Ž . Animal Reproduction Science 57 1999 199–215 www.elsevier.comrlocateranireprosci In vitro capacitation of fresh, chilled and frozen–thawed dog spermatozoa assessed by the chlortetracycline assay and changes in motility patterns A. Rota a,1 , A.I. Pena b , C. Linde-Forsberg a, ) , ˜ H. Rodriguez-Martinez a a Department of Obstetrics and Gynaecology, Centre for ReproductiÕe Biology, Faculty of Veterinary Medicine, Swedish UniÕersity of Agricultural Sciences, P.O. Box 7039, 750 07 Uppsala, Sweden b Area de Reproduccion y Obstetricia, Departamento de Patologıa Animal, Facultad de Veterinaria de Lugo, ´ ´ Lugo, Spain Received 19 August 1998; received in revised form 18 May 1999; accepted 21 July 1999 Abstract The effect of preservation on capacitation status of dog spermatozoa was investigated. Split ejaculates from six dogs were assessed as fresh, chilled for 24 h and rewarmed, and frozen–thawed Ž . samples. Capacitation-like status was assessed using the chlortetracycline CTC -assay and the measurement of sperm motility patterns using a computer-assisted sperm analyzer. Evaluations were performed on washed spermatozoa immediately after dilution in a Tris–fructose–citrate Ž . Ž . buffer TFC or in canine capacitation medium CCM , and at 2-h intervals during 8 h of incubation in 5% CO in air, at 378C. Preservation decreased significantly the proportion of 2 uncapacitated spermatozoa. In TFC, at hour 0, chilled–rewarmed and frozen–thawed samples had a significantly lower proportion of uncapacitated, viable spermatozoa than the fresh samples Ž . P -0.05 according to the CTC-assay. The time course of capacitation was accelerated in the preserved samples, compared to the fresh ones. During incubation in CCM, the mean time from hour 0 to when, according to the CTC-assay, the highest proportion of capacitated spermatozoa ) Corresponding author. Tel.: q46-18-67-2163; fax: q46-18-67-3545; e-mail: catharina.linde-forsberg@og.slu.se 1 Current address: Dipartimento di Clinica Veterinaria, Facolta di Medicina Veterinaria, Universita di Pisa, ` ` Pisa, Italy. 0378-4320r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0378-4320 99 00056-1