Application Study of Intracytoplasmic Sperm Injection for Golden Hamster and Cattle Production

Abstract

Abstract. This paper describes several technical improvements and our results in hamster intracytoplasmic sperm injection (ICSI), hamster round spermatid injection (ROSI) and bovine ICSI. The hamster is the mammalian species in which ICSI was first tried to produce fertilized oocytes. However, until recently, no live offspring following ICSI have ever been obtained. We reported the birth of live offspring following hamster ICSI. Improved points to success were 1) performing hamster ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under microscope with a red light source and 2) injecting sperm heads without acrosomes. Under controlled illumination, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally, cleaved, and developed into morulae. Nine live offspring (19%) were born by transfer of hamster ICSI-derived embryos. Furthermore, we reported the birth of live offspring following hamster ROSI. About 70% of oocytes injected with round spermatids broken before injection were fertilized normally and about half of them developed to morulae and blastocysts. Three (5%) live young were born by transfer of hamster ROSI-derived embryos. On the other hand, in cattle, the main improvements were 1) injection of spermatozoa immobilized by scoring their tail just before injection into oocytes, and 2) additional ethanol activation 4 h after ICSI. About 70% of oocytes injected were activated 4 h after ICSI, and about 30% of them developed to blastocysts. Twenty-four live calves (39%) were born by non-surgical transfer of ICSI-derived embryos. Those results shows that, at present, live offspring are able to be obtained following hamster ICSI, ROSI and bovine ICSI, but further improvement is required due to higher production efficiency of offspring.

Intracytoplasmic Sperm Injection in Cattle

“…Oocyte activation with ethanol alone or in combination with cycloheximide [112,113] or ionomycin [92,114] following ICSI resulted in development of blastocysts. Piezo-driven ICSI more efficient than conventional ICSI [81], and resulted in production of bovine offspring [115,116] when combined with ethanol oocyte activation. Viable calves were efficiently produced by post-ICSI oocyte activation with ethanol, compared to activation with ionomycin alone or a combination of ionomycin + DMAP [117].…”

<b>Assisted Reproductive Technologies in Cattle: Applications in Livestock Production, Biomedical Research and Conservation Biology</b>

“…Although laser-assisted ICSI in which part of the ZP is removed to introduce the needle has been carried out in humans and other mammals, no reports in cattle were found in the present work. Despite several works reporting production of calves with this technique, its effectiveness remains unsatisfactory for commercial application (Horiuchi & Numabe, 1999;Horiuchi, 2006). The main underlying cause of the reduced efficiency of ICSI is the lack of protocols able to induce proper oocyte activation and decondensation of the sperm nucleus (Horiuchi & Numabe, 1999).…”

Formación de pronúcleos mediante la inyección intracitoplasmática de espermatozoides (ICSI) seleccionados por unión a la zona pelúcida en ovocitos de ovino activados químicamente

“…En ovocitos de ovino se ha aplicado EtOH al 7% por 7 minutos (Loi et al, 1998), o la combinación de este tratamiento con 6-DMAP 1.9 mM por 3 horas (Loi et al, 1998), así como EtOH al 7% por 4 minutos después de haberse aplicado Ionomicina (Io) 5 µM durante 4 minutos (Pereyra-Bonnet et al, 2011). En bovinos se ha investigado la aplicación de EtOH al 7% por 5 min, aplicándose 1, 2 o 4 horas después de la ICSI (Emuta y Oriuchi, 2001;Fujinami et al, 2004;Oikawa et al, 2005;Horiuchi, 2006); se ha aplicado este tratamiento después de tratar a los ovocitos con…”