Sexing semen in the dog: preliminary study

Introduction and aim. It is now possible to predetermine the sex of offspring from a number of species before fertilization with an accuracy of 85-95%. The current technology is based on the well-known difference in X- and Y- sperm in the amount of DNA present, and incorporates modified flow cytometric sorting instrumentation to sort X- and Y-bearing sperm. Despite the birth of sex-selected pups has been announced by XY INC. in 2007, no information is available on sorting procedures and conditions for dog semen. In order to produce the number of sorted spermatozoa required for an AI in the dog, many hours of sperm sorting are necessary. The aim of this study was to evaluate which is the more appropriate extender for dog semen conservation at room temperature prior (experiment 1) and post (experiment 2) sorting. Materials and methods. The dogs included in this study were 8 (4 for each experiment). For the ejaculates to be included in exp.2, motility and the percentage of morphologically normal spermatozoa had to be >70%. For the exp.1, the three fractions of an ejaculate were collected from each dog in a calibrated plastic vial by digital manipulation. The volume was measured and for the sperm-rich fraction the sperm concentration was determined with a Bürker chamber. Sperm total motility (%) and movement (scale 0-5)(2) were assessed subjectively using a phase contrast microscope at 37°C and x400 magnification. The semen was divided in 4 aliquotes and diluted 1:1 with 4 different media: a) Tris-fructose-citrate (TFC), b) Tris-glucose-citrate (TGC), c) modified Tyrode’s albumine-lactate-pyruvate medium (mTALP), d) third fraction of the ejaculate (after centrifugation at 5000g for 10 min) (III FR). The diluted samples were kept at room temperature (25°C) for 48 hs and total motility, movement, viability (eosin-nigrosin and SYBR-PI stainings) and acrosomal integrity (Fast Green-Bengal Rose staining) were assessed soon after dilution (t0) and after 2,4,6,8,24, and 48 h. In exp.2, the sperm-rich fraction was rapidly evaluated and diluted with the best extender from exp.1 to obtain a final concentration of 100x106 spermatozoa/ml. For sorting, 1 ml samples were stained with Hoechst 33342 for 1 h at 35° C in the dark. Just prior sorting 25 mg/ml of red food dye was added. A MoFlo SX® sperm sorter (DakoCytomation) was used. Selected spermatozoa were collected in 10 ml tubes containing 500 μl/ml of Tes-Tris plus 2% egg-yolk and then centrifuged at 800g for 10 min and re-suspended in the same 4 extenders of exp. 1. The samples were kept at room temperature for 24 h and the same parameters of exp.1 were assessed at 0, 2, 4 and 24 h. Data were analysed by ANOVA (Statistics for Windows, Stat. Inc., OK, USA). Significance has been assessed for P<0.05. Results. Exp 1: total motility (t24), movement (t4), and viability (t48) were higher in mTALP (P<0.05) compared to the other extenders. No differences were observed for acrosome integrity in the different media (P>0.05). Exp.2: total motility (t4), movement (t4), viability (t4) and acrosome integrity (t2 and t4) were higher in mTALP (p<0.05); furthermore, after 24 hs viability in III FR was significantly lower than in the media (P<0.05). Conclusions. Considering the long time required to obtain a sex-selected inseminating dose in the dog, mTALP can be considered a suitable medium for the conservation of dog semen at room temperature before and after sorting. As showed by the results, the sorting process could involve severe acrosomial damages (at microscopic observation the main alteration found was acrosomial loss) and the choice of an appropriate medium can reduce the number of spermatozoa that loose their fertilizing potential during and after the separation procedures. Further experiments are in progress in our laboratories to test different temperatures for sperm conservation after sorting, in order to preserve sperm parameters for longer periods.