Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 172 Poster Abstracts P438 Correlaction between bovine sperm membrane integrity and mitochondrial cytochemical activity in Bos taurus bulls Zuge, RM 1,2 ; Bertolla, RP 3 ; Nichi, M 1,4 ; Soler, TBS 1,3 ; Cortada, CNM 2 *; Bols, PEJ 4 ; Barnabe, VH 1 1Department of Animal Reproduction (VRA), Faculty of Veterinary Medicine and Zootechny (FMVZ), University of São Paulo (USP), Brazil; 2 Paraná Technology Institute (Tecpar), Brazil; 3 Department of Human Reproduction, Federal University of São Paulo (UNIFESP), Brazil; 4 Laboratory of Veterinary Physiology, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Belgium Introduction Previous studies with varicocele in men and European bulls in tropical region, indicated that heat stress would induce deleterious effects to all structures of the spermatozoa, with special regard to the mitochondria. The disruption of the spermatic mitochondria could have a potential damaging effect not only to an individual cell but to the surrounding cells, specially regarding to the sperm membrane. This could be due to the release of a high amount of reactive oxygen species (ROS) produced in this environment, rich in electrons and oxygen that would lead to an event known as lipid peroxidation. Methods To study whether sperm cells with disrupted mitochondria, would show higher amount of membrane damages probably due to higher levels of ROS attack, semen samples of eleven heat stressed Simmental bulls were collected by electroejaculation, the method of choice when working under field conditions in Brazil. The staining 3- 3’ diamino benzidine (DAB), as an index of mitochondrial activity, the hypo-osmotic swelling test (HOST), as an index of membrane integrity, and the measurement of tiobarbituric acid reactive substances (TBARS), an index of spontaneous lipid peroxidation were used. Data was analysed using the SAS system for Windows. Results Results showed a positive correlation between sperm cells with full mitochondrial activity (DAB class A), and percentage of cells with intact membrane by the HOST (r=0.93, p
16 t h International Congress on Animal Reproduction Poster Abstracts 173 can conclude that treatments of 8 or 9 days of duration would be of election and from practical point of view; the insemination time would be earlier when EB is injected at device removal than 24 h later. Granted by NUMdPlata. P441 Evaluation of different lipid sources in frozen-thawed ram semen Alvarez, M 1 *, Bernardo, J 1 , Boixo, JC 1 , Gomes-Alves, S 1 , Mata-Campuzano, M 2 , Anel, E 1 , De Paz, P 2 , Anel, L 1 1Animal Reproduction and Obstetrics, University of Leon, Spain; 2 Cell Biology, University of Leon, Spain The use of substances free of animal proteins in semen extenders is an important tool to improve the healthy conditions of ovine reproduction programmes. Hygienic control and chemical composition of extenders are important factors that affect the spermatozoa lifespan. Egg yolk provides protection against cold shock and particularly the low density lipoprotein fraction is the responsible of this effect. Other lipoproteins have been demonstrated to be good cryoprotectants (soy bean) but their use is limited. The aim of this study is to test the fertility of frozen-thawed semen from Churra breed rams, cryopreserved with four extenders (different source of lipids). Extenders were made with different sources and concentrations of lipids: egg yolk (UL), low density lipoproteins (LDL), soy lecithin 1% and soy lecithin 2% (granulated commercial soy bean). Semen from ten rams (two consecutive semen collections per session, two sessions) was evaluated (volume, mass motility and spermatozoa concentration). For each ram, the two ejaculates of a session were pooled if both had good quality. Thus, the final ejaculate was divided in 4 aliquots and diluted (1:1) with the 4 freezing extenders: UL (TesT-fructose-10% egg yolk -4% glycerol- antibiotics), LDL8 (TesT-fructose-8% LDL-4% glycerol- antibiotics), SOY1 (TesTfructose-1% soy -4% glycerol- antibiotics) and SOY2 (TesT–fructose- 2% soy-4% glycerol- antibiotics). The samples were cooled at 5ºC (- 0.25ºC/min) and extended to a final concentration of 100x106 spermatozoa/ml. Diluted semen was placed into 0.25 ml straws, sealed and frozen from 5ºC to -100ºC (-20ºC/min) in a programmable cell freezer (Kryo 10, Planer). The straws were plunged into liquid nitrogen until analysis and thawed in a water bath (65ºC, 6 s). The fertilizing capacity was evaluated by laparoscopic intrauterine insemination. The oestrous of the ewes were synchronized using intravaginal sponges with 40 mg fluorogestone acetate (14 days) and 500 UI of eCG (im) at withdrawal. Laparoscopic inseminations were performed at 64 h after the removal of the sponges. The number of inseminated ewes (total 576 from four farms) with each extender was: UL (144), LDL8 (146), SOY1% (146) SOY2% (140). Fertility was not significantly affected by freezing extender, although UL and LDL seemed to show better results (UL: 43.06%; LDL: 49.32%; SOY1: 41.78% and SOY2: 40.71%). The lipid sources assayed are suitable for freezing the ram semen applied by intrauterine insemination. This work was supported in part by CYCYT (AGL2005-07601/GAN), Ovigen, Diputación de León and ASSAF.E. P442 Effect of different thawing rates on post-thaw sperm viability, kinematic parameters and motile sperm subpopulations structure of bull semen Peña, AI 1 *; Muiño, R 1 ; Rivera, MM 2 ; Rigau, T 2 ; Rodriguez-Gil, JE 2 1Faculty of Veterinary Medicine, University of Santiago de Compostela, Spain; 2 Faculty of Veterinary Medicine, Autonomous University of Barcelona, Spain The aim of the present study was to evaluate three thawing rates for bull semen frozen in 0.25 ml-straws: placing the straws in a water bath at 37ºC for 40 s, at 50ºC for 15 s or at 70ºC for 5 s. In a first experiment, the 3 thawing rates were compared in relation to postthaw sperm motility, determined subjectively, and sperm plasma and acrosomal membrane integrity, examined by flow cytometry, after 0 and 5 h of incubation at 37ºC. In a second experiment, the three thawing rates were evaluated based on post-thaw sperm motility, determined using a CASA system, after 0 and 2 h of incubation at 37ºC. In addition, for the motile spermatozoa, the individual motility descriptors were analysed using a multivariate clustering procedure to test the presence of separate sperm subpopulations with specific motility characteristics in the thawed bull semen samples. Finally, it was investigated if the thawing rate had any influence on the relative frequency distribution of spermatozoa within the different subpopulations. In terms of overall post-thaw motility or plasma and acrosomal sperm membrane integrity there were no significant differences between the 3 thawing methods evaluated. The statistical analysis clustered all the motile spermatozoa into 4 separate subpopulations with defined patterns of movement: 1) moderately slow and progressive sperm (27%); 2) “hyperactivated-like” sperm (15.4%); 3) poorly motile non progressive sperm (34.3%) and 4) fast and progressive sperm (23.3%). The thawing rate had no significant influence on the frequency distribution of spermatozoa within the 4 subpopulations, but there was a significant effect (P
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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