reviewed and approved by the Animal Ethics Committee of the University of Extremadura and were performed in accordance with Spanish and European guidelines for research on animals. Human semen was obtained from four men (20–40 year-old), as approved by the institutional review board and the ethics committee of the University of Extremadura, as well as in accordance with the Declaration of Helsinki. Written informed consent was obtained from all of the participants. Reagents Chemical salts (NaCl, KCl, MgSO4, KPO4, 20 HEPES, PVP, PVA, sodium pyruvate, sodium lactate, CaCl2, NaHCO3, formaldehyde, Triton X-100, deoxycholate, EGTA, EDTA, Na3VO4), poly-L-lysine, glucose, bovine serum albumin (BSA) and phosphate-buffered saline (PBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA), INRA 96 (IMV, L’Aigle, France). LIVE/ DEAD Sperm Viability Kit, Dead Cell Apoptosis Kit, Annexin-V, propidium iodide (PI), 4′,6-diamidino-2phenylindole, dihydrochloride (DAPI), merocyanine

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540 and anti-rabbit IgG antibody labelled with the fluorescent probe Alexa 488 were obtained from Molecular Probes (Eugene, OR, USA). Complete, EDTA-free, protease inhibitor cocktail was from Roche Diagnostics (Penzberg, Germany). Bradford reagent, Tris/glycine/ SDS buffer (109) and Tris/glycine buffer (109) were from Bio-Rad (Richmond, CA, USA). Anti-rabbit monoclonal Ab cleaved Caspase 3 (#9664) was purchased from Cell Signaling (Beverly, CA, USA). Antirabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibody and enhanced chemiluminescence detection reagents were from Pierce (Rockford, IL, USA). Hyperfilm ECL was from Amersham (Arlington Heights, IL, USA), and nitrocellulose membrane was obtained from Schleicher and Schuell (Keene, NH, USA). Semen collection and incubation media Semen (one ejaculate per stallion) was obtained from a total of 14 Andalusian horses individually housed at the Veterinary Teaching Hospital of the University of Extremadura, Caceres, Spain. Stallions were maintained according to institutional and European regulations and were collected on a regular basis (two collections/week) during the 2012 breeding season. All animals were healthy donors, and no selection of the ejaculates was performed for incubation or cryopreservation purposes. Ejaculates from stallions (in the age range of 4–20 years) were collected using a Missouri model artificial vagina with an inline filter to eliminate the gel fraction and were lubricated and warmed to 45–50°C. Semen was immediately evaluated and processed. Human semen was obtained from men in the age range of 20–40 years. Each subject was confirmed to be in good health by means of their medical histories and a clinical examination including routine laboratory test and screening. Subjects were all non-smokers, were not using any medication and abstained from alcohol. Informed consent was obtained from all of the participants (n = 4). Samples were collected early in the morning by masturbation after 4–5 days of sexual abstinence and were allowed to liquefy for 30 min (37°C, 5% CO2) before smearing onto poly-L-lysinecoated slides. In this study, human samples were used as a positive control to ensure that it was possible to detect apoptotic bodies in semen samples by staining with M540 (red) and DAPI (blue). To perform the incubation of semen samples, for each stallion’s ejaculate, aliquots were centrifuged at 800 9 g for 10 min at room temperature (RT). Seminal plasma was removed, and the sperm pellet resuspended to 100 9 106 spermatozoa/ml in modified Biggers, Whitten and Whittingham (BWW) media consisting of: 91.6 mM NaCl, 4.6 mM KCl, 2.44 mM MgSO4, 1.2 mM KPO4, 20 mM HEPES, 5.6 mM glucose (anhydrous), 0.27 mM sodium pyruvate, 21.55 mM sodium lactate, 1.7 mM CaCl2, 25 mM, NaHCO3 and 7 mg/ml BSA. The pH of the solution (320 mOsm/kg) was adjusted to 7.4. Samples were incubated for 180 min at 37°C.

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For freezing purposes, the ejaculate was extended 1 : 3 (v/v) with INRA 96 and centrifuged at 600 9 g for 10 min. The resulting sperm pellet was re-extended in freezing medium (Ghent, Minit€ ub Iberica, Spain) to a final concentration of 100 9 106 spermatozoa/ml. The spermatozoa were slowly cooled to 4°C within 1 h, loaded in 0.5 ml plastic straws and frozen horizontally in racks placed 4 cm above the surface of liquid nitrogen for 10 min, after which they were directly submerged in liquid nitrogen. After at least 4 weeks of storage, straws were thawed in a water bath at 37°C for 30 s for further analysis. Identification of apoptotic bodies Immediately after semen collection, fifteen microlitres of fresh semen samples (stallion, human) was smeared onto poly-L-lysine-coated slides and allowed to attach for 10 min. Then, semen was fixed with 4% formaldehyde in PBS for 15 min at RT. After fixation, slides were washed three times for 5 min each time in PBS. Immediately, slides were subjected to different staining (Merocyanine and Annexin-V) to carry out the detection of apoptotic bodies. The number of apoptotic bodies was counted using an epifluorescence microscope (Nikon Instrument Europe BV, Bodhoevedorp, the Netherlands). In the same field observed under microscope, the number of spermatozoa and the apoptotic bodies were counted in parallel (randomly fields were observed until 1000 of spermatozoa were reached in the slide). The quantity of apoptotic bodies was expressed as a percentage (number of apoptotic bodies found in seminal plasma every 100 spermatozoa counted). Merocyanine (M540) + DAPI M540 dye was prepared at a final concentration of 1.3 lM in PBS containing PVP and PVA (0.5 mg/ml each). One ejaculate from 14 stallions was incubated with M540 for 15 min at RT in the dark. After incubation, M540 was removed and slides were incubated with DAPI (1 lg/ll in PBS containing PVP and PVA) for 1 min at RT. Finally, to improve the signal, one drop of glycerol was added to the stained samples and covered with a glass cover slip. Samples were observed in an epifluorescence microscope (Nikon Instrument Europe BV). M540 was excited at 510–560 (emission at 590), and DAPI was observed under blueviolet illumination (excitation at 330–380 nm, emission at 420 nm). Annexin-V + Propidium Iodide PS externalization can be detected with the probe Alexa Fluor 488 Annexin-V (Dead Cell Apoptosis Kit, Molecular Probes, Leiden, the Netherlands) and propidium iodide (PI) nucleic acid stain, which is a probe that is able to detect dead cells. The dye was prepared following the manufacturer protocol: 5 ll of Alexa Fluor 488 Annexin-V and 10 ll of PI (1 mg/ml in deionized water) were added to a commercial binding buffer provided in the kit (50 mM HEPES, 700 mM NaCl, 12.5 mM CaCl2, pH 7.4). Ejaculates (one per

animal) from six stallions, previously coated and fixed on the glass slides, were incubated with this solution for 15 min in the dark at RT. Finally, slides were covered with a cover slip and observed in an epifluorescence microscope (Nikon, Eclipse TE 300). Annexin-V was excited at 450–490 nm (emission at 520 nm), and PI was excited at 510–560 (emission at 590). Assessment of stallion spermatozoa viability The LIVE/DEAD Sperm Viability Kit, (Molecular Probes) was used following the indications of the manufacturer. In brief, 1 to 5 9 106 of fresh (n = 14), incubated (n = 7) and thawed (n = 6) samples (one ejaculate per stallion) were resuspended in a final volume of 1 ml of HEPES-buffered saline solution (10 mM HEPES, 150 mM NaCl, 10% BSA, pH 7.4). Sperm was stained with 5 ll SYBR-14 (100 nM) and incubated at 37°C for 10 min in the dark, and then, samples were stained with 5 ll propidium iodide (PI) (12 mM) and incubated for 10 additional minutes before analysis in the flow cytometer. SYBR-14 fluorescence was detected in FL1, and PI fluorescence was detected in FL3. Flow cytometric analyses were carried out with a Coulter EPICS XL (Coulter Corporation Inc., Miami, FL, USA) flow cytometer equipped with standard optics. The system has an argon-ion laser (Cyonics, Coherent, Santa Clara, CA, USA) performing 15 mW at 488 nm and EXPO 2000 software. Subpopulations were divided by quadrants, and the frequency of each subpopulation was quantified. Forward and sideways light scatter was recorded for a total of 10 000 events per sample. Samples were measured at a flow rate of 200–300 cells/s. Green fluorescence (SYBR) was detected in FL1 (525 nm band pass filter), and red fluorescence (PI) was detected in FL3 (620 nm band pass filter). Sperm motility Sperm kinematics was assessed in fresh, incubated and thawed samples using a computer-assisted semen analysis (CASA) system (ISAS 1.0.6; Proiser S.L., Valencia, Spain). Semen, previously diluted to 50 9 106 spermatozoa/ml, was loaded in 20-lm-deep Leja (Amsterdam, the Netherlands) chambers placed in a thermostatized microscope stage (37°C). The analysis was based on the examination of 25 consecutive, digitalized images obtained from a single field using 910 negative phase contrast objective. Images were taken with a time lapse of 1 s; therefore, the image capture speed was one every 40 ms. The number of objects incorrectly identified as spermatozoa was manually minimized on the monitor using the playback function. With respect to the setting parameters for the program, spermatozoa with a velocity 15 lm/s were considered motile. Indirect immunofluorescence and confocal microscopy Fifteen microlitres of fresh semen samples were spread on poly-L-lysine-coated slides and allowed to attach for 10 min. Then, semen was fixed with 4% formaldehyde © 2014 Blackwell Verlag GmbH

Apoptotic Bodies in Stallion Sperm

in PBS for 15 min at RT and permeabilized with 0.2% Triton X-100 (v/v) in PBS for 5 min. Slides were washed three times for 10 min with PBS and incubated in PBS supplemented with 5% BSA (w/v) for 90 min to block non-specific sites. After blocking, slides were incubated with anti-cleaved Caspase 3 overnight at 4°C diluted 1/ 100 in PBS containing 5% BSA (w/v). The following day, samples were extensively washed with PBS and further incubated for 45 min at RT with the appropriate secondary antibody diluted to 1/500 in PBS containing 5% BSA (w/v), consisting of an anti-rabbit IgG antibody conjugated with the Alexa 488 fluorescent dye (Molecular Probes). Finally, the slides were thoroughly washed with PBS and examined in a Bio-Rad MRC1024 confocal microscope with a 609 objective in oil immersion. Samples were excited at 488 nm with an argon laser, and emission was recorded using a 515-nmlong pass filter set. The absence of non-specific staining was assessed by processing the samples (fresh semen) without primary antibody. Western blotting To separate the proteins according to their apparent molecular masses, SDS-PAGE was performed according to Laemmli (Laemmli 1970). Samples (1 ml) were washed with phosphate-buffered saline (PBS), resuspended in lysis buffer (50 mM Trizma base, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 1 mM EGTA, 0.4 mM EDTA) supplemented with a protease inhibitor cocktail and phosphatase inhibitor (0.2 mM Na3VO4) and sonicated for 5 s at 4°C. The homogenate was centrifuged for 15 min at 10 000 9 g at 4°C, and the supernatant containing soluble proteins in non-ionic and ionic detergents was used for further protein analysis. Proteins were denatured by boiling for 5 min at 95°C in a loading buffer supplemented with 5% mercaptoethanol. The protein content was calculated using the Bradford assay (Bradford 1976). Fifteen micrograms of protein extract of spermatozoa was loaded and resolved by SDS-PAGE on a 10% polyacrylamide gel. The proteins were then transferred to a nitrocellulose membrane, which was subsequently blocked with blocking buffer [5% non-fat dry milk, in a Tris buffered saline Tween-20 (TBST) containing 10 mM Trizma base, 100 mM NaCl and 0.05% Tween 20] for 1 h at RT. Immunoblotting was performed by incubating the membranes in blocking buffer overnight at 4°C with Cleaved Caspase 3 antibody diluted at 1/1,000. Membranes were then washed in TBST and incubated with goat anti-rabbit horseradish peroxidaseconjugated secondary antibody for 45 min at RT. Following 3 washes for 10 min each with TBST, the signal was visualized using a SuperSignal West Pico Chemiluminescent Substrate Kit according to the manufacturer’s instructions. Band intensity was quantified using the software SCION IMAGE for Windows, version 4.02 (Scion Corp., Frederick, MD). Statistical analysis Data were first examined using the Kolmogorov– Smirnov test to determine their distribution. The © 2014 Blackwell Verlag GmbH

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Spearman’s nonparametric test was used to study the correlations between the quantity of apoptotic bodies found in the samples and the results of the sperm analysis. Significance was set at p < 0.05. All analyses were performed using SPSS version 17.0 for Windows (SPSS Inc., Chicago, IL, USA).

Results Identification of apoptotic bodies Several spheroidal bodies were observed under confocal microscope. The diameter of these bodies was variable (3.433, 3.355, 3.511 and 3.904 lm) and similar in size to the diameter of the stallion sperm head (3.133) (Fig. 1). In human samples, used as positive control, apoptotic bodies were stained with M540 (Fig. 2a), but not with the nuclear probe DAPI (Fig. 2b), indicating the absence of nuclei (Gandini et al. 2000). As shown in Fig. 2, these spheroidal bodies were also stained with M540 (Fig. 2c). Moreover, nuclei of the spermatozoa were stained with DAPI, but no signal was observed in these bodies, indicating that they were devoid of nuclei (Fig. 2d). Evaluation of PS externalization To assess whether these spheroidal bodies may present signs of being apoptotic bodies, slides were stained with Annexin-V, which detects translocation of PS. As shown in Fig. 3 and also in Fig. S1, these spheroidal bodies were also stained with Annexin-V (green; Fig. 3a), indicating PS externalization. As expected, only the nuclei of spermatozoa were PI positive (red; Fig. 3b). Detection of Caspase activity To assess whether these bodies have active Caspase 3, immunofluorescence studies were carried out using an antibody that specifically recognizes the active form of this protein. Active Caspase 3 was found either in the acrosome region of the spermatozoa or in the spheroidal bodies

Fig. 1. Identification of apoptotic bodies. Image obtained with BioRad MRC1024 confocal microscope equipped with a 609 objective. Short arrows are pointing apoptotic bodies. Long arrows show the diameter of the apoptotic bodies and length of the sperm head. Numbers are indicating the length in micrometres

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(a)

(b)

(c)

(d)

Fig. 4. Detection of Caspase activity. Active Caspase 3 appears stained in green. Apoptotic bodies are pointed with white arrows. Samples were examined in a Bio-Rad MRC1024 confocal microscope equipped with a 609 objective. Bar = 20 lm. N = 6 Fig. 2. Identification of apoptotic bodies. Human samples were used as positive controls (a,b). (c,d) Representative images from 14 stallions. (a,c) white arrows indicate apoptotic bodies (spheroidal structures) stained with M540 (red). (b,d) nuclei of spermatozoa stained with DAPI (blue); white arrows point out the localization of apoptotic bodies. Samples were examined with a 609 objective in an epifluorescence microscope (Nikon, Eclipse TE 300). Bars = 20 lm for all panels

(a)

(b)

Fig. 5. Assessment of Caspase activity in stallion ejaculates. Each lane represents one stallion (total = 14 individuals). Values of active Caspase 3 in each column represented in the graphic are normalized with respect to tubulin Fig. 3. Evaluation of phosphatidylserine (PS) externalization. (a) Apoptotic bodies (white arrows) appear stained with Annexin-V (green). (b) nuclei of stallion spermatozoa stained with IP (red); white arrows point the localization of apoptotic bodies. Samples were examined with a 609 objective in an epifluorescence microscope (Nikon, Eclipse TE 300). Bars = 40 lm for all panels. N = 6

(Fig. 4). Although the quantity of bodies was variable among stallions, all of them presented Caspase 3 activity. Detection of Caspase activity in stallion ejaculates To measure the activity of Caspase 3, proteins coming from ejaculates of 14 different stallions were resolved by SDS-PAGE, and immunoblotting was carried out using an antibody that specifically recognizes the active form of this protein. Our results showed that there was a large variability in the Caspase 3 activity among the 14 stallions studied (Fig. 5). Caspase 3 activity was not detected in stallions

3, 5, 7 and 10. While spermatozoa from stallions 1, 2, 4, 6 and 14 presented low Caspase 3 activity, stallions 8, 9, 11, 12 and 13 displayed high Caspase 3 activity. Correlation between the quantity of apoptotic bodies and semen parameters in fresh ejaculates Several semen parameters were measured in fresh samples from 14 different stallions (one ejaculate per stallion). Volume of ejaculates ranged from 19 to 90 ml, concentration (spermatozoa/ml) ranged from 100 to 560, total motility (TM) (% of motile spermatozoa) ranged from 46% to 90%, progressive motility (PG) ranged from 30% to 67% and viability (% of alive spermatozoa) ranged from 53% to 85%. In parallel, in the same samples, the quantity of apoptotic bodies in seminal plasma was assessed. Quantity of apoptotic bodies varied among stallions ejaculates: 7.30, 0.85, 0.15, 0.20, 0.10, 0.25, 1.15, 0.15, 0.50, 0.55, 0.50, 2.20, 0.25 and 4.00. © 2014 Blackwell Verlag GmbH

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Statistical analysis was carried out (as described in Materials and Methods) to assess whether a correlation between quantity of apoptotic bodies in seminal plasma and semen parameters and Caspase 3 activity existed. After statistical analysis, no correlation was found between the quantity of apoptotic bodies in seminal plasma and fresh semen parameters such as total volume of the ejaculate, concentration, motility or viability. However, there was a significant positive correlation between the quantity of apoptotic bodies and the activity of Caspase 3 of the spermatozoa from the same ejaculate (Table 1). Correlation between the quantity of apoptotic bodies in fresh ejaculates and semen parameters in incubated ejaculates Next, we studied whether a correlation between the quantity of apoptotic bodies in fresh samples and spermatozoa parameters existed after incubation for 3 h at 37°C. No significant changes were observed in viability or progressive motility after 3 h of incubation; yet, the percentage of motile spermatozoa decreased significantly after 3 h of incubation from 84%  7.06 in fresh samples to 50%  6.90 (p = 0.021). We did not observe a statistical correlation between the quantity of apoptotic bodies found in fresh samples and the PM (% of spermatozoa with a progressive motility) and viability (% of alive spermatozoa) of spermatozoa after 3 h of incubation at 37°C (Table 2). Yet, a statistically positive correlation was found between the quantity of apoptotic bodies in fresh samples and TM (% of motile spermatozoa) after 3 h of incubation at 37°C. Influence of the quantity of apoptotic bodies in fresh semen and the freezability of ejaculates Finally, we aimed to study whether the presence of apoptotic bodies could be related to the resistance of the ejaculate to be frozen. Semen parameters such as progressive/total motility and viability decreased significantly (p < 0.001) after thawing. Despite the decrease observed in all of the parameters studied, our results show a high negative correlation between the quantity of apoptotic bodies found in ejaculates before freezing and their viability and TM (% of motile spermatozoa) after thawing (Table 2); however, no correlation was observed with the percentage of spermatozoa showing progressive motility (PM). Table 1. Correlation between percentage of apoptotic bodies and semen parameters in equine fresh ejaculates Parameters

Mean  SEM

Volume (ml) Concentration (spz/ml) TM (%) PM (%) Viability (%) Caspase 3 activity

56.81 262.50 83.94 50.50 64.66 0.89

     

6.94 45.05 4.54 3.41 5.29 0.35

Correlation quantity of AP 0.057 0.147 0.013 0.093 0.057 0.716**

TM, total motility; PM, progressive motility; AP, apoptotic bodies. **p ≤ 0.01. N = 14.

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Discussion Seminal plasma is a mixture of secretions produced in the testes, epididymides and accessory sex glands, whose constituents vary between individual stallions and have frequently been used as potential fertility markers (Kareskoski and Katila 2008). Among these constituents, we found round structures of different sizes whose proportion varied among stallions and were stained with M540, according to those elements found by Muratori et al. (2004) in human semen. The same group, some years later, characterized M540 bodies as apoptotic bodies by evaluating a series of apoptotic markers (Marchiani et al. 2007b). Therefore, according with those previously showed, our results might be indicating the presence of apoptotic bodies in equine semen. However, we can no discard that those round structures found in equine semen also might be the called ‘residual bodies’ (RB). RBs are made of the cytoplasmic portions of elongated spermatids that are shed during extrusion of differentiated sperm into the lumen of the seminiferous tubule (Kerr and de Kretser 1974). RB display many features of apoptotic bodies: PS externalization and expression levels of Caspase 1, c jun, p53 and p21 (Blanco-Rodriguez and Martinez-Garcia 1999). In the present study, the round structures found in equine semen also presented apoptotic markers such as PS externalization and active Caspase 3, being compatible with both apoptotic bodies and RB. Both hypotheses are feasible, as it has been suggested that the mechanism responsible for the formation of residual bodies is similar to that for apoptotic bodies (Kerr and de Kretser 1974; Blanco-Rodriguez and Martinez-Garcia 1999). Yet, to our knowledge, the presence of residual bodies has been restricted to the seminiferous epithelium (Smith and Lacy 1959; Kerr and de Kretser 1974; Blanco-Rodriguez and Martinez-Garcia 1999), while apoptotic bodies have been found in seminal plasma (Marchiani et al. 2007b). Therefore, although further research work should be done to clarify the nature of such bodies, results obtained in the present work seem to indicate that those round structures found in equine seminal plasma might be apoptotic bodies more than residual bodies. It has been suggested that the quantity of apoptotic bodies in human semen may be considered as a semen marker of altered testis function due to the positive correlation with poor semen quality (Lotti et al. 2012). M450 body levels have been negatively correlated with sperm number/ejaculate, progressive motility and normal morphology (Lotti et al. 2012). Although morphological studies were not performed in the present study, we did not observe any statistical correlation between equine semen quality and the quantity of apoptotic bodies in fresh samples. Further work might be performed to study the relationship between sperm morphology and quantity of apoptotic bodies. An explanation of the absence of correlation observed in this study may be the quantity of apoptotic bodies found in equine semen. There is certain controversy in the literature with respect to the number of apoptotic bodies found in subfertile human patients (Lotti et al. 2012; Gomez-Lopez et al. 2013). The quantity of

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Table 2. Correlation between quantity of apoptotic bodies in fresh semen and semen parameters after incubation at 37°C (n = 7) and after freezing and thawing (n = 7) in the equine specie Post incubation Parameters (%) TM PM Viability

Post freezing/thawing

Mean  SEM (%)

Correlation quantity of AP

Mean  SEM (%)

50  6.90 21  5.92 61  1.67

0.829* 0.429 0.429

26.8  5.85 17.7  4.87 35.6  5.63

Correlation quantity of AP 0.997** 0.808 0.954*

TM, total motility; PM, progressive motility; AP, apoptotic bodies. *p ≤ 0.05. **p ≤ 0.01.

apoptotic bodies found in equine semen was lower than those reported previously (Lotti et al. 2012), in which there was an important statistical correlation between subfertile patients and apoptotic bodies. However, and in agreement with our results, when a lower quantity of apoptotic bodies was detected, there was an absence of statistical correlation between apoptotic bodies and semen quality (Gomez-Lopez et al. 2013) in human samples. Another explanation for the absence of correlation between apoptotic bodies and semen quality in fresh samples may be due to the stallions used in this study, which did not present any pathology, disease or surgery that could affect the reproductive capability, in contrast to all of the previously published studies (Marchiani et al. 2007b; Lotti et al. 2012). Apoptosis in the testis has an important role not only in maintaining an appropriate germ cell to Sertoli cell ratio, but also in removing defective germ cells, both required to maintain the overall quality of sperm production (Shukla et al. 2011). In fact, when testes are subjected to insults such as heat stress, scrotal heating or varicocele, ejaculates presented an increase in the proportion of sperm with characteristics of apoptosis and abnormal morphology (Sinha Hikim et al. 2003; Chen et al. 2004; Paul et al. 2008; Wu et al. 2009). The presence of apoptotic markers in germ cells in fresh ejaculates has been associated with low fertility, either in animals or humans (Shukla et al. 2011; Caballero et al. 2012; Dogan et al. 2012). Therefore, the evaluation of either the presence of apoptotic bodies or apoptotic markers in spermatozoa could help to establish the rate of apoptosis in the testis (Marchiani et al. 2007a; Lotti et al. 2012). The quantity of apoptotic bodies in fresh equine semen showed no correlation with semen quality, as commented earlier; yet, there was a positive correlation between the quantity of apoptotic bodies and Caspase 3 activity in the spermatozoa from the same ejaculate. Moreover, due to the large differences observed, not only in the quantity of apoptotic bodies but also in the Caspase 3 activity observed among individuals, the rate of apoptosis seems to be different among stallions. Apoptosis in the testis may occur through both the death receptor or extrinsic pathway and the mitochondrial or intrinsic pathway (Tapia and Pena 2009). In the intrinsic pathway, in response to apoptotic stimuli, several proteins are released from the intermembrane space of mitochondria into the cytoplasm (Schultz and Harrington 2003). Some of the most well-characterized proteins include cytochrome c, which mediates the activation of Caspase 9 (Schultz and Harrington 2003),

triggering a cascade of caspase activation, including Caspase 3, which promotes cellular apoptosis. Protein level of cytochrome c is significantly increased during capacitation in boar sperm (Choi et al. 2008). The induction of cytochrome c activation in boar sperm significantly increased tyrosine phosphorylation in sperm cells, which is required for capacitation and acrosome reaction, while the blockage of cytochrome c release into the cytosol inhibited the tyrosine phosphorylation of sperm proteins and capacitation (Choi et al. 2008). In human spermatozoa, cytochrome c activation has been associated with motility (Firestone et al. 2012). Firestone et al. demonstrated that exposing human samples to a 30-s infrared laser pulse of 50 mW/cm2 at 905 nm (a wavelength thought to increase light-sensitive cytochrome c oxidase in the mitochondrial electron transport chain) showed a significant increase in motility, most prominent in oligospermic and asthenospermic samples (85% increase), which contain a high quantity of apoptotic bodies (Marchiani et al. 2007b). In our study, those samples with a higher quantity of apoptotic bodies in fresh semen displayed higher Caspase 3 activity. When these samples were incubated for 3 h in a capacitating medium, we observed a positive correlation between the quantity of apoptotic bodies in fresh samples and sperm motility after incubation. This higher increase in motility could be explained by the higher activation of cytochrome c. However, further studies are required to investigate this hypothesis. Sperm cryopreservation is widely used in the context of assisted reproductive techniques. Yet, this procedure inflicts irreversible injury on spermatozoa including damage to plasma membranes, osmotic stress, oxidative stress, premature ageing or capacitation-like changes and phase transitions in the plasmalemma, among others (Ortega-Ferrusola et al. 2008, 2009; Said et al. 2010). Evidence shows a significant increase in some apoptosis markers following cryopreservation and thawing (Said et al. 2010), contributing to sperm death or, if surviving, to reduce their lifespan. In the present study, we observed that those ejaculates with a higher percentage of apoptotic bodies (also higher Caspase 3 activity) were more sensitive to the stress caused during the cryopreservation and thawing process, generating a greater decrease in motility and viability compared to those ejaculates with a low percentage of apoptotic bodies. As AI is permitted by most stud books in the equine species, there is an increasing need for more intense research in equine freezing technologies (Aurich and © 2014 Blackwell Verlag GmbH

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Aurich 2006). Despite there being growing evidence of development in this area, the highly stallion-dependent freezability makes the standardization of protocols difficult (Metcalf 2007). Although higher number of stallions should have been included, we believe that the negative correlation found among quantity of apoptotic bodies and semen quality after thawing could be used, in a future, to estimate the resistance of an ejaculate to support cryopreservation. Acknowledgements The generous collaboration of the Service of Equine Breeding of the Spanish Army is acknowledged. Miro-Moran, A. was supported by a predoctoral fellowship from the Ministerio Ciencia e Innovaci on (BES-2008-002106). We thank Mrs. Mercedes G omez-Blazquez for her excellent technical assistance. This work was supported by Junta de Extremadura-FEDER (GR10010, PCE1002 and DE12006) and by Ministerio de Ciencia e Innovaci on-FEDER, Grants BFU2011-30261 and AGL 2010-20758.

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Conflict of interest None of the authors have any conflict of interest to declare. There has been no financial support for this work that could have influenced its outcome.

Author contributions The study was designed by IMA and JAT. The experiments were performed by ABC, AMM, AMR and JGB. Semen samples were collected and prepared by COF, AMR and JGB. The data were analysed by IMA, JAT, GMS and FJP. Finally, the manuscript was written and corrected by IMA, JAT, GMS and FJP.

Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 Representative image comparing the size between apoptotic bodies found in plasma seminal and cytoplasmic droplet attached to the tail of the sperm..

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Submitted: 24 Jul 2013; Accepted: 10 Nov 2013 Author’s address (for correspondence): IM Aparicio, Veterinary Faculty, Physiology Department, Avda. de la Universidad s/n, 10003 Caceres, Spain. E-mail: imad@ unex.es

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