Sperm-mediated gene transfer–treated spermatozoa maintain good quality parameters and in vitro fertilization ability in swine

Bacci, M.L.; Zannoni, A.; De Cecco, M.; Fantinati, P.; Bernardini, C.; Galeati, G.; Spinaci, M.; R.Giovannoni,; Lavitrano, M.; Seren, E.; Forni, M.

doi:10.1016/j.theriogenology.2009.06.033

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Volume 72, Issue 9 , Pages 1163-1170, December 2009

Sperm-mediated gene transfer–treated spermatozoa maintain good quality parameters and in vitro fertilization ability in swine

M.L. Bacci
- Department of Veterinary Morphophysiology and Animal Production–DIMORFIPA, University of Bologna, Bologna, Italy
- Corresponding author. Tel.: +39 051 2097912/993; fax: +39 051 2097899.
A. Zannoni
- Department of Veterinary Morphophysiology and Animal Production–DIMORFIPA, University of Bologna, Bologna, Italy
M. De Cecco
- Department of Veterinary Morphophysiology and Animal Production–DIMORFIPA, University of Bologna, Bologna, Italy
P. Fantinati
- Department of Veterinary Morphophysiology and Animal Production–DIMORFIPA, University of Bologna, Bologna, Italy
C. Bernardini
- Department of Veterinary Morphophysiology and Animal Production–DIMORFIPA, University of Bologna, Bologna, Italy
G. Galeati
- Department of Veterinary Morphophysiology and Animal Production–DIMORFIPA, University of Bologna, Bologna, Italy
M. Spinaci
- Department of Veterinary Morphophysiology and Animal Production–DIMORFIPA, University of Bologna, Bologna, Italy
R.Giovannoni
- Department of Surgical Science–DISC, University of Milano Bicocca, Monza, Italy
M. Lavitrano
- Department of Surgical Science–DISC, University of Milano Bicocca, Monza, Italy
E. Seren
- Department of Veterinary Morphophysiology and Animal Production–DIMORFIPA, University of Bologna, Bologna, Italy
M. Forni
- Department of Veterinary Morphophysiology and Animal Production–DIMORFIPA, University of Bologna, Bologna, Italy

Received 20 March 2009; received in revised form 29 May 2009; accepted 22 June 2009. published online 22 September 2009.

Abstract

A simple and efficient method for producing multitransgenic animals is required for medical and veterinary applications. Sperm-mediated gene transfer (SMGT) is an effective method for introducing multiple genes into pigs (Sus, Sus scrofa). The major benefits of this technique are the high efficiency, low cost, and ease of use compared with that of other methods: Sperm-mediated gene transfer does not require embryo handling or expensive equipment. The aim of this study was to investigate the influence of SMGT treatment and exogenous DNA uptake on sperm quality. Even after a coincubation with a 20-fold larger amount (100μg/mL) of DNA than usual (5μg/mL), sperm quality parameters were not significantly affected, confirming the hypothesis that the SMGT protocol itself or the amount of bound DNA do not compromise the possibility of an extended employment of SMGT. More importantly, we found that semen used for in vitro fertilization 24h after DNA uptake gave good cleavage (60% vs. 58%, treated vs. control) and developmental rates definitely positive (41% vs. 48%, treated vs. control). These good results are connected to a competitive efficiency of transformation (62%) due to the numerous improvements in SMGT technique. We demonstrate that SMGT-treated spermatozoa retain good quality and fertilization potential for at least 24h, expanding the possibility to apply transgenesis in field conditions in swine, where the greatest hurdles are fertilization timing and plain procedure.