Introduction
Long-term storage of spermatozoa cryopreserved in liquid nitrogen (−196 °C) has been carried out in mammals for almost half a century since the first report (cattle, Stewart, Reference Stewart1951; sheep, Salamon & Lightfoot, Reference Salamon and Lightfoot1967; pig, Pursel & Johnson, Reference Pursel and Johnson1975; Westendorf et al., Reference Westendorf, Richter and Treu1975; human, Bunge & Sherman, Reference Bunge and Sherman1953). Storage of semen in frozen form is as useful method of preserving the germplasm from genetic resources of important economic traits and genetic diversity. However, this method requires expenditure on liquid nitrogen and space for storing containers. It has long been expected that the adaptation of freeze-drying technology for sperm preservation should enable sperm storage at ambient temperatures or at 4 °C. Freeze-drying has been used for preserving viruses, bacteria, yeasts and fungi, because freeze-dried materials are easy to store and transport without special equipment (Day & McLellan, Reference Day and McLellan1995). However, freeze-dried spermatozoa lose their motility even after rehydration (Kusakabe et al., Reference Kusakabe, Szczygiel, Whittingham and Yanagimachi2001) and intracytoplasmic sperm injection (ICSI) is required for successful fertilization or embryo development. Viable offspring have been produced by ICSI of freeze-dried spermatozoa in mice (Wakayama & Yanagimachi, Reference Wakayama and Yanagimachi1998; Kusakabe et al., Reference Kusakabe, Szczygiel, Whittingham and Yanagimachi2001; Kaneko et al., Reference Kaneko, Whittingham and Yanagimachi2003a, Reference Kaneko, Whittingham, Overstreet and Yanagimachib), rabbits (Liu et al., Reference Liu, Kusakabe, Chang, Suzuki, Schmidt, Julian, Pfeffer, Bormann, Tian, Yanagimachi and Yang2004) and rats (Hirabayashi et al., Reference Hirabayashi, Kato, Ito and Hochi2005). However, in pigs (Kwon et al., Reference Kwon, Park and Niwa2004) and cattle (Keskintepe et al., Reference Keskintepe, Pacholczyk, Machinicka, Norris, Curuk, Khan and Brackett2002), it has been reported only that oocytes resulting from ICSI with freeze-dried spermatozoa have developed to the blastocyst stage.
The ICSI procedure renders immotile spermatozoa able to fertilize. However, there is currently a debate about the risk of sperm with abnormalities achieving fertilization (completion to the male pronucleus), because physiological selection processes such as binding to the zona pellucida, acrosomal reaction and fusion to the ooplasm, are bypassed (Sun et al., Reference Sun, Jurisicova and Casper1997; Lopes et al., Reference Lopes, Sun, Jurisicova, Meriano and Casper1998). DNA fragmentation in human spermatozoa is one of the causes of failure of embryonic development and successful pregnancy (Henkel et al., Reference Henkel, Hajimohammad, Stalf, Hoogendijk, Mehnert, Menkveld, Gips, Schill and Kruger2004). This suggests that the presence of structurally intact DNA in the sperm is quite important for normal embryogenesis after fertilization.
It has been suggested that endonucleases are among the causes of DNA fragmentation in spermatozoa (Kusakabe et al., Reference Kusakabe, Szczygiel, Whittingham and Yanagimachi2001). Sperm endonucleases are released from plasma membrane-damaged spermatozoa during freeze-drying or freezing without cryoprotectant (Kusakabe et al., Reference Kusakabe, Szczygiel, Whittingham and Yanagimachi2001) and activated with divalent cation such as Ca2+ and Mg2+ (Sotolongo et al., Reference Sotolongo, Huange, Isenberger and Ward2005). However, activation of endonucleases is inhibited by the addition of chelating agents such as ethylene glycol–bis[beta-aminoethyl ether]-N,N,N′,N′-tetraacetic acid (EGTA) to freeze-drying buffer (Kusakabe et al., Reference Kusakabe, Szczygiel, Whittingham and Yanagimachi2001), with the result that chromosome stability is maintained and the rate of development to offspring after ICSI is improved in mice (Kaneko et al., Reference Kaneko, Whittingham and Yanagimachi2003a). This improved effect has been reported only in mice. No experiments have been designed in other species, including pigs, to clarify the relationship between sperm endonucleases and DNA fragmentation of freeze-dried spermatozoa.
We therefore examined the effect of the addition of chelating agents such as EGTA or ethylenediamine-N,N,N′,N′-tetraacetic acid, disodium salt (EDTA) to freeze-drying buffer in preventing fragmentation of boar sperm nuclear DNA. Furthermore, we investigated the relationship between DNA fragmentation in spermatozoa and in vitro or in vivo development of porcine oocytes following injection with freeze-dried boar sperm heads.
Materials and methods
Sperm collection and freeze-drying
Ejaculated semen was collected from a boar of the Landrace breed. It was centrifuged for 10 min at 600 g and the supernatant was discarded. The sperm pellet was resuspended in Modena solution (Weitze Reference Weitze1991). The spermatozoa were then sonicated for 1 min to isolate the sperm heads from the tails. The spermatozoa were centrifuged again for 2 min at 600 g and were resuspended in four different types of freeze-drying buffer: (1) 50 mM EGTA (no. 346–01312; Dojindo Laboratories) was added to the basic solution (50 mM NaCl and 10 mM Tris–HCl) (Kusakabe et al., Reference Kusakabe, Szczygiel, Whittingham and Yanagimachi2001) (50 mM EGTA); (2) 50 mM EDTA (no. 345–01865; Dojindo Laboratories) was added to the basic solution (50 mM EDTA); (3) 10 mM EDTA and 0.117 M sorbitol were added to the basic solution (10 mM EDTA); and (4) 0.15 M sorbitol was added to the basic solution (non-chelated medium). As a control, the spermatozoa were resuspended in a pig fertilization medium (Pig-FM; Suzuki et al., Reference Suzuki, Asano, Eriksson, Niwa, Nagai and Rodriguez-Martinez2002). The osmolality and pH of the buffers 1–4 were 265 mOsm/kg and pH 8.0, respectively. Those of the control buffer were to 305 mOsm/kg and pH 7.4, respectively. The concentration of spermatozoa in all buffers was adjusted to 3 × 108/ml. Sperm from each group were placed as a 100 µl suspension into a glass ampoule (ϕ 8 mm × 150 mm, Nakayamashoji). Each ampoule was then precooled at −40 °C for 6 h and attached to a freeze-drying system (DuraDry µP, FTS Systems) for 6 h. The ampoule was then closed by heat from a gas burner and stored at 4 °C.
Rehydration and incubation of freeze-dried spermatozoa
Freeze-dried sperm samples were rehydrated in 100 µl distilled water. The sperm suspension was centrifuged for 2 min at 600 g and the supernatant was removed. The pelleted spermatozoa were resuspended and incubated in Pig-FM for 0–180 min, according to the experimental design, at ambient temperatures.
TUNEL assay
DNA fragmentation in spermatozoa was assessed by using a detection kit (In situ Cell Death Detection Kit, Fluorescein, Roche Applied Science). In accordance with the manufacturer's instructions the spermatozoa were centrifuged, fixed and permeabilized. After two washings with 200 µl PBS, spermatozoa were incubated for 15 min at 37 °C in 50 µl of labelling solution containing TdT enzyme and dUTP. Spermatozoa were washed twice with PBS and observed under a standard inverse microscope (Olympus IX71) equipped with appropriate standard fluorescence facilities for green fluorescent protein (GFP) dye at a magnification of × 200 (Fig. 1). For each group, the fluorescence of three independent samples with 300 cells each was evaluated.
Figure 1 Detection of DNA fragmentation in freeze-dried boar spermatozoa. Spermatozoa with fragmented DNA are stained green (arrow). Spermatozoa with intact DNA (arrowhead) are not stained under fluorescence. (a) Phase contrast optics and (b) fluorescence images. Scale bar represents 5 µm.
Oocyte collection and in vitro maturation (IVM)
Protocols for the use of animals were approved by the Animal Care Committee of the National Institute of Agrobiological Sciences and Azabu University, Japan. Ovaries were obtained from prepubertal cross-bred gilts (Landrace, Large White and Duroc breeds) at a local slaughterhouse and transported to the laboratory at 35 °C. Cumulus–oocyte complexes (COCs) were collected from follicles 2–6 mm in diameter in TCM199 (with Hank's salts; Sigma) supplemented with 10% (v/v) fetal bovine serum (Gibco, Life Technologies), 20 mM HEPES (Dojindo Laboratories), 100 IU/ml penicillin G potassium (Sigma) and 0.1 mg/ml streptomycin sulfate (Sigma). Maturation culture was performed as reported previously (Kikuchi et al., Reference Kikuchi, Onishi, Kashiwazaki, Iwamoto, Noguchi, Kaneko, Akita and Nagai2002, Nakai et al., Reference Nakai, Kashiwazaki, Takizawa, Hayashi, Nakatsukasa, Fuchimoto, Noguchi, Kaneko, Shino and Kikuchi2003, Reference Nakai, Kashiwazaki, Takizawa, Maedomari, Ozawa, Noguchi, Kaneko, Shino and Kikuchi2006). In brief, about 40 COCs were cultured for 20–22 h in 4-well dishes (Nunclon Multidishes; Nalge Nunc International), each well contains 500 µl of maturation medium. The medium was a modified North Carolina State University (NCSU)–37 solution (Petters & Wells Reference Petters and Wells1993) containing 10% (v/v) porcine follicular fluid, 0.6 mM cysteine, 50 µM β-mercaptoethanol, 1 mM dibutyl cAMP (dbcAMP; Sigma), 10 IU/ml eCG (PMS 1000 Tani NZ; Nihon Zenyaku Kogyo) and 10 IU/ml hCG (Puberogen 1500 U; Sankyo). The COCs were subsequently cultured for 24 h in maturation medium without dbcAMP and without hormones. Maturation culture was carried out at 39 °C under conditions of CO2, O2 and N2 adjusted to 5, 5 and 90%, respectively (5% O2). After maturation culture, cumulus cells were removed from the oocytes by treatment with 150 IU/ml hyaluronidase (Sigma) and gentle pipetting. Denuded oocytes with the first polar body were harvested under a stereomicroscope and served as IVM oocytes.
Procedure of sperm head injection and oocyte stimulation
Two solutions were prepared for ICSI; (1) for oocytes, a modified NCSU-37 without glucose but supplemented with 0.17 mM sodium pyruvate, 2.73 mM sodium lactate, 4 mg/ml BSA, 50 µM β-mercaptoethanol (IVC-PyrLac; Kikuchi et al., Reference Kikuchi, Onishi, Kashiwazaki, Iwamoto, Noguchi, Kaneko, Akita and Nagai2002) and supplemented with 20 mM HEPES of which the osmolality was adjusted to 285 mOsm/kg (IVC–PyrLac–HEPES; Nakai et al., Reference Nakai, Kashiwazaki, Takizawa, Hayashi, Nakatsukasa, Fuchimoto, Noguchi, Kaneko, Shino and Kikuchi2003, Reference Nakai, Kashiwazaki, Takizawa, Maedomari, Ozawa, Noguchi, Kaneko, Shino and Kikuchi2006) and (2) for sperm, IVC–PyrLac–HEPES supplemented with 4% (w/v) polyvinyl pyrrolidone (MW 360,000; Sigma) (IVC–PyrLac–HEPES–PVP). Sperm heads were injected as described previously (Nakai et al., Reference Nakai, Kashiwazaki, Takizawa, Hayashi, Nakatsukasa, Fuchimoto, Noguchi, Kaneko, Shino and Kikuchi2003, Reference Nakai, Kashiwazaki, Takizawa, Maedomari, Ozawa, Noguchi, Kaneko, Shino and Kikuchi2006). Immediately before ICSI, the sperm suspension was again centrifuged for 2 min at 600 g and resuspended in IVC–PyrLac–HEPES–PVP. About 20 oocytes were transferred into a 20 µl drop of IVC–PyrLac–HEPES. The solution containing the mature oocytes was placed on the cover of a plastic dish (Falcon 35–1005; Becton Dickinson and Company). A small volume (0.5 µl) of the freeze-dried sperm head suspension was transferred to a 2 µl drop of IVC–PyrLac–HEPES–PVP, which was prepared close to the drops used for the oocytes. All drops were covered with paraffin oil (Paraffin Liquid; Nakarai Tesque Inc.). A single sperm head was aspirated from the suspension into an injection pipette and the pipette was moved to the drop containing the oocytes. The sperm head was injected into the ooplasm by using a piezo-actuated micromanipulator (PMAS-CT150; Prime Tech Ltd). One hour after the injection, the sperm head-injected oocytes (20 oocytes) were transferred to an activation solution consisting of 0.28 M d-mannitol, 0.05 mM CaCl2, 0.1 mM MgSO4 and 0.1 mg/ml BSA and washed once. They were then stimulated with a direct current pulse of 1.5 kV/cm for 20 µs by using a somatic hybridizer (SSH-10; Shimadzu).
In vitro culture of sperm head-injected oocytes
Sperm head-injected oocytes before and after electrical stimulation were cultured in vitro. Two types of in vitro culture (IVC) medium were prepared (Kikuchi et al., Reference Kikuchi, Onishi, Kashiwazaki, Iwamoto, Noguchi, Kaneko, Akita and Nagai2002). The first was IVC–PyrLac. The second contained 5.55 mM glucose, as used in the original NCSU-37 medium reported and was also supplemented with 4 mg/ml BSA and 50 µM β-mercaptoethanol (IVC–Glu). IVC–PyrLac was used from day 0 (the day of injection and electrical stimulation) up to day 2. The medium was changed once, to IVC–Glu, on day 2 and this medium was used for subsequent culture. IVC was carried out at 38.5 °C under 5% O2.
Assessment of embryonic development
Embryos cultured for 6 days were mounted on glass slides and fixed in 25% (v/v) acetic acid in ethanol, stained with 1% (w/v) orcein in 45% (v/v) acetic acid and examined under a phase-contrast microscope. We examined the rate of blastocyst formation and mean number of cells per blastocyst.
Statistical analysis
The percentage of spermatozoa with DNA fragmentation was scored. Embryonic development to the blastocyst stage (rate of blastocyst formation and mean number of cells per blastocyst) was evaluated. The percentage data were arcsine transformed (Snedecor & Cochran Reference Snedecor and Cochran1989). The data were subjected to analysis of variance (ANOVA) using the General Linear Model procedure and were then analysed by Duncan's multiple range test (Statistical Analysis System Institute).
Experimental design
Experiment 1: effect of chelating agents on DNA fragmentation after rehydration of freeze-dried spermatozoa
We examined the influence of chelating agents (EDTA and EGTA) on spermatozoal DNA fragmentation. After rehydration of the spermatozoa, the proportion with DNA fragmentation in each group was assessed by the TUNEL method as soon as the sperm had been centrifuged and resuspended in Pig-FM. Fresh ejaculated spermatozoa were also assessed. Furthermore, to examine the influence of duration of incubation after rehydration on the effects of chelating agents on sperm DNA fragmentation, spermatozoa in each group were incubated in Pig-FM at room temperature for 0, 60, 120 or 180 min after rehydration. The proportions of spermatozoa with DNA fragmentation were then examined.
Experiment 2: IVC of freeze-dried sperm head-injected oocytes
We examined the influences of time elapsed after rehydration and chelating agents on the in vitro developmental ability of oocytes injected with freeze-dried sperm heads from each group. Spermatozoa from each group were incubated in Pig-FM at room temperature for 0–60, 60–120 or 120–180 min and then injected into IVM oocytes. The oocytes were electrically stimulated 1 h after the injection, cultured in vitro for 6 days as described above and fixed. Three replicated trials, using a total of 55–134 oocytes, were carried out for each group.
Experiment 3: transfer of freeze-dried sperm head-injected oocytes
To evaluate the in vivo developmental ability of oocytes injected with freeze-dried sperm heads, we transferred oocytes injected with a rehydrated sperm, which had been incubated in Pig-FM or 50 mM EGTA for 0–60 min, to the oviducts of two synchronized recipients for each group (84–124 oocytes per recipient). For the 50 mM EGTA group, we also transferred parthenogenetic oocytes with the sperm-injected oocytes to one of the recipients to increase the chance of pregnancy (King et al., Reference King, Dobrinsky, Zhu, Finlayson, Bosma, Harkness, Ritchie, Travers, McCorqquodale, Day, Dinnyes, De Sousa and Wilmut2002). Parthenogenetic embryos were generated by electrostimulation with a direct current pulse of 2.2 kV/cm for 30 µs and incubated in IVC–PyrLac–HEPES supplemented with 10 µg/ml cytochalasin B (Sigma) at 37 °C for 3 h. Estrus in the recipient non-pregnant gilts was synchronized by an injection of 1000 IU of eCG and, 72 h later, an injection of 500 IU of hCG, as described previously (Kikuchi et al., Reference Kikuchi, Kashiwazaki, Noguchi, Shimada, Takahashi, Hirabayashi, Shino, Ueda and Kaneko1999; Kashiwazaki et al., Reference Kashiwazaki, Kikuchi, Suzuki, Noguchi, Nagai, Kaneko and Shino2001; Nakai et al., Reference Nakai, Kashiwazaki, Takizawa, Hayashi, Nakatsukasa, Fuchimoto, Noguchi, Kaneko, Shino and Kikuchi2003). The sperm-injected or parthenogenetic oocytes were transported to the operating room at 37 °C. Three hours after stimulation, the oocytes were transferred to both oviducts of the estrus-synchronized recipient gilts, in which ovulation was confirmed. Pregnancy was diagnosed in the recipients by using an ultrasound pregnancy detector (Medeta System Ltd) at day 30 after oocyte transfer.
Results
Experiment 1
The proportions of spermatozoa with DNA fragmentation immediately after rehydration are shown in Fig. 2. The percentage of sperm with DNA fragmentation in the Pig-FM group was significantly higher (p < 0.05) than in fresh group, 50 mM EGTA, 50 mM EDTA and 10 mM EDTA groups. The fluctuations in the percentages of DNA-fragmented spermatozoa during the incubation after rehydration are shown in Fig. 3. The percentage of spermatozoa with DNA fragmentation in the Pig-FM group increased gradually with incubation time. On the other hand, the rates were not significantly different among other groups. The rate of DNA fragmentation in the Pig-FM group at 180 min (12.2%) was significantly higher (p < 0.05) than those in the other groups (0.7–4.1%).
Figure 2 Sperm DNA fragmentation after rehydration. Mean percentages ± SEM in TUNEL-positive boar freeze-dried sperm heads are presented. Different superscripts (a, b) indicate values that are significantly different (p < 0.05).
Figure 3 Sperm DNA fragmentation after incubation. After rehydration, spermatozoa from each group were incubated in Pig-FM at room temperature for 0, 60, 120 and 180 min. All samples were evaluated by TUNEL assay. Mean percentages ± SEM of TUNEL-positive boar freeze-dried sperm heads are presented. Different superscripts (a, b) indicate values that are significantly different (p < 0.05).
Experiment 2
The percentages of blastocysts in each group cultured in vitro after injection with the freeze-dried sperm heads incubated in Pig-FM for 0–60, 60–120 or 120–180 min are shown in Fig. 4a. At 120–180 min, the percentage in the Pig-FM group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA group and the 10 mM EDTA group (23.1% and 22.6%, respectively). The average numbers of cells in the blastocysts are shown in Fig. 4b. At 120–180 min, the number in the 10 mM EDTA group (17.8 cells) was significantly lower (p < 0.05) than that in the 50 mM EGTA group (33.1 cells). The 50 mM EGTA group had the highest number, regardless of the incubation time after rehydration.
Figure 4 Embryonic development in vitro to the blastocyst stage of oocytes injected with freeze-dried sperm heads. Oocytes were incubated in Pig-FM at room temperature for 0–60, 60–120 or 120–180 min after rehydration and were cultured in vitro for 6 days. Mean values ± SEM of (a) percentages of blastocysts and (b) mean cell numbers per blastocyst are presented. Different superscripts (a, b) are significantly different (p < 0.05).
Experiment 3
The results of transfer of the embryos generated after freeze-dried sperm head injection to the recipients are shown in Table 1. In the Pig-FM group, both recipients were judged as being pregnant on day 29 after transfer. One of the pregnant recipients miscarried two fetuses on day 39 after the transfer. One seemed to have normal fetal development before the miscarriage (Fig. 5). In the 50 mM EGTA group, one recipient miscarried on day 29 after oocyte transfer (we confirmed that the placenta had been excreted). The other was not diagnosed as being pregnant on day 29.
Figure 5 Two fetuses were derived from ICSI of a freeze-dried boar sperm head in the Pig-FM group. A pregnant recipient miscarried on day 39 after oocyte transfer. The head of the upper fetus was separated from the body; this might have been caused by damage between the time of expulsion of the fetuses and their discovery. The separation does not seem to have occurred before the miscarriage.
Table 1 In vivo development of porcine oocytes after injection with freeze-dried sperm head
a Sperm was incubated for 0–60 min after rehydration.
b Pregnancy was diagnosed on day 30 after the oocytes transfer.
c Parthenogenetic oocytes were also transferred to increase the chance of pregnancy.
Discussion
Intactness of sperm DNA is important for embryonic development. Fatehi et al. (Reference Fatehi, Bevers, Schoevers, Roelen, Colenbrander and Gadella2006) suggested that DNA fragmentation in spermatozoa reduces the rate of blastocyst formation of ICSI oocytes in humans and that structural integrity of sperm DNA is important for embryonic development. Our study suggested, in pigs, that DNA fragmentation decreases ability to develop in vitro to the blastocyst stage. This was emphasized by the fact that the rate of blastocyst formation from the use of longer incubated (120–180 min) sperm in the Pig-FM group, whose rate of DNA fragmentation was significantly higher than those of the other groups, was lower than in the other groups. It also suggested that the level of DNA fragmentation that occurred in spermatozoa during freeze-drying treatment depended on the solution used for treatment, because fragmentation was not observed in fresh ejaculated sperm (Fig. 2). Sperm DNA fragmentation is caused by the action of endonucleases (Kusakabe et al., Reference Kusakabe, Szczygiel, Whittingham and Yanagimachi2001) or oxidative stress (Twigg et al., Reference Twigg, Irvine and Aitken1998). The endonucleases are released from plasma membrane-damaged spermatozoa after freeze-drying or freezing procedures (Kusakabe et al., Reference Kusakabe, Szczygiel, Whittingham and Yanagimachi2001). Therefore, we examined the influence on sperm DNA fragmentation of supplementation with two kinds of chelating agents in freeze-drying buffers. The percentage of spermatozoa with DNA fragmentation in the Pig-FM group was significantly higher than in the 50 mM EGTA, 50 mM EDTA, 10 mM EDTA or fresh groups (Fig. 2). These results show that even the lack of divalent cation in a freeze-drying medium is effective to prevent the increase of sperm DNA fragmentation during freeze-drying process and incubation after rehydration and the addition of chelating agents makes it more stable.
When sperm diluent was not included divalent cation by adding with EGTA or EDTA, the proportions of spermatozoa with DNA fragmentation did not increase during incubation after rehydration. There may be explained that most of the active endonucleases has been released from spermatozoa just after sperm rehydration and removed by replacement of sperm diluent. However, considering the results that the DNA fragmentation increased in Pig-FM group (Fig. 3), the active endonucleases may exist or released even after centrifugation and resuspension with Pig-FM. It may be possible that chelating agents remain on the surface of the sperm after replacement of sperm diluent with Pig-FM, resulting in the relatively lower level of the sperm DNA fragmentation after rehydration and incubation. It is also possible that, when whole or part of the sperm membrane is damaged, agents that do not have membrane permeability – such as the agents used in here – can enter the sperm through the damaged area, thus preventing fragmentation of the sperm DNA. On the other hand, the proportion of sperm with DNA fragmentation gradually increased with time in the Pig-FM group, because no agent was included. However, in the non-chelated group, which did not have a chelating agent either, the proportion of sperm with DNA fragmentation did not increase. Although this phenomenon is not easily explained, the reason could be as follows. We added sorbitol to this diluent for non-chelated group to adjust the osmotic pressure. Sorbitol is used as a cryoprotectant for human sperm and protects the sperm membrane (Alvarez & Storey, Reference Alvarez and Storey1993). As mentioned above, endonucleases are released from sperm with damaged membranes (Sotolongo et al., Reference Sotolongo, Huange, Isenberger and Ward2005) and easily injure the DNA (Ward et al., Reference Ward, Kaneko, Kusakabe, Biggers, Whittingham and Yanagimachi2003). The concentration of sorbitol in non-chelating medium may be low to act as an effective cryoprotectant. However, these facts suggest that sperm DNA fragmentation might have been inhibited by supplementation with sorbitol in the non-chelated group because the integrity of the sperm membrane was maintained during freeze-drying. Another hypothesis about the effect of sorbitol is still open for discussion.
We evaluated chelating agents and treatments for their suitability in freeze-drying of porcine spermatozoa. The percentages of sperm with DNA fragmentation in the 50 mM EDTA and 10 mM EDTA groups were almost the same as in the 50 mM EGTA group (Fig. 3). These results suggest that EDTA and EGTA are equally effective in inhibiting endonucleases. However, the rates of blastocyst formation and the mean number of cells per blastocyst in the 50 mM EDTA and 10 mM EDTA groups were lower than those in the 50 mM EGTA group. The explanation for this discrepancy has not yet been clarified; however, there seems to be a possibility that a different mechanism participates in the selection of ions, depending on the agent. Divalent cations such as Ca2+ or Mg2+ play an important part in the control of metabolism in cells (Rubin, Reference Rubin1975). Considering the possibility that EGTA or EDTA remains on the surface of the sperm even after washing and replacement of sperm diluent with Pig-FM, an agent or agents may enter the ooplasm in association with the sperm head. EDTA chelates both ions easily; in contrast, EGTA chelates only Ca2+ (Sanui & Pace, Reference Sanui and Pace1967; Azuma et al., Reference Azuma, Kondo, Ikeda, Imai and Yamada2002). An imbalance of these ions in sperm-injected oocytes may cause the discrepancy between the DNA fragmentation and developmental effects of the two agents. The influences of divalent cations on the metabolism and developmental ability of embryos should be further investigated. EGTA is a suitable chelating agent for adding to freeze-drying buffers for boar sperm, as reported in a previous study in mice (Kusakabe et al., Reference Kusakabe, Szczygiel, Whittingham and Yanagimachi2001).
In the present study, although the difference in the rate of sperm with DNA fragmentation was small (Fig. 3), there was the large difference in the rate of blastocyst between Pig-FM and 50 mM EGTA groups in 120–180 min (Fig. 4a). The explanation for this discrepancy has not yet been completed, but the damage on the DNA at the time of ICSI may be resulted in the enhanced decrease in the developmental rates after the culture. Recently, it is reported, in human, that sperm DNA fragmentation relates ‘late’ (such as development to the blastocyst stage), but not ‘early’ (such as abnormality at the zygote and early cleavage stages), paternal effect on embryo development (Tesarik et al., Reference Tesarik, Greco and Mendoza2004). Some paternal factors, except for sperm DNA fragmentation, such as abnormal chromatin packaging (Acharyya et al., Reference Acharyya, Kanjilal and Bhattacharyya2005) or protamine deficiency (Nasr-Esfahani et al., Reference Nasr-Esfahani, Salehi, Razavi, Anjomshoa, Rozbahani, Moulavi and Mardani2005) and sperm aneuploidy (Petit et al., Reference Petit, Frydman, Benkhalifa, Aboura, Fanchin, Frydman and Tachdjian2005) have also been suggested. The further studies should be needed to clarify for participation of many other factors in developmental ability of embryos.
In the embryo transfer experiment, we obtained two fetuses of 39 day in the 0–60 min Pig-FM group, but in the 50 mM EGTA group one recipient aborted on day 29. Kure-bayashi et al. (Reference Kure-bayashi, Miyake, Okabe and Kato2000) has already confirmed that parthenogenetic porcine diploid embryos have the ability to develop up to day 29 after transfer. Our results suggest that the aborted fetuses in the Pig-FM group might have developed from fertilization by ICSI, but that the abortion in the 50 mM EGTA group might have been caused by implantation of only the parthenogenetic embryos. However, in this group, the origin of the embryos (oocytes after ICSI or oocytes activated parthenogenetically) could not be confirmed from our results. These facts suggest that a short period (0–60 min) of incubation of boar sperm after freeze-drying, even without a chelating agent, enables fertilization and in vivo development to fetuses. After incubation for 0–120 min, there were no significant differences in DNA fragmentation and blastocyst development between Pig-FM and 50 mM EGTA groups; this might have been the reason behind our result that fertilization and fetal development to day 39 were obtained by chance only in the Pig-FM group. However, more careful attention needs to be paid to the effect of chelating agents on fetal development after embryo transfer.
In conclusion, our results suggest that fragmentation of DNA in freeze-dried sperm decreases the developmental ability of injected oocytes. EGTA would be suitable chelating for addition to freeze-drying buffer. Furthermore, our results clearly suggest that oocytes injected with freeze-dried sperm heads have the competence to grow to day 39 after oocyte transfer. However, there is still a problem in obtaining viable offspring from freeze-dried boar spermatozoa.
Acknowledgements
The authors would like to thank Dr J. Kurisaki, Dr A. Onishi, Dr M. Iwamoto, Dr M. Fahrudin, Dr N.W.K. Karja and Dr T. Somfai for critical discussion on the present study and also Ms T. Aoki and Ms C. Terui for technical assistance.
