Intracytoplasmic Sperm Injection Using Cryopreserved, Fixed, and Freeze-Dried Sperm in Eggs of Nile Tilapia Germa ´n A. Poleo, 1 * Robert A. Godke, 2 Terrence R. Tiersch 1 1 Aquaculture Research Station, Louisiana Agricultural Experimental Station, Louisiana State University Agricultural Center, 2410 Ben Hur Road, Baton Rouge, LA 70803, U.S.A 2 Department of Animal Sciences, Louisiana Agricultural Experimental Station, Louisiana State University Agricultural Center, Baton Rouge, LA 70803, U.S.A. Received: 22 December 2003 / Accepted: 20 May 2004 / Online publication: 24 March 2005 Abstract Gamete preservation techniques are essential in animal husbandry as well as in assisted reproduc- tion for humans. In this research we attempted to use 3 different sperm preservation techniques in combination with newly developed techniques for intracytoplasmic sperm injection (ICSI) to fertilize eggs of a teleost fish, the Nile tilapia (Oreochromis niloticus). Of 47 eggs injected with fresh sperm, 11 (23%) were fertilized, 5 developed abnormally, and 4 developed normally and hatched; from these, one grew to adulthood. Nuclear DNA content of 4 of the abnormal embryos indicated that they were diploid. Flow cytometric analysis of a blood sample from the surviving ICSI fish collected 2 months after fertilization indicated that the fish was dip- loid. Of 45 eggs injected with cryopreserved sperm, 9 (20%) developed to the blastula stage. Of 40 eggs injected with sperm preserved in 70% methanol, none were fertilized. No injections were possible with freeze-dried Nile tilapia sperm owing to technical difficulties during manipulation. Al- though the findings described here are limited, they provide the first steps toward using sperm preser- vation methods in addition to cryopreservation for fertilization in fishes. Key words: ICSI — Nile tilapia — fertilization — sperm — eggs — cryopreservation Introduction Sperm storage began to be applied commercially in livestock shortly after the first calf produced from cryopreserved semen was born in 1951 (reviewed by Curry, 2000). Currently, industries such as dairy are based on the use of artificial insemination and frozen sperm. Although artificial insemination with cryopreserved sperm provides advantages over natu- ral fertilization, not all farm animal industries have incorporated this assisted reproductive technology. In aquaculture, for example, cryopreservation has been studied in an estimated 200 species of fish (Rana, 1995), but it has not become established in research or commercial applications (Tiersch, 2000). This will likely change in the near future given that aquaculture is the fastest growing sector of agricul- ture and overfishing and human development are threatening dozens of species of fish (Hutchings, 2000; Pauly et al., 2002). The establishment of repositories for genetic material is seen as a way of maintaining biological diversity, developing brood- stocks, and reducing space used in hatcheries for maintenance of males (Holt, 1997; Tiersch, 2000; Cloud et al., 2000). At present cryopreservation is the only reliable way to preserve fish sperm indefinitely; however, it is expensive and potentially unreliable because it requires a constant supply of liquid nitrogen. A good alternative would be the use of freeze-dried sperm or sperm preserved in alcohol, each of which could be stored at 4°C or at room temperature (25°C). The concept of using freeze-drying as a sperm storage technique was first introduced 50 years ago with reports of unsuccessful attempts to obtain viable avian sperm after vitrification and dehydration (Polge et al., 1949). It was not until 1957 that the first live births of rabbits were reported from freeze-dried *Present address: Estacio ´ n de Piscicultura, Universidad Centroc- cidental ‘‘Lisandro Alvarado,’’ Apartado 400, Barquisimeto, Estado Lara, Venezuela Correspondence to: Terrence R. Tiersch; E-mail: ttiersch@agctr. lsu.edu 104 DOI: 10.1007/s10126-004-0162-5  7, 104–111 (2005)  Ó Springer Science+Business Media, Inc. 2005