Evaluation of Plasmid Delivery by Electroporation as a Means of Increasing Gonadotropin-Releasing Hormone Production in Stallions

Abstract 

A plasmid delivery system validated in other species was assessed for its potential for inducing long-term expression of gonadotropin-releasing hormone (GnRH) in stallions. The efficacy of this technique was demonstrated using two plasmids: pSEAP, expressing secreted embryonic alkaline phosphatase (SEAP), and pGnRH, expressing GnRH. In experiment 1, geldings were used as a model to test the effect of muscle of injection (splenius, pectoralis, and semitendinosus; n = 3 for each site) on the expression of the reporter plasmid, pSEAP. Concentrations of SEAP rose (P < .01) in jugular plasma samples, indicating uptake and expression of the pSEAP plasmid. Concentrations of SEAP were greatest (P < .05) and most consistent after pectoralis injection, and this site was chosen for injection and electroporation in the subsequent experiment. In ex-periment 2, stallions were treated with pGnRH (2 mg, n = 3; and 4 mg, n = 3) or 2 mg of pSEAP (control; n = 4) to determine the effects on the reproductive axis. Treatment with pGnRH (day 0) resulted in higher (P < .05) plasma testosterone concentrations from day 35 to 56 and increased the luteinizing hormone (LH) (P < 0.01) and testosterone (P < .1) responses to GnRH challenge on day 21. Daily semen characteristics from days 31 to 36 showed no effect (P > .1) of pGnRH treatment on seminal characteristics. It was concluded that delivery by electroporation of plasmids encoding peptide hormones may serve as a means of long-term in vivo production of peptides in the horse. Increases in LH and testosterone secretion after GnRH were observed in pGnRH-treated stallions; however, optimal conditions for expression need to be determined in future experiments.

Keywords: Electroporation, GnRH, Plasmid, Reproduction, Stallion