Equine spermatozoa stored in the epididymis for up to 96 h at 4 °C can be successfully cryopreserved and maintain their fertilization capacity

Animal Reproduction Science 136 (2013) 280–288

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Equine spermatozoa stored in the epididymis for up to 96 h at 4 ◦ C can be successfully cryopreserved and maintain their fertilization capacity L.A. Vieira, J. Gadea, F.A. García-Vázquez, K. Avilés-López, C. Matás ∗ Department of Physiology, Faculty of Veterinary, University of Murcia, Murcia 30071, Spain

a r t i c l e

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Article history: Received 25 July 2012 Received in revised form 4 October 2012 Accepted 5 October 2012 Available online 1 November 2012 Keywords: Stallion Epididymal Spermatozoa Storage time Cryopreservation

a b s t r a c t After injury or death of a valuable male, recovery of epididymal spermatozoa may be the last chance to ensure preservation of its genetic material. The objective of this research was to study the effect of sperm storage, at 4 ◦ C up to 96 h, in the epididymides obtained from castrated horses and its effect on different functional sperm parameters. Aims were to study the effect of (1) sperm storage on viability and chromatin condensation; (2) pre-incubation of recovered epididymal sperm in the freezing extender, prior cryopreservation, on viability and chromatin condensation; and (3) freezing–thawing on viability, chromatin condensation, ROS generation, protein tyrosine phosphorylation and heterologous fertilization rate (ICSI and IVF using bovine oocytes) of sperm recovered from the epididymis up to 96 h post castration. The average volume (720 ± 159 ␮L) and the concentration (6.5 ± 0.4 × 109 spermatozoa/mL) of sperm recovered from the epididymis were not affected by storage. Sperm viability after refrigeration at 4 ◦ C for up to72 h was similar (P < 0.01). The effect of sperm dilution in the freezing media showed similar values up to 48 h, while viability was preserved up to 72 h (P < 0.01). Cryopreserved spermatozoa show similar viability between different storage times. Chromatin condensation was not affected by storage time; however, incubation for 30 min in freezing medium and freezing–thawing process induced an increase in the chromatin decondensation. ROS generation was not affected by storage up to 96 h. Epididymal storage did not affect sperm protein tyrosine phosphorylation patterns; although the pattern of phosphorylation changed to strong staining of the equatorial segment when the sperm where capacitated in sperm–TALP. Finally, successful and similar pronuclear formation (analyzed by ICSI) and in vitro penetration (evaluated with bovine zone free oocyte) was observed using cryopreserved sperm obtained from prolong epididymal storage at 4 ◦ C. In conclusion, cryopreservation of epididymal stallion sperm stored for up to 72 h in the epididymis at 4 ◦ C, maintain both viability and ability to fertilize in vitro. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Traumatic injuries, colic conditions or unexpected death can end the breeding careers of many stallions and under those circumstances owners may want to recover sperm from these animals in view of preserving valuable genetics

∗ Corresponding author. Tel.: +34 868 887256; fax: +34 868 884147. E-mail address: [email protected] (C. Matás). 0378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2012.10.027

(Cary et al., 2004). Some techniques have been developed to collect the sperm after terminal cases, such as electroejaculation or epididymal sperm extraction. Electroejaculation is not an effective method for obtaining more viable sperm mainly due to urine contamination (Cary et al., 2004). However, epididymal sperm collection is a successful technique in different species, including stallions (Barker and Gandier, 1957; Sharma et al., 1997; Blash et al., 2000). Moreover, pregnancies were achieved in goats, red deer, dogs, pigs and humans using recovered epididymal sperm

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(Holtz and Smidt, 1976; Marks et al., 1994; Sharma et al., 1997; Zomborszky et al., 1999; Blash et al., 2000; Hewitt et al., 2001). Stallion epididymides contain large numbers of morphologically normal and viable spermatozoa and therefore they represent an available source of germplasm (Bruemmer, 2006; Papa et al., 2008; Monteiro et al., 2011). Therefore, research into recovery of viable epididymal spermatozoa from dead or castrated stallions is essential to enable the preservation of gametes from these animals that may die unexpectedly or are castrated before they show valuable genetic characteristics. In mammals, epidydimal sperm cells can survive for some time in the epididymides of dead animals, but deterioration of sperm quality usually correlates with body decomposition and increases as the postmortem interval increases (Songsasen et al., 1998). Facilities trained to recover and preserve epididymal sperm after castration or death are not always available or in proximity, therefore, epididymides storage at low temperatures allows extra time needed for sperm recovery and processing (Hoodbhoy and Talbot, 1994; Baumber et al., 2003). To date, results of studies that have examined sperm quality upon recovery after various days of epididymal storage at 4 ◦ C have shown that percentage of sperm motility decreases as storage interval increases (Songsasen et al., 1998). Monteiro et al. (2011) showed that stallion sperm recovered from the epididymal cauda, either immediately after castration or after 24 h storage at 4 ◦ C in the epididymis, have similar fertility capacity as ejaculated sperm. Other studies have shown that stallion spermatozoa can be successfully cryopreserved (Barker and Gandier, 1957; Barker, 1962; Jimenez, 1987; James et al., 2002; Bruemmer et al., 2003; Papa et al., 2008; Heise et al., 2010). In 1957, Barker and Gandier achieved the first pregnancy in a mare inseminated with frozen–thawed epididymal stallion spermatozoa. However, to date they are still limited studies regarding fertility capacity of frozen–thawed epididymal stallion sperm (Barker and Gandier, 1957; Morris et al., 2002; Melo et al., 2008; Heise et al., 2010). Taken together this indicates that storage of stallion sperm in the epididymis for extensive long periods of time at low temperature can be cryopreserved and can be used safely in order to preserve spermatozoa from highly genetically valued animals. The aim of the current study is to evaluate the effect of long periods of sperm storage (up to 96 h) at 4 ◦ C in the epididymides obtained from castrated horses and its effect on different functional sperm parameters before and/or after cryopreservation. For this purpose, sperm viability, chromatin condensation, ROS generation, protein tyrosine phosphorylation and acrosome integrity were evaluated. In addition, fertilization capacity was also evaluated by heterologous intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) using zone-free bovine oocytes.

2. Material and methods All reagents were obtained from Sigma–Aldrich Química, S.A. (Madrid, Spain) unless otherwise indicated.

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2.1. Animals and epididymal sperm collection Testicles and epididymides (82 units) from sexually mature stallions were immediately collected after animal castration conducted by four different veterinary surgeons. The organs were transported to the laboratory in an insulated container at room temperature within 1 h after collection. After transport to the laboratory, the testis and epididymides were washed with physiological saline solution (0.9% NaCl). Epididymides were wrapped in foil to prevent desiccation and stored in a 4 ◦ C refrigerator. Each epididymal sample was randomly assigned to an experimental group for storing during 0, 24, 48, 72 or 96 h at 4 ◦ C. After storage, the cauda region of the epididymis was dissected and the spermatozoa were obtained by retrograde air flushing in a 4 ◦ C refrigerated chamber. For this purpose, a 21-G needle attached to a 5 mL syringe filled with air was introduced into the vas deferens. Spermatozoa were then flushed in a retrograde direction from the vas deferens through the cauda epididymis into a 1.5 mL eppendorf tube. Epididymal fluid volume, sperm concentration, viability and chromatin condensation were measured. The rest of the epididymal fluid was used for cryopreservation. 2.2. Freezing procedure Lactose-EDTA-egg yolk medium was used as sperm freezing extender. The composition of this extender was 100 mL deoinized water (Milli-Q, Millipore), 11% (w/v) lactose, 0.1% (w/v) EDTA, 0.089% (w/v) sodium bicarbonate, 3 mL egg yolk and 3.5 mL of glycerol, pH = 7.4 (Tischner, 1979). After preparation the extender was stabilized at 4 ◦ C. This medium was freshly prepared on the day of use. Spermatozoa were extended in the freezing media at 4 ◦ C (final concentration 100 × 106 cells/mL) for 30 min. After that time an aliquot was taken to assess sperm viability and chromatin condensation before cryopreservation. Sperm samples were loaded in polyvinyl chloride straws (0.5 mL, IMV, France) at 4 ◦ C, frozen over nitrogen vapor plugged (4 cm) for 15 min, and stored in liquid nitrogen until analyzed. 2.3. Thawing procedure Straws were thawed in a water bath at 37 ◦ C for 30 s and the content diluted in 4 mL Ca2+ and Mg2+ -free PBS (phosphate buffer saline) (Merkies and Buhr, 1998) previously warmed at 37 ◦ C. 2.4. Sperm evaluation procedures 2.4.1. Sperm concentration and viability The sperm concentration was evaluated by haemocytometer (Neubauer counting chamber; VWR International, Haasrode, Belgium). Sperm membrane integrity was assessed by incubating sperm in a solution containing 20 ␮L of carboxifluorescein diacetate (CFD, 0.46 mg/mL), to which 20 ␮L of propidium iodide (PI, 200 mg/mL), 10 ␮L of formaldehyde saline solution (1%), 100 ␮L of the

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sample of semen and 900 ␮L of physiological saline solution were added (Harrison and Vickers, 1990). At least 200 cells per sample were evaluated using a microscope equipped with epi-fluorescence (100× magnification, Leica® DMLS). Spermatazoa were classified into two groups: (1) cells with green fluorescence: intact membrane integrity; and (2) cells with red fluorescence: altered membrane integrity.

2.4.2. Analysis of seminal parameters by flow cytometry Flow cytometric analyses were performed on a Coulter Epics XL cytometer (Beckman Coulter Inc., Miami, FL, USA). A 15 mW argon ion laser operating at 488 nm excited the fluorophores. Data from 10,000 events per sample were collected in list mode, and four measures per sample were recorded. Flow cytometric data were analyzed using the program Expo32ADC (Beckman Coulter Inc.) using a gate in forward and side scatter to exclude eventual remaining debris and aggregates from the analysis. Flow cytometry was used for assessment of chromatin condensation, acrosomal status, viability, and ROS generation. The frozen–thawed samples were washed by centrifugation (700 × g for 3 min), the supernatant was removed, and the pellet was resuspended in 1 mL of fresh PBS medium before sperm assessment.

2.4.2.1. Production of reactive oxygen species (ROS). Production of ROS was measured by incubating the spermatozoa in the presence of 2 ,7 -dichlorodihydrofluorescein diacetate (H2 DCFDA) (0.5 ␮M in thawing medium PBS) at 37 ◦ C (Gadea et al., 2008). H2 DCFDA is a stable cell-permeable non-fluorescent probe commonly used to detect cellular ROS production. It is de-esterified intracellularly and turns to highly fluorescent 2 ,7 -dichlorofluorescin upon oxidation. Green fluorescence was collected with an FL1 sensor using a 525 nm band-pass filter. Measurements were expressed as the mean green intensity fluorescence units (mean channel in the FL1) and this was used as index of ROS generation. ROS generation was measured after 30 and 60 min of incubation at 37 ◦ C.

2.4.2.2. Determination of chromatin condensation. Sperm chromatin was stained with PI for the determination of sperm chromatin condensation (Gadea et al., 2011). Samples were incubated in a solution of ethanol and phosphate buffered saline (PBS) (70/30, v/v) for 30 min for sperm membrane permeabilization and kept at −80 ◦ C until use. After thawing and process as was indicated above, the samples were incubated with a PI solution (10 mg/mL). Samples were maintained in the darks at room temperature for 1 h before flow cytometric analysis. PI fluorescence (red) was collected with an FL3 sensor using a 650 nm bandpass filter. Measurements were expressed as the mean red intensity fluorescence units (mean channel in the FL3) and this was used as index of the state of the chromatin condensation, as it is directly related to the PI uptake by DNA.

2.5. Localization of proteins phosphorylated in tyrosine residues after capacitation Immunofluorescence was employed to determine the localization of proteins phosphorylated in tyrosine residues as previously has been described (Matas et al., 2011). Frozen–thawed spermatozoa were washed by Percoll 45/60% density gradient and then were re-suspended in PBS or in sperm–TALP (109 mM NaCl, 3.15 mM KCl, 1.99 mM NaHCO3 , 0.347 mM NaH2 PO4 , H2 O, 8.468 mM Hepes, 8.3 mM PVP, 10 mM sodium lactate, 0.199 mM sodium piruvate, 0.068 mM streptomycin and 0.086 mM penicillin) (McPartlin et al., 2009). The spermatozoa resuspended in sperm–TALP were incubated under 5% CO2 for 15 min at 37 ◦ C and the spermatozoa re-suspended in PBS were incubated for 15 min at 37 ◦ C in air. Samples were centrifuged for 3 min at 700 × g. Spermatozoa were fixed in 2% formaldehyde solution for 60 min at 4 ◦ C. Then the samples were washed once in PBS, blocked with 4% (w/v) BSA–PBS and incubated overnight at 4 ◦ C. The spermatozoa were washed and re-suspended in PBS, smeared onto a microscope slide, and allowed to air dry. Slides were then incubated for 1 h with anti-phosphotyrosine monoclonal antibody at 4 ◦ C (1:200, clone 4G10, Millipore, Madrid, Spain) rinsed with PBS, and incubated for an additional 1 h with fluorescein-conjugated goat anti-mouse antibodies (1:400, Bio-Rad Laboratories, Madrid, Spain). After rinsing with PBS, samples were mounted on the slides with 90% glycerol/PBS (v/v). Sperm were observed using a microscope equipped with epi-fluorescence (100× magnification, Leica® DMLS) for anti-phosphotyrosine antibody labeling. Sperm were classified into two groups according to the localization of the anti-phosphotyrosine monoclonal antibody signal (Pommer et al., 2003): (A) fluorescent presents in the sub-equatorial region and, (B) fluorescence signal in the flagellum (Fig. 1). A minimum of two slides per sample was evaluated, counting a minimum of 200 spermatozoa per sub-sample. 2.6. Heterologous ICSI and IVF with cattle oocytes The medium used for in vitro maturation (IVM) bovine oocytes was TCM-199 with Earle’s salts, 10% (v/v) fetal bovine serum (FBS), 2 mM l-glutamine, 0.2 mM sodium pyruvate, 50 mg/mL gentamicin, 10 IU/mL eCG (Foligon; Intervet International BV), and 10 IU/mL hCG (Veterin Corion Divasa Farmavic). The IVF medium for this species consisted of IVF-TALP as previously described by Ikawa et al. (2010). 2.6.1. In vitro maturation of cattle oocytes IVM of cattle oocytes were performed as previously described (Coy et al., 2008). Cow cumulus oocyte complexes (COCs) were collected by aspiration of follicles (2–6 mm in diameter) of ovaries from the slaughterhouse. COCs were then washed twice in TCM – previously equilibrated for 5 h at 38.5 ◦ C and 5% CO2 . Groups of 30–40 oocytes were cultured in 500 mL maturation medium for 24 h at 38.5 ◦ C and 5% CO2 .

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Fig. 1. Frozen–thawed epididymal stallion spermatozoa classified into two groups according to the localization of the anti-phosphotyrosine monoclonal antibody signal: (A) sub-equatorial segment and (B) flagellum.

2.6.2. Intracitoplasmatic sperm injection (ICSI) of bovine oocytes Spermatozoa were obtained by centrifugation of frozen–thawed semen on a 45/60 discontinuous Percoll (Pharmacia) gradient for 20 min at 700 × g. The pellet was re-suspended in sperm–TALP medium and washed again for 5 min at 700 × g. The pellet was finally re-suspended in 1 mL sperm–TALP media. In vitro matured cattle oocytes with zona pellucida were injected using frozen–thawed epididymal spermatozoa. The sperm injection was performed as previously has been described (García-Vázquez et al., 2009, 2010). After ICSI, the oocytes were transferred to IVF-TALP medium and incubated at 38.5 ◦ C and 5% CO2 . Eighteen to 20 h post-injection, injected oocytes were washed in PBS and fixed for evaluation. The oocytes were fixed for 30 min (0.5% glutaraldehyde in PBS), stained for 15 min by Hoechst 33342 (1% in PBS), washed in PBS containing 1 mg/mL polyvinylpyrrolidone, mounted on glass slides, and examined under an epifluorescence microscope for evidence of sperm status and the pronuclear formation (Fig. 2A). 2.6.3. In vitro fertilization with zona-free cattle oocytes In vitro fertilization were performed as previously described (Coy et al., 2008). Briefly, after the oocytes IVM, 30–40 oocytes were transferred to a well containing a solution of 0.3% (w/v) pronase to solubilize the ZP surrounding the oocyte. Zona-free oocytes were washed three times in IVF-TALP medium and groups of 25–30 oocytes were transferred to a well of four-well Nunc multidishes (Nunc, Roskilde, Denmark) containing 250 ␮L of IVF-TALP medium previously equilibrated at 38.5 ◦ C, 5% CO2 . Spermatozoa were obtained by centrifugation of frozen–thawed semen on a 45/60% discontinuous Percoll (Pharmacia) gradient for 20 min at 700 × g. The pellet was re-suspended in sperm–TALP medium and washed again

for 5 min at 700 × g. The pellet was finally re-suspended in 500 ␮L IVF-TALP with procaine (5 mM) (McPartlin et al., 2009). The sperm concentration was adjusted to 2 × 105 cells/mL and maintained for 15 min at 38.5 ◦ C in humidified atmosphere with 5% CO2 before IVF was performed. Sperm suspensions (250 ␮L) were added to the fertilization wells to obtain a final concentration of 105 cells/mL. Eighteen to 20 h post-fertilization, zona-free oocytes were washed in PBS and fixed for evaluation, as described above, in order to evaluate the pronucleus formation (Fig. 2B). 2.7. Experimental design A total of 82 epididymides were used. The epididymides were stored at 4 ◦ C for 0, 24, 48, 72 and 96 h. After a corresponding time of storage in the epididymides, sperm was collected and (i) volume, concentration, viability and sperm chromatin decondensation were analyzed, (ii) sperm viability and chromatin decondensation was assessed in sperm that were further incubated for 30 min in the freezing extender and (iii) viability, chromatin decondensation, ROS production (after 30 and 60 min of sperm incubation in sperm–TALP), protein tyrosine phosphorylation pattern and heterologous fertilization rate (IVF and ICSI) were finally evaluated in post frozen–thawed sperm. 2.8. Statistical analysis Data are expressed as the mean ± SEM and analyzed by ANOVA, considering the specific sperm treatment (storage time) as the main variable. When ANOVA revealed a significant effect, values were compared by the least significant difference pairwise multiple comparison post hoc test (Tukey). Differences were considered statistically significant at P < 0.05.

Fig. 2. Pronuclear formation after 18–22 h post-injection or insemination evaluated by Hoescht staining. (A) Cattle oocytes injected with frozen–thawed epididymal stallion spermatozoa. (B) Zona-free cattle oocytes incubated with frozen–thawed epididymal stallion spermatozoa.

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Table 1 Concentration (×109 cells/mL) of spermatozoa obtained from epididymides stored at 4 ◦ C for up to 96 h. No differences were found between storage times. Storage time (h)

Concentration (×109 cells/mL)

0 24 48 72 96

5.3 6.3 6.0 7.7 7.7

Total

6.4 ± 0.3

± ± ± ± ±

0.4 1.1 0.9 0.8 0.9

Table 3 Viability of frozen–thawed spermatozoa obtained from epididymides stored at 4 ◦ C up to 96 h. Viability evaluated after 0, 30 and 120 min of incubation at 37 ◦ C. No differences were found between storage times. Storage time (h)

Sperm viability (%) 0 min ± ± ± ± ±

30 min

0 24 48 72 96

35.3 35.8 32.6 37.9 36.5

Total

35.7 ± 1.4a

a,b,c

4.2 5.0 1.7 3.9 2.4

23.0 22.1 20.7 20.3 26.0

± ± ± ± ±

120 min 3.1 3.6 3.1 2.3 2.3

22.8 ± 1.2b

17.8 12.7 14.5 13.4 17.6

± ± ± ± ±

2.9 2.7 2.3 1.1 1.5

15.5 ± 0.9c

with different superscripts differ (P < 0.05).

3. Results 3.1. Sperm evaluation Spermatozoa were successfully recovered from epididymides using retrograde flushing with air technique. The epididymal fluid collected had a mean volume of 720 ± 159 ␮L, a mean sperm concentration of 6.5 ± 0.4 × 109 spermatozoa/mL and a total mean number of spermatozoa obtained from each epididymis 4.7 ± 0.7 × 109 cells. There were no differences between these variables and sperm storage time in the epididymis prior collection (up to 96 h) (Table 1, P > 0.05). The viability of the samples obtained was greater than 85% when they were recovered from the epididymides on the same day of castration (0 h) (Table 2), and viability was well conserved in stored samples at 4 ◦ C during the first 72 h with values greater than 80%. Only samples stored in the epididymides for 96 h had a significantly lesser viability compared with the other groups (Table 2, P < 0.01,). Viability of sperm diluted in freezing media followed by refrigeration at 4 ◦ C for 30 min was also well conserved in stored samples during the first 48 h (Table 2). However, viability significantly decreased in sperm retrieved after 72 and 96 h compared to the 0 h group (Table 2, P < 0.05). In contrast, viability post frozen–thawed was not affected by the cryopreservation in epididymal sperm retrieved at any time up to 96 h (Table 3, P > 0.05), with mean values close to 35% immediately after thawing (Table 3). The chromatin condensation of the raw samples were not affected by the storage of sperm in the epididymides (Fig. 3, P > 0.05). However, the cold shock occurring during the cooling (dilution in the freezing media and maintained 30 min at 4 ◦ C) and freezing process induced an increase in the chromatin condensation (less PI uptake) which was

similar in all the experimental groups analyzed (Fig. 3, P < 0.05). Sperm recovered from the epididymides and that underwent a freezing and thawing cycle were not different in ROS generation when incubated for either 30 or 60 min in sperm–TALP (Fig. 4, P > 0.05). 3.2. Sperm tyrosine phosphorylation pattern An objective in this section was to analyze the influence of the sperm storage in epididymides kept at 4 ◦ C for up to 96 h on patterns of protein sperm tyrosine phosphorylation. The results from frozen–thawed spermatozoa incubated in PBS (non-capacitating medium) had a similar pattern of phosphorylation in all the experimental groups (Fig. 5, P > 0.05). When spermatozoa were incubated in capacitating medium (sperm–TALP medium), an increase in the percentage of tyrosine phosphorylation in relation to the control group was observed only in the

Table 2 Viability of spermatozoa obtained from epididymides stored at 4 ◦ C for up to 96 h. Raw samples obtained from epididymides by retrograde flushing with air. Cooled samples were extended in freezing media and maintained at 4 ◦ C for 30 min. Storage time (h)

Raw samples

0 24 48 72 96 P value

86.5 ± 84.4 ± 84.0 ± 81.1 ± 77.2 ± 0.01

a

1.3 1.3ab 2.0ab 2.0ab 2.6b

Cooled at 4 ◦ C for 30 min 84.9 ± 79.1 ± 80.0 ± 76.0 ± 67.6 ± <0.01

2.0a 1.7ab 2.2ab 2.4bc 2.8c

Fig. 3. Chromatin condensation of spermatozoa obtained from epididymides stored at 4 ◦ C for up to 96 h. (a) White bars: raw samples; (b) diagonal bars: samples extended in freezing media and cooled to 4 ◦ C for 30 min and; (c) black bars: frozen–thawed samples. No differences were found between storage times.

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3.3. Heterologous ICSI and IVF In an heterologous ICSI system a total of 59 cattle oocytes were injected with frozen–thawed spermatozoa from each of the different experimental groups (0–96 h storage in the epididymides). Spermatozoa from all the experimental groups induced egg activation and pronucleus formation (Table 5). In an heterologous IVF system, a total of 209 zona-free cattle oocytes were incubated with spermatozoa from the different experimental groups. Effective penetration and both female and male pronuclear formation was detected (Table 5). 4. Discussion

Fig. 4. ROS production by frozen–thawed spermatozoa obtained from epididymides stored at 4 ◦ C for up to 96 h. ROS production measured after 30 min (diagonal bar) and 60 min (black bars) of incubation. No differences were found between storage times.

Fig. 5. Protein tyrosine phosphorylation observed in frozen–thawed spermatozoa obtained straight after storage at 4 ◦ C for up to 96 h. No differences were found between storage times.

sub-equatorial segment of the sperm head (Table 4, P < 0.05). In contrast, there were no differences in the signal observed in the flagellum between both experimental groups (Table 4, P = 0.53).

Table 4 Protein tyrosine phosphorylation observed in frozen–thawed epididymal spermatozoa incubated in sperm–TALP (capacitating media). Signal pattern

Control Sperm–TALP a,b

Flagellum

Sub-equatorial segment

No signal

9.4 ± 2.3 7.5 ± 1.5

2.7 ± 0.5a 33.5 ± 5.2b

68.8 ± 3.5a 46.4 ± 4.3b

with different superscripts differ (P < 0.05).

Cryopreservation of recovered epididymal spermatozoa may be the last opportunity to preserve the genome of an animal of high genetic value or at risk of extinction (Bruemmer, 2006). As cryopreservation facilities are not always in proximity to where an animal dies or where castration is performed, it is, therefore, important to determine how long can a spermatozoa be stored in the epididymides (at low temperature), prior cryopreservation, before it loses its ability to fertilize an oocyte. Because cryopreservation protocols, including media and methodology, for domesticated animals are made specifically for ejaculated spermatozoa of a specific species, it is therefore important to identify alternative methods for storing epididymal spermatozoa of genetic value or endangered species. In the present study the techniques and methods were developed to recover and cryopreserve stallion spermatozoa from the epididymides up to 96 h post castration before spermatozoa lose fertilizing capacity. The quality of the raw and frozen–thawed samples in the present study was maintained during at least 72 h. This is of great interest as it allows for the potential production of offsprings using assisted reproductive technologies (AI, IVF or ICSI). Several methods of sperm recovery from the cauda region of the epididymis have been described including aspiration, slicing of the tissue, incubation with physiological media (floating methodology) and retrograde flush of the epididymal tubule from the vas deferens with the use of extenders (Cary et al., 2004). The number of spermatozoa recovered from our methodology averaged 4.7 × 109 spermatozoa similar to those described by other studies using extender media for the flushing procedure (4.0, 4.6 and 4.5 × 109 spermatozoa, respectively) (Jimenez, 1987; James et al., 2002; Cary et al., 2004) or by floating procedure (4.9 × 109 spermatozoa) (Cary et al., 2004; Fioratti et al., 2008). However, other studies using flushing procedures recovered significantly more sperm (12–30 × 109 spermatozoa) (Bruemmer, 2006; Granemann et al., 2006; Monteiro et al., 2011). This difference could simply be due to some others factors such as sexual maturity, testicular volume, and weight (Fioratti et al., 2008). Moreover, the retrograde flushing with air used in methodology of the present study has several advantages in comparison with the other techniques used previously (Bruemmer, 2006; Granemann et al., 2006; Monteiro et al., 2011). This methodology simplifies the recovery process because it

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Table 5 Pronuclear formation in intact bovine oocytes injected (ICSI) with epididymal stallion spermatozoa or incubated (IVF) with zona free bovine oocytes. Epididymal spermatozoa were recovered from epididymides stored at 4 ◦ C for up to 96 h. Storage time (h)

ICSI

IVF

Number of oocytes

Pronuclear formation (%)

Number of oocytes

Pronuclear formation (%)

0 24 48 72 96

22 13 5 10 9

2 (18.1) 1 (7.6) 1 (20) 4 (40) 5 (55.5)

106 72 7 11 13

3 (2.8)a 7 (9.7)a 4 (57.1)b 1 (9.0)a 2 (15.3)a

Total

59

9 (15.25)

209

a,b

17 (8.13)

Different superscripts in the same column indicate different values (P < 0.05).

prevents: (i) dilution of the sample with an extender avoiding equilibration period to obtain correct temperature and pH, therefore saving time in the process and (ii) subsequent centrifugation to concentrate the sample before freezing which could damage the spermatozoa (Alvarez et al., 1993; Matás et al., 2007). In addition, the technique used is not complicated and after training, epididymal fluid can be obtained from most samples (100% in the current study). Similar to previous reports, the recovery of sperm was not affected by the storage time in the epididymides (James et al., 2002). Sperm viability was greater than 75% in all the groups studied and a significant decrease was only detected after 96 h of storage. However, cryopreservation did not further affect sperm viability in sample stored at 4 ◦ C in the epididymis post-castration up to 96 h, similarly to other studies (Bruemmer et al., 2002; James et al., 2002; James, 2004). Although sperm motility, viability and morphology are important variables for successful sperm–oocyte interaction to occur. Spermatozoa must also have a resistant and undamaged chromatin (Dias et al., 2004). Previous studies in human and domestic animals have shown that a cycle of freezing and thawing induce important changes in the sperm chromatin, resulting in greater compactness (Hamamah et al., 1990; Gadea et al., 2005, 2008, 2011). In the present study, it was confirmed that the compactness of the chromatin occurs in the first phase of the freezing procedure during the cooling phase but storage time did not further affect the compactness of the chromatin. Another important point to take into consideration when freezing stallion spermatozoa is the generation of ROS agents that could unbalance the antioxidant defense system as previously was described in different species (Bilodeau et al., 2000; Gadea et al., 2004, 2011). Seminal plasma is a potent source of ROS scavengers, which protect the spermatozoa from the adverse effects of ROS. However epididymal spermatozoa are therefore more prone to damage by ROS as epididymal fluid appears to have limited amounts of scavengers (Dacheux et al., 2005). In the present study, there were no differences in ROS generation between storage times. The oxygen tension and conventional substrates such as glucose are less in the epididymis and, therefore, reduces sperm metabolism (Lone et al., 2011). The low temperature used to preserve the epididymides could explain the lesser amounts of ROS generated by sperm. Further studies need to be developed to analyze changes induced by cryopreservation on the antioxidant

system of the epididymal spermatozoa and their effect on sperm structure and functionality (membrane lipoperoxidation, DNA fragmentation, etc.). In the present study, the evaluation of the protein tyrosine phosphorylation was used as a tool for monitoring the sperm functionality after the capacitation process. Protein tyrosine phosphorylation has an important role in intracellular signaling, transport or cellular cycle progression. Stallion spermatozoa display an increase in protein tyrosine phosphorylation when they are capacitating in vitro. Pommer et al. (2003) reported an increase of phosphorylation in the mid- and principal piece of the sperm flagellum when freshly ejaculated stallion sperm were capacitated for 3 h in the presence of dbcAMP, caffeine (phosphodiesterase inhibitor) or methyl ␤-cyclodextrin (cholesterol acceptor). In contrast, the majority of spermatozoa stored in the stallion cauda region of the epididymis appear to be incapable (or less able) of undergoing capacitation and acrosome reaction (Sostaric et al., 2008). This fact, at least in part, explains the poor pregnancy rates obtained and/or the large sperm numbers required to obtain pregnancies when fresh or frozen–thawed stallion epididymal spermatozoa used for artificial insemination (Morris et al., 2002; Heise et al., 2010). In addition, the freezing process promotes sperm capacitation-like processes (Watson, 1995; Bailey et al., 2000), hence this might reduce the longevity of cryopreserved spermatozoa in the female reproductive tract. However, there was no previous information about the effect of epididymal sperm storage over time on capacitation. In the present study, it was observed that the detection of protein tyrosine phosphorylation in the sub-equatorial segment was very low in all the experimental groups (<4%) and no differences were found between them (Fig. 5). When spermatozoa were incubated under capacitation conditions (sperm–TALP), protein tyrosine phosphorylation increase in sub-equatorial segment (>30%). Sperm cryopreservation reduces sperm functional parameters compared to that of freshly ejaculates sperm (Thomas et al., 1998; Bilodeau et al., 2000). However, a reduction in sperm quality does not necessarily directly or proportional translate into a reduction in fertility. Unfortunately, it is generally not possible to conduct large-scale fertility trials or homologous IVF studies because the animals or gametes are not available, even in domestic species, such trials can be expensive and time-consuming (Roth et al., 1999). Sperm penetration assays can assess a number of sperm functions simultaneously (e.g. motility, ability

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to undergo acrosome reaction, oocyte penetration and DNA decondensation) and may be a better assay to assess sperm quality (Gadea, 2005). Therefore, heterologous IVF have been used to predict the fertility of spermatozoa in different species including equine (Choi et al., 2002, 2003; Garcia-Alvarez et al., 2009; Clulow et al., 2010). In the current study, ICSI and IVF was performed in a heterologous system to evaluate the quality of frozen–thawed epididymal spermatozoa, recovered from postmortem epididymides stored at 4 ◦ C over time. Spermatozoa from all the experimental groups (0, 24, 48, 72 and 96 h) were able to induce egg activation and develop female and male pronuclei (Table 5). However, penetration rates were very low compared to other studies developed with stallion or donkey ejaculated spermatozoa (Choi et al., 2003; Taberner et al., 2010). One possible cause for these low rates could be related to a different pattern of capacitation in epididymal spermatozoa in comparison to ejaculated spermatozoa, as it was previously reported in boar spermatozoa (Matas et al., 2010). That could need changes in the capacitating media and protocols to be modified and adapted for epididymal spermatozoa. Penetration rates can vary by more than 40% depending on the medium used (Choi et al., 2003). The results in ICSI or IVF systems obtained between spermatozoa recovered from the different storage time groups seem to be contradictory because more pronuclear formation is observed after a prolong storage time than in control group (ICSI at 96 h and IVF at 48 h; Table 5, P > 0.05). Although the difference is not statistically different, this discrepancy could be explained by the fact that each storage time refers to a different male and it is known that there is an intrinsic fertility and cryopreservation variation between males (Barbas and Mascarenhas, 2009). In addition, collection of sperm was done throughout the year and it is well known that sperm quality is affected by seasonal variation (Hoffmann and Landeck, 1999). An interesting fact observed was that in all cases, stallion frozen–thawed epididymal spermatozoa were able to activate the oocyte (formation of male and female pronucleus). This demonstrates the integrity of the factors necessary for this event to occur. A sperm factor, sensitive to heat and proteases has been determined, to be responsible for the generation of IP3 and Ca2+ oscillations. This factor is an isoform of phospholipase C, named PLCz (Yoneda et al., 2006) and it is now widely accepted that PLCz activates the oocyte (Kashir et al., 2010). In frozen–thawed spermatozoa, a process that damages the sperm membrane, the amount of this PLCz in spermatozoa decreases (Kashir et al., 2011). Nakai et al. (2011) studied the effect on the male pronucleus formation after ICSI of the treatment of the spermatozoa by various methods to break or modify membranes (sonication, Triton X-100 and three cycles of freezing and thawing). Oocytes injected with complete spermatozoa had greater pronuclear formation than oocytes injected with treated sperm (under the conditions described above) suggesting that these sperm contain lower amount of PLCz. In conclusion, the storage of sperm in the epididymes at 4 ◦ C for up to 96 h could be an efficient system for the salvage of gametes from genetically important stallion that may die unexpectedly or are castrated. The retrograde flushing by air followed by cryopreservation ensure

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the preservation of a number of functional sperm which can be used to successfully fertilize heterologous oocytes using techniques such as IVF and ICSI. However, further evaluation using artificial insemination to produce viable offsprings is necessary to confirm the fertilization competence of the conserved gametes. Acknowledgments The authors appreciate the generous effort and work from the veterinary surgeons José Aldebarán Rubio, Pedro Sánchez, Carlos Galves and Natividad González, who helped us in recovering the testes and epididymides derived from castrations. We would also like to thank Dr Linda Lefièvre for reviewing this manuscript. This work was supported by Fundación Seneca project 08752/PI/08. References Alvarez, J.G., Lasso, J.L., Blasco, L., Nunez, R.C., Heyner, S., Caballero, P.P., Storey, B.T., 1993. Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime. Hum. Reprod. 8, 1087–1092. Bailey, J.L., Bilodeau, J.F., Cormier, N., 2000. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J. Androl. 21, 1–7. Barbas, J.P., Mascarenhas, R.D., 2009. Cryopreservation of domestic animal sperm cells. Cell Tissue Bank 10, 49–62. Barker, C.A., 1962. Long-term survival of frozen equine epididymal spermatozoa. Can. Vet. J. 3, 221–222. Barker, C.A., Gandier, J.C., 1957. Pregnancy in a mare resulting from frozen epididymal spermatozoa. Can. J. Comp. Med. Vet. Sci. 21, 47–51. Baumber, J., Ball, B.A., Linfor, J.J., Meyers, S.A., 2003. Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa. J. Androl. 24, 621–628. Bilodeau, J.F., Chatterjee, S., Sirard, M.A., Gagnon, C., 2000. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol. Reprod. Dev. 55, 282–288. Blash, S., Melican, D., Gavin, W., 2000. Cryopreservation of epididymal sperm obtained at necropsy from goats. Theriogenology 54, 899–905. Bruemmer, J., Moore, A., Squires, E., 2003. Freezing epididymal sperm from transported testicles. In: Squires, E., Wade, J.F. (Eds.), Proceedings of a Workshop on Transporting Gametes and Embryos. R & W Publications, Brewster, MA, USA, pp. 61–62. Bruemmer, J., Reger, H., Zibinski, G., Squires, E., 2002. Effect of storage at 5 C on the motility and cryopreservation of stallion epididymal spermatozoa. Theriogenology 58, 405–407. Bruemmer, J.E., 2006. Collection and freezing of epididymal stallion sperm. Vet. Clin. North Am. Equine Pract. 22, 677–682. Cary, J.A., Madill, S., Farnsworth, K., Hayna, J.T., Duoos, L., Fahning, M.L., 2004. A comparison of electroejaculation and epididymal sperm collection techniques in stallions. Can. Vet. J. 45, 35–41. Clulow, J.R., Evans, G., Maxwell, W.M., Morris, L.H., 2010. Evaluation of the function of fresh and frozen–thawed sex-sorted and non-sorted stallion spermatozoa using a heterologous oocyte binding assay. Reprod. Fertil. Dev. 22, 710–717. Coy, P., Grullon, L., Canovas, S., Romar, R., Matas, C., Aviles, M., 2008. Hardening of the zona pellucida of unfertilized eggs can reduce polyspermic fertilization in the pig and cow. Reproduction 135, 19–27. Choi, Y., Love, C., Love, L., Varner, D., Brinsko, S., Hinrichs, K., 2002. Developmental competence in vivo and in vitro of in vitro-matured equine oocytes fertilized by intracytoplasmic sperm injection with fresh or frozen–thawed spermatozoa. Reproduction 123, 455. Choi, Y.H., Landim-Alvarenga, F.C., Seidel Jr., G.E., Squires, E.L., 2003. Effect of capacitation of stallion sperm with polyvinylalcohol or bovine serum albumin on penetration of bovine zona-free or partially zonaremoved equine oocytes. J. Anim. Sci. 81, 2080–2087. Dacheux, J.L., Castella, S., Gatti, J.L., Dacheux, F., 2005. Epididymal cell secretory activities and the role of proteins in boar sperm maturation. Theriogenology 63, 319–341. Dias, A.J., Maia, M.S., Retamal, C.A., Lopez, M.L., 2004. Identification and partial characterization of alpha-1,4-glucosidase activity in equine epididymal fluid. Theriogenology 61, 1545–1558.

288

L.A. Vieira et al. / Animal Reproduction Science 136 (2013) 280–288

Fioratti, E.G., Melo, C.M., Villaverde, A.I.S.B., Martin, I., Felício, G.B., Ferreira, H.N., Oliveira, J.V., Alvarenga, M.A., Papa, F.O., 2008. Correlation of testicular volume and weight with sperm recovery from stallion epididymis. Anim. Reprod. Sci. 107, 322. Gadea, J., 2005. Sperm factors related to in vitro and in vivo porcine fertility. Theriogenology 63, 431–444. Gadea, J., García-Vázquez, F., Matas, C., Gardon, J.C., Canovas, S., Gumbao, D., 2005. Cooling and freezing of boar spermatozoa: supplementation of the freezing media with reduced glutathione preserves sperm function. J. Androl. 26, 396–404. Gadea, J., Gumbao, D., Canovas, S., García-Vázquez, F.A., Grullon, L.A., Gardon, J.C., 2008. Supplementation of the dilution medium after thawing with reduced glutathione improves function and the in vitro fertilizing ability of frozen–thawed bull spermatozoa. Int. J. Androl. 31, 40–49. Gadea, J., Molla, M., Selles, E., Marco, M.A., García-Vázquez, F.A., Gardon, J.C., 2011. Reduced glutathione content in human sperm is decreased after cryopreservation: effect of the addition of reduced glutathione to the freezing and thawing extenders. Cryobiology 62, 40–46. Gadea, J., Selles, E., Marco, M.A., Coy, P., Matas, C., Romar, R., Ruiz, S., 2004. Decrease in glutathione content in boar sperm after cryopreservation. Effect of the addition of reduced glutathione to the freezing and thawing extenders. Theriogenology 62, 690–701. Garcia-Alvarez, O., Maroto-Morales, A., Martinez-Pastor, F., FernandezSantos, M.R., Esteso, M.C., Perez-Guzman, M.D., Soler, A.J., 2009. Heterologous in vitro fertilization is a good procedure to assess the fertility of thawed ram spermatozoa. Theriogenology 71, 643–650. García-Vázquez, F.A., García-Roselló, E., Gutiérrez-Adán, A., Gadea, J., 2009. Effect of sperm treatment on efficiency of EGFP-expressing porcine embryos produced by ICSI-SMGT. Theriogenology 72, 506–518. García-Vázquez, F.A., Ruiz, S., Matas, C., Izquierdo-Rico, M.J., Grullon, L.A., De Ondiz, A., Vieira, L., Aviles-Lopez, K., Gutierrez-Adan, A., Gadea, J., 2010. Production of transgenic piglets using ICSI-sperm-mediated gene transfer in combination with recombinase RecA. Reproduction 140, 259–272. Granemann, L., Weiss, R., Kozicki, L., Muradas, P., Treml, T., 2006. Total number of spermatozooa from stud-horses collected by means of artificial vagina and through feedback flow of the epididymis tail. Arch. Vet. Sci. 11, 73–77. Hamamah, S., Royere, D., Nicolle, J.C., Paquignon, M., Lansac, J., 1990. Effects of freezing–thawing on the spermatozoon nucleus: a comparative chromatin cytophotometric study in the porcine and human species. Reprod. Nutr. Dev. 30, 59–64. Harrison, R.A.P., Vickers, S.E., 1990. Use of fluorescent probes to assess membrane integrity in mammalian spermatozoa. J. Reprod. Fertil. 88, 343–352. Heise, A., Kahn, W., Volkmann, D.H., Thompson, P.N., Gerber, D., 2010. Influence of seminal plasma on fertility of fresh and frozen–thawed stallion epididymal spermatozoa. Anim. Reprod. Sci. 118, 48–53. Hewitt, D.A., Leahy, R., Sheldon, I.M., England, G.C., 2001. Cryopreservation of epididymal dog sperm. Anim. Reprod. Sci. 67, 101–111. Hoffmann, B., Landeck, A., 1999. Testicular endocrine function, seasonality and semen quality of the stallion. Anim. Reprod. Sci. 57, 89–98. Holtz, W., Smidt, D., 1976. The fertilizing capacity of epididymal spermatozoa in the pig. J. Reprod. Fertil. 46, 227–229. Hoodbhoy, T., Talbot, P., 1994. Mammalian cortical granules: contents, fate, and function. Mol. Reprod. Dev. 39, 439–448. Ikawa, M., Inoue, N., Benham, A.M., Okabe, M., 2010. Fertilization: a sperm’s journey to and interaction with the oocyte. J. Clin. Invest. 120, 984–994. James, A., Green, H., Hoffman, S., Landry, A., Paccamonti, D., Godke, R., 2002. Preservation of equine sperm stored in the epididymis at 4 ◦ C for 24, 48, 72 and 96 hours. Theriogenology 58, 401–404. James, A.N., 2004. Preservation of sperm harvested from the rat, caprine, equine and bovine epididymis. Doctoral Thesis. The Interdepartmental Program of Animal and Dairy Sciences, Texas A&M University. Jimenez, C., 1987. Effects of Equex STM and equilibration time on the pre-freeze and postthaw motility of equine epididymal spermatozoa. Theriogenology 28, 773–782. Kashir, J., Heindryckx, B., Jones, C., De Sutter, P., Parrington, J., Coward, K., 2010. Oocyte activation, phospholipase C zeta and human infertility. Hum. Reprod. Update 16, 690–703. Kashir, J., Heynen, A., Jones, C., Durrans, C., Craig, J., Gadea, J., Turner, K., Parrington, J., Coward, K., 2011. Effects of cryopreservation and density-gradient washing on phospholipase C zeta concentrations in human spermatozoa. Reprod. Biomed. Online 23, 263–267.

Lone, F.A., Islam, R., Khan, M.Z., Sofi, K.A., 2011. Effect of transportation temperature on the quality of cauda epididymal spermatozoa of ram. Anim. Reprod. Sci. 123, 54–59. Marks, S., Dupuis, J., Mickelsen, W., Memon, M., Platz Jr., C., 1994. Conception by use of postmortem epididymal semen extraction in a dog. J. Am. Vet. Med. Assoc. 204, 1639–1640. Matás, C., Decuadro, G., Martínez-Miró, S., Gadea, J., 2007. Evaluation of a cushioned method for centrifugation and processing for freezing boar semen. Theriogenology 67, 1087–1091. Matas, C., Sansegundo, M., Ruiz, S., García-Vázquez, F.A., Gadea, J., Romar, R., Coy, P., 2010. Sperm treatment affects capacitation parameters and penetration ability of ejaculated and epididymal boar spermatozoa. Theriogenology 74, 1327–1340. Matas, C., Vieira, L., García-Vázquez, F.A., Aviles-Lopez, K., Lopez-Ubeda, R., Carvajal, J.A., Gadea, J., 2011. Effects of centrifugation through three different discontinuous Percoll gradients on boar sperm function. Anim. Reprod. Sci. 127, 62–72. McPartlin, L.A., Suarez, S.S., Czaya, C.A., Hinrichs, K., Bedford-Guaus, S.J., 2009. Hyperactivation of stallion sperm is required for successful in vitro fertilization of equine oocytes. Biol. Reprod. 81, 199–206. Melo, C., Papa, F., Fioratti, E., Villaverde, A., Avanzi, B., Monteiro, G., Dell’Aqua, J., Pasquini, D., Alvarenga, M., 2008. Comparison of three different extenders for freezing epididymal stallion sperm. Anim. Reprod. Sci. 107, 331-331. Merkies, K., Buhr, M.M., 1998. Epididymal maturation affects calcium regulation in equine spermatozoa exposed to heparin and glucose. Theriogenology 49, 683–695. Monteiro, G.A., Papa, F.O., Zahn, F.S., Dellaqua Jr., J.A., Melo, C.M., Maziero, R.R., Avanzi, B.R., Alvarenga, M.A., Guasti, P.N., 2011. Cryopreservation and fertility of ejaculated and epididymal stallion sperm. Anim. Reprod. Sci. 127, 197–201. Morris, L., Tiplady, C., Allen, W., 2002. The in vivo fertility of cauda epididymal spermatozoa in the horse. Theriogenology 58, 643–646. Nakai, M., Ito, J., Sato, K., Noguchi, J., Kaneko, H., Kashiwazaki, N., Kikuchi, K., 2011. Pre-treatment of sperm reduces success of ICSI in the pig. Reproduction 142, 285–293. Papa, F.O., Melo, C.M., Fioratti, E.G., Dell’aqua Jr., J.A., Zahn, F.S., Alvarenga, M.A., 2008. Freezing of stallion epididymal sperm. Anim. Reprod. Sci. 107, 293–301. Pommer, A.C., Rutllant, J., Meyers, S.A., 2003. Phosphorylation of protein tyrosine residues in fresh and cryopreserved stallion spermatozoa under capacitating conditions. Biol. Reprod. 68, 1208–1214. Roth, T.L., Bush, L.M., Wildt, D.E., Weiss, R.B., 1999. Scimitar-horned oryx (Oryx dammah) spermatozoa are functionally competent in a heterologous bovine in vitro fertilization system after cryopreservation on dry ice, in a dry shipper, or over liquid nitrogen vapor. Biol. Reprod. 60, 493–498. Sharma, R.K., Padron, O.F., Thomas, A.J., Agarwal, A., 1997. Factors associated with the quality before freezing and after thawing of sperm obtained by microsurgical epididymal aspiration. Fertil. Steril. 68, 626–631. Songsasen, N., Tong, J., Leibo, S.P., 1998. Birth of live mice derived by in vitro fertilization with spermatozoa retrieved up to twenty-four hours after death. J. Exp. Zool. 280, 189–196. Sostaric, E., Aalberts, M., Gadella, B.M., Stout, T.A., 2008. The roles of the epididymis and prostasomes in the attainment of fertilizing capacity by stallion sperm. Anim. Reprod. Sci. 107, 237–248. Taberner, E., Morato, R., Mogas, T., Miro, J., 2010. Ability of Catalonian donkey sperm to penetrate zona pellucida-free bovine oocytes matured in vitro. Anim. Reprod. Sci. 118, 354–361. Thomas, C.A., Garner, D.L., DeJarnette, J.M., Marshall, C.E., 1998. Effect of cryopreservation of bovine sperm organelle function and viability as determined by flow cytometry. Biol. Reprod. 58, 786–793. Tischner, M., 1979. Evaluation of deep-frozen semen in stallions. J. Reprod. Fertil. Suppl. 27, 53–59. Watson, P.F., 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod. Fertil. Dev. 7, 871–891. Yoneda, A., Kashima, M., Yoshida, S., Terada, K., Nakagawa, S., Sakamoto, A., Hayakawa, K., Suzuki, K., Ueda, J., Watanabe, T., 2006. Molecular cloning, testicular postnatal expression, and oocyte-activating potential of porcine phospholipase Czeta. Reproduction 132, 393–401. Zomborszky, Z., Zubor, T., Toth, J., Horn, P., 1999. Sperm collection from shot red deer stags (Cervus elaphus) and the utilization of sperm frozen subsequently thawed. Acta Vet. Hung. 47, 263–270.