Bovine blastocyst development from oocytes injected with freeze-dried spermatozoa.
Pronuclear formation, and the chromosomal constitution and developmental capacity of bovine zygotes formed by intracytoplasmic sperm injection with freeze-dried (lyophilized) spermatozoa were evaluated. Frozen-thawed spermatozoa were selected, freeze-dried, and stored at 4 degrees C until use. After 22-24 h of in vitro maturation oocytes were denuded and injected singly with a lyophilized spermatozoon. Injected oocytes were activated by treatment with 10 microM ionomycin (5 min) alone and in combination with 1.9 mM 6-dimethylaminopurine (DMAP) for 4 h. Ionomycin plus DMAP activation treatment resulted in a significantly higher proportion of sperm-injected oocytes with two pronuclei than was found after activation with ionomycin alone (74% vs. 56%; P < 0.03). The rates of cleavage, morula, and blastocyst development of sperm-injected oocytes treated with ionomycin plus DMAP were higher than after activation with ionomycin alone (63.3%, 34.2%, and 29.6% vs. 44.7%, 18.7%, and 10.6%, respectively; P < 0.05). Seventy-three percent of blastocysts produced with lyophilized sperm were diploid. These results demonstrate that in vitro-matured bovine oocytes can be fertilized with freeze-dried sperm cells, and that resultant zygotes can develop into karyotypically normal blastocysts.
Transgenic and Embryonic Stem Cell Core, IMMAG, Medical College of Georgia, Augusta, GA 30912, USA. lkeskintepe@sherinstitute.com
Levent
L
Keskintepe
Gabriela
G
Pacholczyk
Anna
A
Machnicka
Karen
K
Norris
M Akif
MA
Curuk
Iqbal
I
Khan
Benjamin G
BG
Brackett
D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D001755:Blastocyst / Q000502:physiology*; D002417:Cattle; D003470:Culture Media; D003584:Cytological Techniques; D005314:Embryonic and Fetal Development / Q000502:physiology; D005260:Female; D005612:Freeze Drying; D066298:In Vitro Techniques; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007621:Karyotyping; D008297:Male; D008845:Microinjections; D009028:Morula / Q000502:physiology; D009865:Oocytes / Q000502:physiology*; D011247:Pregnancy; D012732:Sex Determination Analysis; D020554:Sperm Injections, Intracytoplasmic; D013094:Spermatozoa / Q000502:physiology*
D003470:Culture Media; D007476:Ionophores; D015759:Ionomycin; C001020:N(6),N(6)-dimethyladenine; D000225:Adenine