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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

Published online by Cambridge University Press:  01 February 2007

M. Nakai ,
N. Kashiwazaki ,
A. Takizawa ,
N. Maedomari ,
M. Ozawa ,
J. Noguchi ,
H. Kaneko ,
M. Shino  and
K. Kikuchi
M. Nakai
Affiliation:
Division of Animal Sciences, National Institute of Agro-biological Sciences, Tsukuba, Ibaraki 305–8602, Japan. Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229–8501, Japan.
N. Kashiwazaki
Affiliation:
Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229–8501, Japan.
A. Takizawa
Affiliation:
Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229–8501, Japan.
N. Maedomari
Affiliation:
Division of Animal Sciences, National Institute of Agro-biological Sciences, Tsukuba, Ibaraki 305–8602, Japan. Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229–8501, Japan.
M. Ozawa
Affiliation:
Division of Animal Sciences, National Institute of Agro-biological Sciences, Tsukuba, Ibaraki 305–8602, Japan.
J. Noguchi
Affiliation:
Division of Animal Sciences, National Institute of Agro-biological Sciences, Tsukuba, Ibaraki 305–8602, Japan.
H. Kaneko
Affiliation:
Division of Animal Sciences, National Institute of Agro-biological Sciences, Tsukuba, Ibaraki 305–8602, Japan.
M. Shino
Affiliation:
Laboratory of Animal Reproduction, School of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa 229–8501, Japan.
K. Kikuchi*
Affiliation:
Division of Animal Sciences, National Institute of Agro-biological Sciences, Tsukuba, Ibaraki 305–8602, Japan.
*
All correspondence to: K. Kikuchi, Division of Animal Sciences, Reproductive Biology Research Unit (Kannondai), Kannondai 2–1-2, Tsukuba, Ibaraki 305–8602, Japan, Tel: +81 298 38 7447. Fax: +81 298 38 7408. e-mail: kiku@nias.affrc.go.jp
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Summary

Successful offspring production after intracytoplasmic injection of freeze-dried sperm has been reported in laboratory animals but not in domesticated livestock, including pigs. The integrity of the DNA in the freeze-dried sperm is reported to affect embryogenesis. Release of endonucleases from the sperm is one of the causes of induction of sperm DNA fragmentation. We examined the effects of chelating agents, which inhibit the activation of such enzymes, on DNA fragmentation in freeze-dried sperm and on the in vitro and in vivo developmental ability of porcine oocytes following boar sperm head injection. Boar ejaculated sperm were sonicated, suspended in buffer supplemented with (1) 50 mM EGTA, (2) 50 mM EDTA, (3) 10 mM EDTA, or (4) no chelating agent and freeze-dried. A fertilization medium (Pig-FM) was used as a control. The rehydrated spermatozoa in each group were then incubated in Pig-FM at room temperature. The rate of DNA fragmentation in the control group, as assessed by the TUNEL method, increased gradually as time after rehydration elapsed (2.8% at 0 min to 12.2% at 180 min). However, the rates in all experimental groups (1–4) did not increase, even at 180 min (0.7–4.1%), which were all significantly lower (p < 0.05) than that of the control group. The rate of blastocyst formation after the injection in the control group (6.0%) was significantly lower (p < 0.05) than those in the 50 mM EGTA (23.1%) and 10 mM EDTA (22.6%) groups incubated for 120–180 min. The average number of blastocyst cells in the 50 mM EGTA group (33.1 cells) was significantly higher (p < 0.05) than that in the 10 mM EDTA group (17.8 cells). Finally, we transferred oocytes from 50 mM EGTA or control groups incubated for 0–60 min into estrous-synchronized recipients. The two recipients of the control oocytes became pregnant and one miscarried two fetuses on day 39.

The results suggested that fragmentation of DNA in freeze-dried boar sperm is one of the causes of decreased in vitro developmental ability of injected oocytes to the blastocyst stage. Supplementation with EGTA in a freeze-drying buffer improves this ability.

Keywords

DNA fragmentationEndonucleaseFreeze-dryICSIPig
Type
Research Article
Information
Zygote , Volume 15 , Issue 1 , February 2007 , pp. 15 - 24
Copyright
Copyright © Cambridge University Press 2007

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