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The ability of sperm to bind to zonae pellucidae (ZP) has been correlated with fertilizing capacity of sperm in several species. Limited numbers of equine oocytes are available to perform such assays. Therefore, use of heterologous ZP to perform gamete binding tests with stallion sperm would be useful. We have found that addition of 10% of skim milk-based extender with glucose [EZ-Mixin®, Animal Reproduction Systems, Chino, USA;; (EZ)] to TALP significantly increased the number of stallion sperm bound to bovine ZP. Objectives of the present experiments were to determine: (1) if stallion sperm bind in similar numbers to equine and bovine ZP, and (2) the effects of skim milk, milk proteins and glucose on sperm binding to ZP. Denuded bovine (immature) and equine (mature) oocytes were stored at 5°C in salt solution (1.5M MgCl2, 40mM HEPES, 0.1% PVP). In Experiment I, 4 ejaculates from 2 stallions were centrifuged at 300g for 6min, and sperm pellets were resuspended in 1mL of TALP or EZ. Sperm were stained with Hoechst 33342, centrifuged, and resuspended to 2×106 sperm mL−1. Oocytes were placed into droplets of 45μL of TALP (7 to 10 oocytes/trt/ejac). Extended sperm (5μL) were added to oocytes, resulting in 2times105 sperm mL−1, and the mixutre was incubated for 2h at 38.5°C. Oocytes then were pipetted in TALP to remove loosely attached sperm and observed with fluorescence microscopy;; mean numbers of sperm bound to bovine and equine ZP for TALP were 29±1.9 and 36±2.6 (P&amp;gt;0.1) and for EZ, 149±5 and 152±6.3 (P&amp;gt;0.1), respectively. More sperm bound to ZP with EZ than to ZP with TALP (P&amp;lt;0.001). Experiment II used 4 ejaculates from 4 stallions. After initial centrifugation, sperm were resuspended in 1mL of each of six extenders: TALP, EZ, TALP containing 89.5mM glucose (TG), TALP containing 163.5mM glucose (THG), TALP containing 2.4mgmL−1 of skim-milk powder (TSM), and INRA 96® (IMV Technologies, L’Aigle, France) that contains 27mgmL−1 of native phosphocaseinate. Hoechst 33342-stained sperm and bovine oocytes were processed as described for Experiment I. Treatments containing milk proteins resulted in more sperm binding (P&amp;lt;0.01) than those without milk proteins (Table 1). In conclusion, use of bovine oocytes led to similar results for equine and bovine oocytes;; therefore, bovine oocytes can be used for binding assays with stallion sperm. High concentrations of glucose increased numbers of sperm bound to ZP;; however, presence of milk or milk proteins was more effective in enhancing binding of sperm to ZP. INRA96 contains relatively low glucose (67mM) and one milk protein. Therefore, we hypothesize that native phosphocaseinate may cause increased sperm binding to ZP.Table 1Mean sperm bound per ZP±SEM (n=38–40/group)

263BINDING OF STALLION SPERM TO EQUINE AND BOVINE ZONAE PELLUCIDAE: EFFECT OF MILK, MILK PROTEINS AND GLUCOSE