ANIREP 3632 1–59 22 Abstracts / Animal Reproduction Science xxx (2008) xxx–xxx 21 Application of fluorescence microscopy in the routine analysis of equine spermatozoa S. Gamboa a,∗ , M.S. Rodrigues a , P. Cortez a , R. Rebord˜ ao a , J. Ramalho-Santos b a Animal Reproduction Laboratory, Department of Zootechnic Sciences, Agricultural School, Polytechnic Institute of Coimbra, Bencanta, 3040-316 Coimbra, Portugal b Department of Zoology, Center for Neuroscience and Cell Biology of Coimbra, University of Coimbra, 3004-517 Coimbra, Portugal E-mail address: scgamboa@esac.pt (S. Gamboa). Spermatozoa are highly polarized cells and their efficiency is dependent of an intricate network that evolves cellular sub-structures and a poorly understood metabolism. Thus, the functional apti- tude of sperm cells relies on a diversity of factors not taken into account by traditional semen analyses. Several non-fluorescent and fluorescent staining techniques, which can be used sepa- rately or in combination, have been developed to determine the viability, DNA integrity, acrosomal status and metabolism of spermatozoa. Stallions (n = 7) of different breeds were used to study seminal characteristics, structural and functional features of spermatozoa and their capability for fertilization. Following routine laboratory tests were carried out: quantitative (volume, concen- tration, pH) and qualitative [motility, live sperm (eosin–nigrosin stain) and morphology (india ink stain)] in ejaculates (n = 54 semen samples; 7–8 ejaculates/stallion). Hypo-osmotic swelling (HOS) test was also used to evaluate the functional integrity of the sperm membrane. Fluorescent dyes were utilized in a triple stain to assess, by fluorescence microscopy, membrane (IP) and acro- somal integrity (FITC-PSA) together with mitochondrial membrane potential (JC-1). Apoptosis (TUNEL) and lipid peroxidation (C11-Bodipy 581/591 ) were also determined. Ultrasonographic (6.5 MHz probe) pregnancy diagnoses were done 13 d after the last AI. Classical semen parame- ters varied among ejaculates from the same stallion and among males (P < 0.05). The percentage of live sperm obtained with the triple stain (52.5% live sperm, IP/JC-1/PSA) correlated signifi- cantly (r = 0.477, P < 0.01) with the eosin–nigrosin stain (67.1% of live sperm). The percentage of live and acrosome intact sperm with a higher mitochondria potential (IP - JC1 + PSA - ) also showed significant correlations with sperm motility (r = 0.459, P ≤ 0.001) and mid-piece abnor- malities (r = -0.291, P ≤ 0.05). Dead sperm cells presented both higher and lower mitochondrial membrane potential and this last population inversely correlated with sperm motility (r = -0.430, P ≤ 0.001) and live sperm (r = -0.372, P ≤ 0.01). The sperm population that did not show a positive response to HOS test correlated well with dead, acrosome reacted cells with high (IP + JC1 + PSA + , r = 0.318, P < 0.05) or low (IP + JC1 - PSA + , r = 0.286, P < 0.05) mitochondrial mem- brane potential. Discrete levels of lipid peroxidation were found and no evidence of apoptotic mature spermatozoa was established. Semen samples obtained from the 7 stallions were used in an AI breeding program (300 × 10 6 progressive motile sperm) to inseminate 48 mares. The per cycle pregnancy rates varied from 25% to 78.5% and the best results were obtained with semen from stallions with a higher percentage of live and acrosome intact cells with high mitochondrial membrane potential. Structural and functional staining of equine sperm with fluorescent probes may be useful in the routine analysis because they allow us to evaluate different compartments and functions of individual cells, including the plasma and acrosomal membrane integrity and mitochondrial function.