Short Communication A Pilot Comparison of Laser-Assisted vs Piezo Drill ICSI for the In Vitro Production of Horse Embryos K Smits, J Govaere, M Hoogewijs, S Piepers and A Van Soom Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium Contents Intracytoplasmic sperm injection (ICSI) is the method of choice for the in vitro production (IVP) of equine embryos. However, conventional ICSI has been associated with mechanical damage to the oocyte caused by the deformation of the zona pellucida (ZP) and exposure of the oolemma to negative pressure during injection. Introduction of the less traumatic and more efficient piezo drill-assisted ICSI (PDAI) yielded higher cleavage rates and more consistent results. Nevertheless, PDAI is also associated with disadvantages such as the use of mercury and possible DNA damage. This led us to explore an alternative method avoiding oocyte trauma, namely laser-assisted ICSI (LAI), which involves creating a hole in the ZP prior to ICSI. In this pilot study, PDAI and LAI were compared for ICSI in the horse. No significant influences on subsequent embryonic development were observed. Introduction Conventional in vitro fertilization has been largely unsuccessful in the horse, and ICSI is preferred for the in vitro production (IVP) of equine embryos. Conven- tional ICSI is characterized by mechanical penetration of the ZP and the oolemma with a bevelled injection pipette and subsequent injection of an immobilized sperm into the cytoplasm of a mature oocyte. The first pregnancy after ICSI in horses was announced in 1996 (Squires et al. 1996) and was followed by a period of variable results. In 2002, PDAI was introduced in horses and, at the same time, improved cleavage rates were reported (Choi et al. 2002; Galli et al. 2002). However, whether there was a causal relationship between PDAI and the increased cleavage rate was not studied. The aim of this technical study was to introduce laser-assisted ICSI (LAI) and compare it with PDAI for ICSI in horses. During LAI, a hole is made in the ZP prior to ICSI, thereby reducing distortion of the oocyte during injection. This less traumatic approach has been suggested to be beneficial in human patients with fragile oolemmae (Abdelmassih et al. 2002). Materials and methods Horse embryos were produced in vitro in DMEM-F12- based medium as previously described (Smits et al. 2010). Only compact cumulus-oocyte-complexes were used, and oocytes with visible polar bodies were fertilized by means of PDAI (Prime Tech, Ibaraki, Japan) or LAI (XYClone, Hamilton Thorne, Beverly, MA, USA). PDAI was performed with a blunt injection pipette (piezo-6-25; Humagen, Charlottesville, VA, USA). A small amount of mercury was put in the tip of the pipette. A progressively motile sperm was immobilized by piezo pulses, the ZP and the oolemma were penetrated with a piezo intensity setting of 5 and 4, respectively, and a speed of 4 and 3, respectively, and the sperm was injected into the ooplasm. For LAI, one progressively motile sperm was immobilized by crushing its tail. The laser device was used to create multiple adjacent small holes, using maximal power and a pulse time of 150 ls. After this, the oolemma was penetrated mechanically with a bevelled injection pipette (MIC-50- 35; Humagen), in conformity with conventional ICSI. Five replicates were performed; in three of them, PDAI preceded LAI, and in the other two, the sequence was reversed. Injected oocytes were cultured up to the blastocyst stage as previously described (Smits et al. 2010). On day 2.5, the cleavage rate was evaluated and non-cleaved embryos were removed. Half of the medium was changed on day 2.5 and again on day 6 and blastocyst formation was evaluated on day 9. Rates of cleavage and blastocyst formation were compared using a Pearson chi-square test. The mean injection times were compared using a t-test. A p-value <0.05 was consid- ered significant. Results The results are summarized in Table 1. Of the 78 oocytes that were injected with the help of the laser, 63 cleaved (cleavage rate = 81%), and four of these reached the blastocyst stage (6.3%). In the piezo group, 71 of the 104 oocytes cleaved, (68%); 31 of these cleaved piezo embryos were used in another experiment and five of the 40 remaining embryos, used in this study, developed to blastocysts (12.5%). Neither the cleavage rate nor the blastocyst rate differed significantly between the two groups (p > 0.05). The mean time for ICSI, including the handling of the oocytes before and after the procedure, was longer for LAI (4.0 min ⁄ oocyte) than for PDAI (2.9 min ⁄ oocyte) (p > 0.05). After LAI, a persistent hole in the ZP was formed which resulted in blastomere leakage at day 2.5 (Fig. 1) and hatching at the blastocyst stage (Fig. 2). This was predominantly apparent in a preliminary trial where a pulse duration of 200 ls was used; reduction to 150 ls diminished the phenomenon. Discussion The developmental competences of embryos produced by PDAI and LAI were comparable; the cleavage rate Reprod Dom Anim 47, e1–e3 (2012); doi: 10.1111/j.1439-0531.2011.01814.x ISSN 0936-6768 Ó 2011 Blackwell Verlag GmbH