Please cite this article in press as: Jerez-Ebensperger, R.A., et al., Effect of pasteurized egg yolk and rosemary honey supplementation on quality of cryopreserved ram semen. Small Ruminant Res. (2015), http://dx.doi.org/10.1016/j.smallrumres.2015.07.010 ARTICLE IN PRESS G Model RUMIN-4983; No. of Pages 4 Small Ruminant Research xxx (2015) xxx–xxx Contents lists available at ScienceDirect Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres Effect of pasteurized egg yolk and rosemary honey supplementation on quality of cryopreserved ram semen R.A. Jerez-Ebensperger a , V. Lu˜ no a , M. Olaciregui a , N. González a , I. de Blas b , L. Gil a,∗ a Reproduction and Obstetrics Area, Animal Pathology Department, Universidad de Zaragoza, Spain b Infectious Diseases Area, Animal Pathology Department, Universidad de Zaragoza, Spain a r t i c l e i n f o Article history: Received 10 November 2014 Received in revised form 1 July 2015 Accepted 14 July 2015 Available online xxx Keywords: Cryopreservation Pasteurized egg yolk Honey Ram Sperm a b s t r a c t The aim of this study was to determine the effect of pasteurized egg yolk and the supplementation with rosemary honey on ram sperm quality after cryopreservation process. Ejaculates were obtained from three rams (Rasa Aragonesa), and semen samples were processed using straw freezing procedure. In experiment 1, semen samples were frozen in four different extenders: (D1) Fiser egg yolk extender as control, and three experimental extenders: (D2) pasteurized egg yolk in fraction 1 (F1); (D3) rose- mary honey in F1 and fraction 2 (F2); D4) pasteurized egg yolk and rosemary honey in F1 and honey in F2. Total and progressive motility, viability, acrosome integrity and hypoosmotic swelling test response (HOST test) were evaluated post-thawing and 2 h post-thawing. A significant increase in percentage of total motile sperm was observed in the presence of pasteurized egg yolk, alone or in combination with rosemary honey. Acrosome integrity was significantly higher in pasteurized egg yolk extenders (p < 0.05), however rosemary honey decreased HOST positive sperm after thawing. In experiment 2, embryo devel- opment was tested. D2 extender showed that the embryo development rate was significantly (p < 0.05) higher than both D1 and D4 extenders. It could be suggested that Fiser extender composition can be modified replacing fresh egg yolk by pasteurized egg yolk and fructose by honey, without detrimental effects on sperm quality after cryopreservation. © 2015 Published by Elsevier B.V. 1. Introduction Cryopreservation as a technique for storing ram semen has many advantages but during freeze-thawing process thermal, mechanical, chemical and osmotic stress (Holt et al., 1992; Holt and North, 1994; Watson, 1981) induce certain detrimental structural effects on spermatozoa. These changes involve several biochemical and functional damages (Bailey et al., 2000; Watson, 2000) to the spermatozoa that reduce their motility, plasma membrane func- tionality and acrosome integrity (Celeghini et al., 2007; Woelders et al., 1997), leading spermatozoa to a lower fertilizing ability (Medeiros et al., 2002; Salamon and Maxwell, 2000; Tekin et al., 2006). Maintenance semen quality during cryopreservation is the main objective of the process. The design and use of suitable extenders and protocols is an important point to avoid the adverse effects of cold shock, such as intracellular ice crystals formation. The utiliza- ∗ Corresponding author at: C/Miguel Servet 177, 50013 Zaragoza, Spain. Fax: +34 976 761612. E-mail address: lydiagil@unizar.es (L. Gil). tion of egg yolk in the semen extender was reported in 1940, Philips and Lardy (1940) indicated that egg yolk was beneficial for sperm preservation. Currently egg yolk is a usual component in most of the sperm cryopreservation extenders in domestic and exotic ani- mals. Egg yolk has a beneficial effect as stabilizer of the spermatozoa membrane (Salamon and Maxwell, 2000) and acrosome integrity in association with other components (Bogart and Mayer, 1950; Phillips, 1939), and protects against cold shock (Drobnis et al., 1993; Watson, 1981). Furthermore, the addition of sugars to extenders shows a beneficial effect on spermatozoa viability and acrosome integrity (Martín et al., 1980). Rosemary honey provides a combi- nation of large amount of sugars (including glucose, fructose and sucrose) with antioxidant properties (Alamanni, 1994). The combi- nation of both characteristics may protect at spermatozoa against cold shock injuries. In order to determine new alternatives in cryopreservation protocols, we investigated the role of pasteurized egg yolk and rosemary honey on ram sperm quality during cryopreservation. http://dx.doi.org/10.1016/j.smallrumres.2015.07.010 0921-4488/© 2015 Published by Elsevier B.V.