Animal Reproduction Science 168 (2016) 100–109 Contents lists available at ScienceDirect Animal Reproduction Science journal h om epa ge : w ww.elsevier.com/locate/anireprosci The benefits of liposomes for chilling canine sperm for 4 days at 4 ◦ C Redha Belala a,b , Juliette Delay a , Lamia Amirat a , Marie-Hélène Ropers c , Jocya Le Guillou a , Marc Anton c , Eric Schmitt d , Chantal Thorin e , Sandrine Michaud a , Rachid Kaidi b , Daniel Tainturier a , Djemil Bencharif a,∗ a Laboratory of Biotechnology and Pathology of Reproduction, ONIRIS: The National Veterinary, Food Agriculture, and Food Hygiene School of Loire Atlantique, BP 40706, 44307 Nantes, France b Laboratory of Biotechnology of Animal Reproduction, SAAD DAHLAB University of Blida (U.BLIDA1), BP 270, 09000 Blida, Algeria c UR1268 Biopolymères Interactions Assemblages, Equipe Interfaces et Systèmes Dispersés, INRA, F-44316 Nantes Cedex 3, France d IMV Technologies, 10 rue Clemenceau, BP 81, 61302 Aigle Cedex, France e Department of Statistics, ONIRIS: The National Veterinary, Food Agriculture, and Food Hygiene School of Loire Atlantique, BP 40706, 44307 Nantes, France a r t i c l e i n f o Article history: Received 4 September 2015 Received in revised form 29 February 2016 Accepted 29 February 2016 Available online 2 March 2016 Keywords: Canine sperm Refrigeration Liposomes Mechanisms of spermatozoa protection Langmuir-Blodgett trough a b s t r a c t This study comprises 3 experiments exploring the possible benefits and mechanism of action of liposomes for chilling (4 ◦ C) canine sperm over a period of 4 days. In the first experiment, 20 ejaculates collected from 5 Beagle dogs were chilled in an extender containing 6% low density lipoproteins (LDL) (Control), or one of 7 extenders con- taining different concentrations (2, 4, 6, 8, 10, 15, 20%) of liposomes (LIPO). These ejaculates were chilled over 4 days and motility was assessed daily using a Hamilton Thorne analyzer (HTM-IVOS, 14.0). The 2% LIPO obtained the best results (p = 0.038) after four days (72.55% motile spermatozoa and 31.4% progressive spermatozoa). In experiment 2, 10 ejaculates were collected from same 5 dogs and chilled in 6% LDL or 2% LIPO-based extenders. Sperm integrity characteristics were assessed prior to refrigeration and every 48 h for four days (D0, D2, and D4). Acrosome integrity was assessed using the FITC-PSA test (Fluorescein IsoThiocyanate-Pisum Sativum Agglutinin), plasma membrane (PM) integrity using both the hypo-osmotic swelling test (HOSt) and SYBR14/Propidium Iodide test (SYBR14/PI), and DNA integrity using the Acridine-Orange test (AO). The 2% LIPO extender provided equivalent preservation of sperm integrity parameters to the reference extender (6% LDL). In experiment 3, a Langmuir-Blodgett trough was used to evaluate the mechanistic inter- actions between LDL, LIPO, prostatic fluid, and the canine spermatozoal membrane during chilling. Results indicate that LDL and LIPO interact differently with the biomimetic mem- brane. The most likely conclusion of these findings is that LDL and liposomes employ different protective mechanisms during the chilling (4 ◦ C) of canine spermatozoa. © 2016 Elsevier B.V. All rights reserved. ∗ Corresponding author. E-mail address: djemil.bencharif@oniris-nantes.fr (D. Bencharif). 1. Introduction Chilled canine semen was successfully used for the first time in 1954 (Harrop, 1954). It has become increasingly http://dx.doi.org/10.1016/j.anireprosci.2016.02.032 0378-4320/© 2016 Elsevier B.V. All rights reserved.