SPERM MEDIATED GENE TRANSFER IN SHEEP: SELECTING RAMS AS SEMEN DONORS

DÍAZ PUMARA PZ; RAFAELLI P; ABBATE V; ERNESTO JI

Porto de Galinhas

Congreso; XXIV REUNIÃO ANUAL DA SOCIEDADE BRASILEIRA DE TECNOLOGIA DE EMBRIÕES; 2010

SOCIEDADE BRASILEIRA DE TECNOLOGIA DE EMBRIÕES (SBTE) O

Microinjection into the male pro-nucleus, retroviral factors and transfected stem cells was been used to produce transgenic animals. All results judged inefficient as trial/success, expensive and involve complex techniques. Sperm Mediated Gene Transfer (SMGT) is studied since 1989 as a technique capable of solving these problems. Bracket in 1971 show the mammalian sperm’s ability to incorporate DNA through membranes. Lavitrano et. al. (Cell,1989) proved possible to make: the addition of exogenous DNA fractions to epididymal sperm in mouse, his subsequent transfer to the egg during the fertilization process, the transgene expression in a proportion of animals born normally and its transfer to subsequent generations. Also these authors prove that the ejaculated sperm was resistant to this process because of a glycoprotein in seminal plasma. Removing this obstacle, it is possible that the exogenous DNA will be incorporating to ejaculated spermatozoa (SPZ) preserving fertility potential. Subsequently, several researchers reported transgenic animals achieved by this method. In swine ejaculated SPZ capacity to incorporate exogenous DNA is correlated positively with best zootechnical parameters of semen quality. Our goal is develop transgenic sheep expressing heterologous proteins in milk using SMGT. As a preliminary step, we propose to obtain through IVF and AI, founders who have expressed a fluorescent plasmid (pEGFP-N1-Clontech) in cells of the adult organism. Six rams of Milschaff breed, after a health assessment and a clinical examination, were used to donate semen trough artificial vagina. In successive extractions four parameters were evaluated: libido, volume, density and vitality. Libido and vitality in subjective scale, from 0 to 5. Objective scales were used to volume and concentration (cc and SPZ/ mm3). A full amount of 60 attempts produce a total of 48 semen collections. Libido: range amplitude from zero to five. Average 2,95. Volume average was 0.73 cc. rank 0-5 cc. SPZ concentration showed a great extent, among rams and between samples of the same ram. Standard4,7 x 10 6 sperm per mm3 . Range 0.163 x 10 6 to 9.2 x 10 6 SPZ per mm3 . Vitality: motility, vigor and strength showed smaller amplitude. Range 3-5. Average 4,38. Libido, ejaculate volume and concentration were highly variable between rams and between samples. Vitality was less variable. Last parameter showed more relevance at the time of judge the tolerance of semen to the co-incubation with fluorescent plasmid. Results in our trial are consistent with data presented by researchers cited for swine. Only 2 of 6 rams tested (33.3%) were selected at the moment of deciding their coincubation with exogenous ADN.