Search tips
Search criteria 


Logo of jargspringer.comThis journalToc AlertsSubmit OnlineOpen Choice
J Assist Reprod Genet. 2009 October; 26(9-10): 531–536.
Published online 2009 October 24. doi:  10.1007/s10815-009-9358-y
PMCID: PMC2788692

Pronuclear formation of freeze-dried canine spermatozoa microinjected into mouse oocytes



The aim of the present study was to investigate the fertilizing capacity of fresh, frozen-thawed and freeze-dried canine spermatozoa.


After canine spermatozoa were injected into mouse oocytes, the rates of oocyte activation, male pronuclear formation and chromosomal aberrations were investigated.


The rates of oocyte activation were comparable (90.6–100%), no matter the sperm type injected. The percentage of male pronuclear formation was higher (P < 0.001) in the freeze-dried spermatozoa (92.3%) than the fresh (61.5%) and frozen-thawed (69.2%) spermatozoa. However, the chromosomal damage in the oocytes injected with freeze-dried spermatozoa was higher (72.9%: P < 0.001) than with fresh (26.9%) and frozen-thawed (21.4%) spermatozoa.


These data indicate using mouse oocytes that freeze-dried canine spermatozoa may potentially fertilize canine oocytes although chromosomal damage is frequently generated.

Keywords: Dog, Chromosome, Freeze-drying, Intracytoplasmic sperm injection, Spermatozoa


Cryopreservation of spermatozoa has enabled the cost effective preservation and long-distance transportation of genetic resources. As one of the cryopreservation methods in mammalian spermatozoa, freeze-drying has been applied in mouse [18], rat [9, 10], rabbit [11], bull [12], boar [13, 14] and human [15] spermatozoa. An advantage in freeze-drying is to store the spermatozoa without the use of liquid nitrogen for a long period. However, freeze-dried spermatozoon must be microinjected into an oocyte to be fertilized, because it is no longer motile. Since the chromosomal integrity of these spermatozoa is impaired [7], methodological optimization of freeze-drying is still an essential issue for successful sperm preservation [1517].

Canine species are bred worldwide as companion animals and working dogs, including guide dogs for the blind. Assisted Reproductive Technologies (ARTs) in canines, e.g. artificial insemination [18, 19], in vitro fertilization [20, 21] and somatic cell nuclear transfer [22, 23], has been steadily progressing. ARTs seems to have led to the more efficient production of capable working dogs, and may be further advanced by successful preservation of canine spermatozoa. However, to the best of our knowledge, canine spermatozoa have never been freeze-dried. Activation of oocytes by sperm, subsequent formation of male pronucleus (MPN) and their chromosomal integrity can be considered as indicators for fertilizing ability of spermatozoa. Furthermore, these indicators can be monitored by microinjection into mouse oocytes in mouse [24, 25], whale [26] and human [27, 28] spermatozoa.

We report here that freeze-dried canine spermatozoa microinjected into mouse oocytes were able to activate oocytes and form MPN.

Materials and methods

Reagents and media

All chemicals were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) unless specifically stated. The culture medium for mouse oocytes after intracytoplasmic sperm injection (ICSI) was Chatot-Ziomek-Bavister (CZB) [29] supplemented with 5.56 mM D-glucose and 4 mg/ml bovine serum albumin (fraction V; Sigma-Aldrich, St. Louis, MO). Mouse oocyte collection and microinjection were performed in a modified CZB supplemented with 20 mM Hepes-Na, 5 mM NaHCO3, and 0.1 mg/ml polyvinyl alcohol (cold water soluble; Sigma-Aldrich) in place of bovine serum albumin (H-CZB). Sperm preparation was performed in modified Toyoda-Yokoyama-Hoshi (TYH) medium [30] supplemented with 20 mM Hepes, 5 mM NaHCO3, and 0.1 mg/ml polyvinyl alcohol in place of bovine serum albumin (H-TYH). The pH value of both H-CZB and H-TYH was adjusted to approximately 7.4.


(C57BL/6 J × DBA/2)F1 (B6D2F1) mice (CLEA Japan, Inc., Tokyo, Japan) were used to collect the oocytes. Also, spermatozoa were recovered from a Labrador Retriever in our colony. Animal care and all experiments were performed according to the Guiding Principles for the Care and Use of Research Animals of Obihiro University of Agriculture and Veterinary Medicine.

Preparation of oocytes and spermatozoa for ICSI

B6D2F1 female mice, 7–11 weeks of age, were superovulated by i.p. injection of 10 IU equine chorionic gonadotrophin (Asuka Pharmaceutical, Tokyo, Japan) followed by injection of 10 IU human chorionic gonadotrophin (Asuka Pharmaceutical) 48 h later. The oocytes recovered from the oviducts between 14 and 16 h after human chorionic gonadotrophin injection were denuded of their cumulus cells by treatment with 0.1% (w/v) bovine testicular hyaluronidase (Sigma-Aldrich) in H-CZB. The denuded oocytes were repeatedly rinsed in CZB medium and kept at 37°C under 5% CO2 in the same medium until ICSI.

Sperm collection, freezing and freeze-drying procedures

The ejaculated spermatozoa were collected by digital manipulation [31] into a sterile tube (Corning, NY, USA). The first and third fractions (seminal plasma) of the ejaculates were discarded. The sperm-rich second fraction of the ejaculates was used for the experiments. One each of the ejaculates was subjected to a cryopreservation, freezing or freeze-drying, as described below.

Spermatozoa were frozen by using the commercially available semen extender, AndroMed (Minitüb, Tiefenbach, Germany), following the supplier recommended methods. Briefly, the semen was diluted with AndroMed and cooled to 4°C for 3 h. Then, the sperm suspension was loaded into a 0.25 ml straw, and kept in an atmosphere of liquid nitrogen vapor, i.e. placed horizontally 6 cm above the surface of liquid nitrogen in a closed styrene foam box (21.0 cm × 13.0 cm × 13.0 cm), for 15 min. The straws were plunged into liquid nitrogen and stored up to 3 months. When thawing, the straws were immersed into water bath at 37°C and immediately used for ICSI. Percentage of motile spermatozoa after thawing was 46.2%.

Freeze-drying was performed as reported by Kawase et al. [5]. Briefly, the spermatozoa were placed in EGTA Tris-HCl-buffered solution (50 mM EGTA, 50 mM NaCl and 10 mM Tris-HCl, pH 8.0) [17] and kept at 37°C for 10 min. The sperm suspensions (250 μl) were transferred into the amber vacuum vial for freeze-drying (V-2B; Nichiden-rika Glass, Kobe, Japan). The vials were plunged into liquid nitrogen for 5 min and then transferred to a programmable freeze-dryer (BETA2–16; Martin Christ Gefriertrocknungsanlagen, Osterode am Harz, Germany) that had been precooled to −30°C. The freeze-drying conditions consisted of primary drying at a pressure of 0.37 mbar and secondary drying at a pressure of 0.001 mbar. Since the freeze-dried spermatozoa can be kept indefinitely at −80°C or below [4], the vials were stored at −80°C for up to 1 month. The freeze-dried spermatozoa were rehydrated by adding 250 μl of milli-Q water, and immediately used for ICSI.

ICSI procedure

Before injection, a batch of 15 oocytes was transferred into a droplet (5 μl) of H-CZB, which had been prepared beside a sperm-containing droplet in the ICSI chamber covered with paraffin oil (Merck Japan, Tokyo, Japan). A spermatozoon was aspirated into the injection pipette tail first in H-TYH containing 10–12% polyvinyl pyrrolidone (molecular weight: 360000; Nacalai Tesque, Kyoto, Japan), and the tail was cut at the mid-piece by applying a few piezopulses. The tail-cut spermatozoon was individually injected into a mouse oocyte according to the method of Kimura and Yanagimachi [32]. The ICSI series of experiments was finished within 1 h of sperm preparation. Thereafter, the injected oocytes were washed with CZB and transferred into a droplet (30 μl) of the same medium covered with paraffin oil at 37°C under 5% CO2 in air for cultivation.

Chromosome preparation of one-cell oocytes

Six hours after ICSI, the rate of normal morphological oocytes was observed. The deformed oocytes were discarded and morphologically normal oocytes (Fig. 1) were transferred to CZB containing 0.02 μg/ml vinblastine sulfate to inhibit the first cleavage division. At 19–21 h after ICSI, they were treated with 0.5% protease (Kaken Pharmaceuticals, Tokyo, Japan) in Ca2+− and Mg2+−free Dulbecco’s phosphate buffered saline to digest the zona pellucida. Then, they were maintained in hypotonic solution consisting of equal volumes of 1% (w/v) sodium citrate and 30% (v/v) fetal calf serum (Gibco-BRL, Grand Island, NY) for 10 min at room temperature. These samples were prepared by the gradual-fixation/air drying method [33]. The slides were conventionally stained with 2% Giemsa (Merck) in buffered saline (pH 6.8) for 10 min.

Fig. 1
Mouse oocytes injected with frozen-thawed canine spermatozoa at 6 h after ICSI. Arrowheads indicate morphologically normal oocytes. Only morphologically normal oocytes are examined to evaluate the fertilizing capacity of canine spermatozoa. Bar = 50 μm ...

Statistical analysis

All experiments were repeated three to five times. The chi-square test or Fisher’s exact probability test was used for analyses. Differences were considered significant when the P value was less than 0.05.


Freeze-dried spermatozoa are represented in Fig. 2a. The results of oocyte activation, MPN formation and chromosomal integrity are summarized in Table 1. When fresh, frozen-thawed and freeze-dried spermatozoa were injected into mouse oocytes, the proportions of morphologically normal oocytes after 6 h of ICSI significantly changed (P < 0.001) to 29.6, 59.1 and 100% of oocytes, respectively. Almost all oocytes (90.6–100%) were activated successfully, no matter which of the three sperm types was injected. On the other hand, the rate of MPN formation was significantly higher (P < 0.001) in the freeze-dried spermatozoa (92.3%; Fig. 2b) than the fresh (61.5%) and frozen-thawed (69.2%) spermatozoa. The chromosomal integrity was analyzable in a range of 86.4 to 93.4% (Fig. 3). Chromosomal damage was significantly increased (P < 0.001) in the freeze-dried spermatozoa compared with the fresh and frozen-thawed spermatozoa (Table 1).

Fig. 2
Freeze-dried spermatozoon a and the oocytes fertilized with freeze-dried spermatozoa b. Arrowheads indicate male or female pronucleus. Bar = 10 μm
Table 1
Fertilizing capacity of fresh, frozen-thawed and freeze-dried canine spermatozoa microinjected into mouse oocytes
Fig. 3
Chromosomes spread from freeze-dried canine spermatozoa injected into mouse oocytes. a Normal chromosome set. The haploid chromosome number of canine spermatozoa is 39. Minor b and multiple c chromosome aberrations. Arrows indicate the chromosomal or ...


It is well known that the fertilizing capacity of mammalian sperm can be assessed using rodent oocytes [3436]. Moreover, the spermatazoan chromosomes in a variety species can be easily visualized using these oocytes in combination with ICSI techniques [2528]. Therefore, the present study carried out an assessment of the fertilizing capacity of canine spermatozoa using mouse oocytes.

As shown in Table 1, freeze-dried canine spermatozoa were as capable of fertilization as fresh and frozen-thawed spermatozoa. When freeze-dried canine spermatozoa were injected into mouse oocytes, a majority of the nuclei transformed into the MPN. The value was obviously higher than in the other groups. A possible reason is that the plasma membrane of the freeze-dried spermatozoa was completely destroyed [7, 11, 15], which could lead to a more rapid contact between the sperm nuclei and the ooplasm.

Mouse oocytes are hypersensitive to the acrosome enzyme in spermatozoa: mouse oocytes injected with acrosome-intact bull and boar spermatozoa or acrosomal enzyme(s) undergo deformation and never reach the 2-cell stage [37]. In our preliminary experiments, paternal nuclei in all of the deformed oocytes at 19–21 h after ICSI entered arrest at the time of decondensation or pronuclear formation. They never reached the first mitotic stage (data not shown), suggesting that acrosomal contents in spermatozoa are disturbed the progress of fertilization. Therefore, the deformed oocytes were discarded and the remainder, morphologically normal oocytes, was utilized for the assessment of the fertilizing capacity (Fig. 1). Kimura et al. [24] demonstrated that sperm perinuclear material contained a necessary substance (sperm-borne oocyte activating factor: SOAF) to activate the oocytes, and that the SOAF activity could be analyzed using mouse oocytes. As shown in Table 1, it seems that the SOAF activity in canine spermatozoa was not species-specific, and was maintained even when the spermatazoa were freeze-dried. However, a portion of the mouse oocytes injected with freeze-dried and sonicated bull spermatozoa did exhibit an abnormal pattern of calcium oscillations [12]. For a conclusive assessment of the successful oocyte activation, the calcium oscillation pattern induced by freeze-dried canine spermatozoa remains a subject for future investigation.

As shown in Table 1, the chromosomal integrity in fresh canine spermatozoa was relatively low (73.3%) compared with mouse (91.6% [25]) and human (95.7% [28]) spermatozoa. In our preliminary experiment, when frozen-thawed canine spermatozoa were injected into the double-volume (artificially fused) mouse oocytes, the rates of morphologically normal oocytes and MPN formation improved to 87.5 and 91.8%, respectively. Since mouse oocytes are hypersensitive to acrosomal enzyme, the size (volume) of the cytoplasm of the mouse oocyte might be an important factor for an assessment of fertilizing capability in canine spermatozoa.

The freeze-drying procedure generated chromosomal aberrations in canine spermatozoa (Table 1). Since frozen-thawed spermatozoa did not increase chromosomal damage, one of the reasons for the increased damage is likely to be demembranization in freeze-dried spermatozoa. Sperm nuclei with a defective plasma membrane can easily come into contact with endonuclease, leading to the generation of chromosomal nicks. In the mouse, the chromosomes in 30–60% of the freeze-dried spermatozoa were impaired [3, 7, 15], while the percentage of chromosomally damaged canine spermatozoa was 72.9% in the present study. The chromosomal damage of freeze-dried mouse spermatozoa can be decreased by modifying the pH value of the storage media [17], and by pre-treatment with diamide [3] and EGTA [15]. Therefore, it is expected that optimized conditions will subsequently be established for successful freeze-drying of canine spermatozoa.



Canine spermatozoa can be freeze-dried with oocyte activating and male pronuclear forming capacity.


1. Wakayama T, Yanagimachi R. Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nat Biotechnol. 1998;16:639–41. [PubMed]
2. Kaneko T, Nakagata N. Relation between storage temperature and fertilizing ability of freeze-dried mouse spermatozoa. Comp Med. 2005;55:140–4. [PubMed]
3. Kaneko T, Whittingham DG, Overstreet JW, Yanagimachi R. Tolerance of the mouse sperm nuclei to freeze-drying depends on their disulfide status. Biol Reprod. 2003;69:1859–62. [PubMed]
4. Kawase Y, Araya H, Kamada N, Jishage K, Suzuki H. Possibility of long-term preservation of freeze-dried mouse spermatozoa. Biol Reprod. 2005;72:568–73. [PubMed]
5. Kawase Y, Tachibe T, Jishage K, Suzuki H. Transportation of freeze-dried mouse spermatozoa under different preservation conditions. J Reprod Dev. 2007;53:1169–74. [PubMed]
6. Kawase Y, Hani T, Kamada N, Jishage K, Suzuki H. Effect of pressure at primary drying of freeze-drying mouse sperm reproduction ability and preservation potential. Reproduction. 2007;133:841–6. [PubMed]
7. Kusakabe H, Szczygiel MA, Whittingham DG, Yanagimachi R. Maintenance of genetic integrity in frozen and freeze-dried mouse spermatozoa. Proc Natl Acad Sci U S A. 2001;98:13501–6. [PubMed]
8. Ward MA, Kaneko T, Kusakabe H, Biggers JD, Whittingham DG, Yanagimachi R. Long-term preservation of mouse spermatozoa after freeze-drying and freezing without cryoprotection. Biol Reprod. 2003;69:2100–8. [PubMed]
9. Kaneko T, Kimura S, Nakagata N. Offspring derived from oocytes injected with rat sperm, frozen or freeze-dried without cryoprotection. Theriogenology. 2007;68:1017–21. [PubMed]
10. Hochi S, Watanabe K, Kato M, Hirabayashi M. Live rats resulting from injection of oocytes with spermatozoa freeze-dried and stored for one year. Mol Reprod Dev. 2008;75:890–4. [PubMed]
11. Liu JL, Kusakabe H, Chang CC, Suzuki H, Schmidt DW, Julian M, et al. Freeze-dried sperm fertilization leads to full-term development in rabbits. Biol Reprod. 2004;70:1776–81. [PubMed]
12. Abdalla H, Hirabayashi M, Hochi S. The ability of freeze-dried bull spermatozoa to induce calcium oscillations and resumption of meiosis. Theriogenology. 2009;71:543–52. [PubMed]
13. Kwon IK, Park KE, Niwa K. Activation, pronuclear formation, and development in vitro of pig oocytes following intracytoplasmic injection of freeze-dried spermatozoa. Biol Reprod. 2004;71:1430–6. [PubMed]
14. Nakai M, Kashiwazaki N, Takizawa A, Maedomari N, Ozawa M, Noguchi J, et al. Effects of chelating agents during freeze-drying of boar spermatozoa on DNA fragmentation and on developmental ability in vitro and in vivo after intracytoplasmic sperm head injection. Zygote. 2007;15:15–24. [PubMed]
15. Kusakabe H, Yanagimachi R, Kamiguchi Y. Mouse and human spermatozoa can be freeze-dried without damaging their chromosomes. Hum Reprod. 2008;23:233–9. [PubMed]
16. Kaneko T, Nakagata N. Improvement in the long-term stability of freeze-dried mouse spermatozoa by adding of a chelating agent. Cryobiology. 2006;53:279–82. [PubMed]
17. Kaneko T, Whittingham DG, Yanagimachi R. Effect of pH value of freeze-drying solution on the chromosome integrity and developmental ability of mouse spermatozoa. Biol Reprod. 2003;68:136–9. [PubMed]
18. Abe Y, Lee DS, Sano H, Akiyama K, Yanagimoto-Ueta Y, Asano T, et al. Artificial insemination with canine spermatozoa frozen in a skim milk/glucose-based extender. J Reprod Dev. 2008;54:290–4. [PubMed]
19. Suwa Y, Abe Y, Lee DS, Yanagimoto-Ueta Y, Suzuki H: Individual fertility differences in the frozen-thawed spermatozoa among semen donors in the Labrador Retriever. Reprod Med Biol. 2009;8(3):125–29
20. Otoi T, Shin T, Kraemer DC, Westhusin ME. Influence of maturation culture period on the development of canine oocytes after in vitro maturation and fertilization. Reprod Nutr Dev. 2004;44:631–7. [PubMed]
21. Rodrigues Bde A, dos Santos LC, Rodrigues JL. Embryonic development of in vitro matured and in vitro fertilized dog oocytes. Mol Reprod Dev. 2004;67:215–23. [PubMed]
22. Lee BC, Kim MK, Jang G, Oh HJ, Yuda F, Kim HJ, et al. Dogs cloned from adult somatic cells. Nature. 2005;436:641. [PubMed]
23. Kim S, Park SW, Hossein MS, Jeong YW, Kim JJ, Lee E, et al. Production of cloned dogs by decreasing the interval between fusion and activation during somatic cell nuclear transfer. Mol Reprod Dev. 2009;76:483–9. [PubMed]
24. Kimura Y, Yanagimachi R, Kuretake S, Bortkiewicz H, Perry AC, Yanagimachi H. Analysis of mouse oocyte activation suggests the involvement of sperm perinuclear material. Biol Reprod. 1998;58:1407–15. [PubMed]
25. Tateno H, Kamiguchi Y. Evaluation of chromosomal risk following intracytoplasmic sperm injection in the mouse. Biol Reprod. 2007;77:336–42. [PubMed]
26. Watanabe H, Tateno H, Kusakabe H, Matsuoka T, Kamiguchi Y, Fujise Y, et al. Fertilizability and chromosomal integrity of frozen-thawed Bryde's whale (Balaenoptera edeni) spermatozoa intracytoplasmically injected into mouse oocytes. Zygote. 2007;15:9–14. [PubMed]
27. Rybouchkin AV, De Sutter P, Dhont M. Unprotected freezing of human spermatozoa exerts a detrimental effect on their oocyte activating capacity and chromosome integrity. Zygote. 1996;4:263–8. [PubMed]
28. Watanabe S. Frequent structural chromosome aberrations in immotile human sperm exposed to culture media. Hum Reprod. 2004;19:940–7. [PubMed]
29. Chatot CL, Ziomek CA, Bavister BD, Lewis JL, Torres I. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil. 1989;86:679–88. [PubMed]
30. Toyoda Y, Yokoyama M, Hoshi T. Studies on the fertilization of mouse eggs in vitro: I. In vitro fertilization of eggs by fresh epididymal sperm (in Japanese). Jpn J Anim Reprod. 1971;16:147–51.
31. Johnston SD, Root Kustritz MV, Olson PN. Semen collection, evaluation, and preservation. In: Kersey R, LeMelledo D, editors. Canine and feline theriogenology. Philadelphia: Saunders; 2001. p. 287–306.
32. Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod. 1995;52:709–20. [PubMed]
33. Mikamo K, Kamiguchi Y. A new assessment system for chromosomal mutagenicity using oocytes and early zygotes of the Chinese hamster. In: Ishihara T, Sasaki MS, editors. Radiation-Induced Chromosome Damage in Man. New York: Alan R. Liss; 1983. p. 411–32.
34. Yanagimachi R, Yanagimachi H, Rogers BJ. The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Biol Reprod. 1976;15:471–6. [PubMed]
35. Lee JD, Kamiguchi Y, Yanagimachi R. Analysis of chromosome constitution of human spermatozoa with normal and aberrant head morphologies after injection into mouse oocytes. Hum Reprod. 1996;11:1942–6. [PubMed]
36. Rybouchkin A, Dozortsev D, Pelinck MJ, De Sutter P, Dhont M. Analysis of the oocyte activating capacity and chromosomal complement of round-headed human spermatozoa by their injection into mouse oocytes. Hum Reprod. 1996;11:2170–5. [PubMed]
37. Morozumi K, Yanagimachi R. Incorporation of the acrosome into the oocyte during intracytoplasmic sperm injection could be potentially hazardous to embryo development. Proc Natl Acad Sci U S A. 2005;102:14209–14. [PubMed]

Articles from Journal of Assisted Reproduction and Genetics are provided here courtesy of Springer Science+Business Media, LLC