Sperm-mediated gene transfer (SMGT) is a method for the production of transgenic animals based on the intrinsic ability of sperm cells to bind and internalize exogenous DNA molecules and to transfer them into the oocyte at fertilization. Recombinase-A (RecA) proteincoated exogenous DNA has been used previously in pronuclear injection systems increasing integration into goat and pig genomes. However, there are no data regarding transgene expression after ICSI. Here, we set out to investigate whether the expression of transgenic DNA in porcine embryos is improved by recombinase-mediated DNA transfer and if it is possible to generate transgenic animals using this methodology. Different factors which could affect the performance of this transgenic methodology were analyzed by studying 1) the effect of the presence of exogenous DNA and RecA protein on boar sperm functionality; 2) the effect of recombinase RecA on in vitro enhanced green fluorescent protein (EGFP)-expressing embryos produced by ICSI or IVF; and 3) the efficiency of generation of transgenic piglets by RecA-mediated ICSI. Our results suggested that 1) the presence of exogenous DNA and RecA-DNA complexes at 5 mg/ml did not affect sperm functionality in terms of motility, viability, membrane lipid disorder, or reactive oxygen species generation; 2) EGFP-expressing embryos were obtained with a high efficiency using the SMGT-ICSI technique in combination with recombinase; however, the use of IVF system did not result in any fluorescent embryos; and 3) transgenic piglets were produced by this methodology. To our knowledge, this is the first time that transgenic pigs have been produced by ICSI-SGMT and a recombinase.

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We developed a method to evaluate bovine sperm membranes in normal (1G) and simulated microgravity (Sim-µG). Bovine spermatozoa are used as a model system because they have cellular membranes analogous to those of other cell types, and yet are much simpler because they have no cytoplasm and do not participate in DNA transcription or mRNA translation. They can be cultured as single cells and are easily evaluated for membrane characteristics using flow cytometry. These features make the mammalian spermatozoon a useful model for exploring the principles of membrane structure/function in the presence of a variety of environmental challenges such as simulated microgravity. Cryopreserved, washed beef bull sperm (4-8 × 106 mL −1 ) were incubated under non-capacitating conditions (modified glucose-free Tyrode's medium containing low bicarbonate, HEPES buffer, pyruvate and 3 mg mL −1 BSA V; 23 • C in air), and these spermatozoa remained alive for 24-48 h at 1G. To simulate µG, spermatozoa were incubated under the same conditions, in a HARV 10 rotating wall vessel (RWV, Synthecon, Inc, Houston,TX, USA) at 9 rpms. Spermatozoa were incubated in 1G and Sim-µG environments for 2.5-4.5 h and subsequently exposed to 0, 60 or 80 µg mL For further statistical analysis, and incorporation of non-parametric statistical tools (including pattern recognition using Support Vector Machines), the data were processed using a collection of Perl scripts and C programs. Results: Live/dead status: When Sim-µG + 60 µg mL −1 LC sperm were compared to 1G + 60 µg mL −1 LC, and 80 µg mL −1 LC sperm, their profiles were more similar to the 1G 80 µg mL −1 LC profiles. AR status: the Sim-µG + 60 µg mL −1 LC profiles were similar to the 1G + 60 µg mL −1 LC profiles. Mitochondrial Status: the Sim-µG + 60 µg mL −1 LC profiles were more similar to 1G + 80 µg mL −1 LC profiles. Summary: although Sim-µG sperm lost their motility within 3 h, they were alive. Cell profiles indicate that Sim-µG sperm nuclear membranes are less stable and their mitochondria are less functional than the 1G controls, but their acrosomes are intact indicating that fertilizing potential may remain. Additional experiments are needed to determine the time course for Sim-µG, induced changes, and whether Sim-µG sperm can penetrate eggs.