147 CryoLetters 25, 147-154 (2004) Ó CryoLetters, c/o Royal Veterinary College, London NW1 0TU, UK EFFECT OF THE EXTENDER SUPPLEMENT EQUEX-STM ON CRYOPRESERVED SEMEN IN THE ASSAF SHEEP A. Akourki 1 , L. Gil 1* , A. Echegaray 3 , E. Espinosa 1 , A. Josa 1 , I. de Blas 1 , N. Gonzalez 1 , M. Gallegos de la Hoya 2 and L.C. Meque 1 1 Facultad Veterinaria, Depto. de Patolog7a Animal (Reproducci9n), Universidad de Zaragoza, c/ Miguel Servet 177, 50013 Zaragoza, Spain. Email: lydiagil@unizar.es 2 Facultad de Medicina Veterinaria y Zootecnia, Universidad de Ju?rez del Estado de Durango, 34000 Durango, Mexico. 3 Empresa Magapor SL., Ejea de los Caballeros, 50600 Zaragoza, Spain Abstract This study was designed to evaluate the effect of adding the detergent Equex-STM to the extender used to dilute semen for cryopreservation on several indicators of sperm preservation. Two consecutive ejaculates per day were obtained from 5 Assaf sheep on two days out of every week over three alternate months. The freezing protocol involved diluting the semen in Fiser’s extender, to which 0.7 % Equex-STM was added or omitted before cryopreserving the semen in straws by exposure to nitrogen vapor. Equex-STM supplementation gave rise to significantly (p<0.05) improved sperm quality variables after different periods of freezing (0 hours, 1 week and 1 month). The variables examined were: individual motility, viability, acrosome integrity, plasma membrane integrity (HOS test) and morphological anomalies. This improvement was independent of the ram and month of testing. In a second experiment in which we incubated the semen (0 and 6 hours) at 37ºC after thawing, Equex-STM also showed a beneficial effect on sperm quality. Keywords: Assaf sheep, semen, Equex-STM, extender, cryopreservation INTRODUCTION Inadequate cryopreservation of ovine semen is one of the main causes of the low fertility recorded in this species. The problem is particularly associated with the cervical route of insemination, and has been attributed to the stress the sperm undergo during the freezing/thawing process, which even the most widely used cryoprotectant, glycerol, is unable to prevent. The cryoprotective effect of glycerol is related to its ability to preserve the kinetic features of sperm (22), leading to a greater percentage of viable sperm cells with an intact acrosome after the thawing process (3, 12). To avoid undesirable effects, Fiser and Fairfull (8) proposed 3-6 % as the optimal concentration of glycerol in an extender. In an effort to search for an alternative cryoprotecting agent, several chemical compounds are presently being added to the extender used to dilute the semen for cryopreservation. These supplements include proline, taurine and glycine betain, which have achieved improved sperm motility after thawing (22, 24, 23) and trehalose and EDTA, which have resulted in better preserved sperm properties (2). Nevertheless, it has not always been possible to correlate these improvements with results post-insemination (24, 23). In contrast, the addition of Equex-STM