ࡱ>       5@q$bjbj22.(XX~~~~~~~####^$dO$X5X5X5X5X5l9<xޥRW)~(>X5X5(>(>~~X5X5 (>]~X5~X5ޥ(>ޥ&ԣ~~ԣX5$ @^Ӽ#<ԣ0Oԣ]RR]ԣ~~~~ԣ]~ʤ (>(>(>(>(>(>(> A comparison of BoviPure and Percoll on bull sperm separation protocols for IVF M. Samardzija HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "aff1" a,  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "cor1"  INCLUDEPICTURE "http://www.sciencedirect.com/scidirimg/entities/REcor.gif" \* MERGEFORMATINET ,  HYPERLINK "mailto:smarko@vef.hr"  INCLUDEPICTURE "http://www.sciencedirect.com/scidirimg/entities/REemail.gif" \* MERGEFORMATINET , M. Karadjole HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "aff1" a, M. Matkovic HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "aff2" b, M. Cergolj HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "aff1" a, I. Getz HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "aff1" a, T. Dobranic HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "aff1" a, A. Tomaskovic HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "aff1" a, J. Petric HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "aff2" b, J. Surina HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "aff2" b, J. Grizelj HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "aff1" a and T. Karadjole HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "aff1" a aClinic for Obstetrics and Reproduction, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, 10000 Zagreb, Croatia bCentre for Reproduction and Animal Breeding, Planinska 2b, 10000 Zagreb, Croatia Received 18 February 2005; revised 19 April 2005; accepted 29 April 2005. Available online 8 June 2005. Abstract In the present study, we have examined the effect of density gradient preparations BoviPure and Percoll on bull sperm separation and the in vitro fertilization (IVF) and culture (IVC) results. Frozen/thawed semen from five simmental bulls were pooled. Sperm quality parameters such as sperm motility, concentration, membrane activity (HOS assay), membrane integrity (SYBR-14/PI assay) and acrosomal status (EthD-1/FITC-PSA assay) were evaluated before and after sperm processing for IVF using BoviPure and Percoll density gradient separations. The results of the evaluated parameters before sperm processing were: motility 50%, concentration 82.33נ106spz/mL, membrane activity 39.05%, membrane integrity 42.97% and the acrosomal status 46.90% of the live spermatozoa with intact acrosomes. After sperm processing with BoviPure and Percoll the motility was 66.67 and 64.17%, the concentration was 25.50נ106 and 27.67נ106spz/mL, the membrane activity was 53.78 and 56.58%, the membrane integrity was 70.85 and 68.76% of and the acrosomal status was 74.16 and 67.46% of the live spermatozoa with intact acrosomes, respectively. Percentages were referred to the total number of spermatozoa. There were significant differences (P<0.05) between the evaluated parameters before and after sperm processing for both separation protocols. We found no significant differences (P>0.05) regarding sperm evaluation parameters between the protocols. A total of 492 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA in six replicates. The cleavage (D2) and blastocysts (D7) rate were significantly higher (P<0.05) for the BoviPure group compared to the Percoll group: 75.80 and 28.21%; 61.58 and 20.83%, respectively. However, the number of hatched blastocysts (D10) did not differ significantly between sperm separation protocols (P>0.05). Our results indicate that both protocols gave suitable sperm for IVF. This finding and the similarity between those two density gradient preparations suggests that BoviPure is a good alternative for sperm separation in bovine IVF. Keywords: Bull; Sperm; BoviPure; Percoll; IVM; IVF; IVC  INCLUDEPICTURE "http://www.sciencedirect.com/scidirimg/sci_dir/line.gif" \* MERGEFORMATINET  Article Outline 1.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "SECX1" Introduction 2.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "SECX2" Material and methods 2.1.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "SECX3" General approach 2.2.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "SECX4" BoviPure gradient 2.3.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "SECX5" Percoll gradient 2.4.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "SECX6" Sperm quality parameters asssessment 2.5.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "SECX7" Collection of cumulusoocyte complexes (COC) and in vitro maturation (IVM) 2.6.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "SECX8" In vitro fertilization and culture (IVF and IVC) 2.7.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "SECX9" Statistical analyses 3.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "SECX10" Results 4.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "SECX11" Discussion  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bibl001" References 1. Introduction Sperm separation procedures are able to significantly improve the sperm quality enhancing progressive motility and morphological normal spermatozoa. For this reason in the IVF, sperm separation methods have a very important role. Such selection of spermatozoa separates motile sperm from nonmotile, remove seminal plasma, cryoprotective agents, other background materials and debris ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib32" Zavos, 1992), and also at the same time initiates the capacitation of the sperm ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib1" Centola et al., 1998). One method of sperm separation is selective fractionation by density-gradient centrifugation. Percoll is one commercial medium for the density-gradient centrifugation of cells, viruses and subcellular particles also widely used in IVF. It is composed of colloidal silica particles (1530nm in diameter) coated with nondialysable polyvinylpyrrolidone (PVP). Percoll density-gradient fractionation clearly separates spermatozoa from foreign material such as extender particles, cells and bacteria. The morphological selection of spermatozoa in the prepared population varies, with most tail, and midpiece defects being primarily excluded ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib20" Rodriguez-Martinez et al., 1997). The problem is that some batches of Percoll have endotoxic effect so it was discarded for use in assisted reproduction technics in human medicine ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib2" Chen and Bongso, 1999). There have been reports that batches of Percoll differ in composition and this variation may affect cleavage rates and embryo development ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib18" Mendes et al., 2003). As a result of Percoll endotoxcity many pharmaceutical companies researched for a good quality substitute for Percoll. Recently, BoviPure, a sperm separation and purification product formulated specifically for use with bull sperm became available. BoviPure is an iso-osmotic salt solution containing colloidal silica particles coated with silane. At this time, very few studies have been conducted to evaluate BoviPure for in vitro production of bovine embryos ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib22" Sieren and Youngs, 2001). The control of sperm quality after commercial freezing/thawing of bull semen is still restricted to the subjective assessment of sperm motility, despite its low correlation with fertility ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib23" Sderquist et al., 1991 and  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib15" Kjaestad et al., 1993). The integrity of the plasma membrane reflects the viability of spermatozoa and can be assessed by dual staining of the membrane with permanent nucleic acid stain, SYBR-14, combined with propidium iodide.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib4" Correa and Zavos, 1994 and  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib3" Correa and Zavos, 1996 and  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib21" Rota et al. (2000) have used hypoosmotic swelling (HOS) test for the evaluation of the functional integrity of the frozen/thawed bovine sperm membrane. Capacitation is a sequence of biochemical changes leading to destabilization of the sperm membranes which enables the sperm to become able for fertilization ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib28" Van Soom and de Kruif, 1996). Capacitation involves sperm plasma membrane events that lead to an increased cellular calcium influx, fusion and vesiculation of the plasma and outer acrosomal membranes, and the loss of the acrosomal protein matrix in a process termed acrosome reaction ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib31" Yanagimachi, 1994). The normal acrosome reaction of spermatozoa is an essential requirement for mammalian fertilization ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib12" Henkel et al., 1993). In addition, flourescein-conjugated plant lectins have been shown to be useful in selective acrosomal staining of human ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib5" Cross et al., 1986), stallion ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib7" Farlin et al., 1992), ram ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib26" Sukardi et al., 1997) and bovine spermatozoa ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib30" Way et al., 1995). The aim of this research was to compare the recently available commercial product BoviPure with Percoll, based on the sperm quality parameters and results of IVF and IVC of bovine embryos. 2. Material and methods 2.1. General approach For the purpose of our research a group of five simmental bulls with proven fertility was chosen. The frozen-thawed sperm of all the five bulls was mixed-up making a pool and then the sperm parameters were estimated. All the components of the media for IVM/IVF/IVC used in this investigation were supplied by Sigma Chemical Co. (St. Louis, MO, USA). 2.2. BoviPure gradient Sperm preparation for IVF on BoviPure gradient was accomplished according to producer's directions (Nidacon Laboratories AB, Gthenborg, Sweden). BoviPure works at room temperature. In a 10mL centrifuge tube 2mL of BoviPure Bottom Layer Medium was placed and then carefully layered with 2mL of BoviPure Top Layer Medium. Aliquots of 400L of thawed semen were gently placed into a warm test tube and diluted with BoviPure buffer in 1:1 ratio. The amount of 800L of the prepared semen was gently loaded onto the top of the gradient and centrifugated for 20min at 300g. After centrifugation, the fluid above the sperm pellet was carefully removed. The pellet was resuspended with 5mL of BoviPure wash and centrifugated for 10min at 500g. This final pellet was resuspended in 150L of mIVF, and the final concentration was adjusted to 1נ106spz/mL. 2.3. Percoll gradient Percoll gradient was made according to  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib19" Parrish et al. (1995). Isotonic Percoll solution was used for preparation of 90 and 45% gradients with HEPES-TALP medium. The Percoll density gradient was made by layering 1.5mL of 45% Percoll solution on the 1.5mL of 90% solution in 15mL Falcon tubes. On the top of the gradient 400L of thawed semen was layered and then tubes were centrifugated for 15min at 700g. The pellets were resuspended in the same amount of HEPES-TALP media and centrifugated for 7.5min at 300g. Afterwards the pellets were resuspended in mIVF and the final concentration was adjusted to 1נ106spz/mL. 2.4. Sperm quality parameters asssessment The sperm quality parameters were evaluated immediately after thawing and after sperm preparation for IVF. Sperm concentration was determined with a Thoma chamber. Progressive motility of semen was subjectively assessed by visual estimation under microscope. The functional integrity of bovine sperm membrane was determined by hypoosmotic swelling test (HOS) and dual staining with SYBR-14/PI. The hypoosmotic swelling test was performed according to  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib14" Jeyendran et al. (1984) with the exception that osmolarity was adjusted to 100mOsm/kg as described for frozen-thawed bovine spermatozoa by  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib4" Correa and Zavos (1994). The assay was performed mixing 50L of semen with 1mL of hypoosmotic solution and incubating at 37C for 60min. A total of 400 cells were evaluated in at least five different fields under 400 magnification. Spermatozoa with changes were denoted as swelled or HOS positive (HOS+). To assay the sperm viability we used a SYBR-14/PI staining as described by  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib13" Januskauskas et al. (1999). Aliquots of 50L thawed semen were diluted in 150L of mTALP containing 3L PI and 2L SYBR-14 (Live/Dead Sperm Viability Kit, Molecular Probes Inc., USA). Incubation and staining procedures of the samples were performed according to the method described by  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib9" Garner and Johnson (1995) with minor modifications. The nuclei of SYBR-14-stained spermatozoa were bright green, while dead sperm nuclei were stained red with PI (propidium iodide). A total of 300 spermatozoa were counted under 400 magnification in two replicates, and the mean values were then used for the analysis. For acrosome staining, a slightly modified procedure described by  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib13" Januskauskas et al. (1999) was used. Aliquots (15L) of ethidium homodimer (EthD-1) counter-stained semen were smeared onto microscope slides, air dried, fixed and permeabilized with 96% ethanol for 30s. The unbounded dye of EthD-1 was removed using centrifugation at 200g for 5min twice, preventing that excess of dye stain live spermatozoa after permeabilization with ethanol. We kept smear for 15min at "20C and then eliminated the ethanol. Twenty microliters of FITC-labeled pisum sativum agglutinin (FITC-PSA) solution (100g/mL) in PBS were spread over each smear and incubated in moist chamber at 37C for 7min. They were subsequently agitated in destilled water to remove unbound dye, air dried and mounted with 15L of anti-fade solution. Three hundred morphologicaly normal spermatozoa were assessed under 1000 magnification in each smear and then classified according to the method of  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib26" Sukardi et al. (1997) in one of four categories, based on their FITC-PSA and EthD-1 staining patterns: (a) live, acrosome intact sperm; (b) dead, acrosome intact sperm; (c) live, acrosome reacted sperm; (d) dead, acrosome reacted sperm. 2.5. Collection of cumulus oocyte complexes (COC) and in vitro maturation (IVM) Ovaries were collected within 2h after slaughter and transported to the laboratory in physiological saline (0.9%) with antibiotics (100 I.U. penicillin and 100g streptomycin/mL) at 37C. Immature bovine oocytes were aspirated from 2 to 8mm diameter follicles using 18G needles attached to a vacuum pump. Only oocytes with homogenous ooplasm and intact cumulus investment were selected for the further procedure. They were washed two times in TCM 199 medium buffered with 15mM HEPES supplemented with 10% of FCS and then two times in IVM medium. Cumulusoocyte complexes were matured in vitro in TCM 199 bicarbonate medium supplemented with 10% FCS, FSH/LH (Pergonal 75/75 I.U./mL, Serono), 1g/mL estradiol-17 and 100M cysteamine. Oocytes were incubated in 50L droplets of maturation media under mineral oil at 39C and with 5% CO2 for 24h. 2.6. In vitro fertilization and culture (IVF and IVC) The expanded COCs were washed in TALP-HEPES medium supplemented with 3g/mL BSA-FAF and transfered in 40L droplets of IVF medium under mineral oil. The IVF medium was modified Tyrode's bicarbonate buffered solution supplemented with 10g/mL heparin, 0.5g/mL hypotaurine, 0.5g/mL epinephrine and 6mg/mL BSA. The sperm suspension was then added at a volume of 10L to the droplets with oocytes. After sperm-oocytes co-incubation at 39C and with 5% CO2 for 18 24h, the fertilized oocytes were denuded from cumulus cells and spermatozoa and then washed three times in HEPES-TALP medium and in culture medium. Synthetic oviductal fluid (SOF) with amino acids and 8mg/mL BSA, according to  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib6" Edwards et al. (1997) was used. Fertilized oocytes were cultured in vitro in SOF medium without glucose for 48h and then transfered in SOF with 1.5mM glucose and cultured in vitro until Day 10 at 39C in 5% CO2, 7% O2 in 88% N2, according to  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib8" Furnus et al. (1997). The medium was changed every 2nd day. Bovine embryos were evaluated according to the IETS standards: on the 2nd day of culture we registered the number of cleaved embryos, on the 7th day the number of morulas and blastocysts and on the 10th day the number of hatched blastocysts ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib16" Manual of IETS, 1998). 2.7. Statistical analyses The statistical analyses between protocols were done by ANOVA (StatSoft, Statistica, 5.1 version 1984 1996) using the arcsin transformation (arcsin"P/100) of the percent values, comparisons by the Tukey's tests post hoc analysis and correlation analyses between sperm parameters before and after processing and IVF and IVC results. 3. Results The sperm motility before processing was (503.65%) and after sperm processing were (66.674.01%) for BoviPure and (64.173.75%) for Percoll. Comparing the results of sperm motility before processing with the results after sperm processing it was found out that there were significant differences (P<0.05) between them. However, there were no significant differences (P>0.05) in the motility values between the sperm preparation methods. The sperm concentration before processing was (82.331.73נ106spz/mL) and after sperm processing was (25.501.73נ106spz/mL) for BoviPure and for Percoll (27.671.52נ106spz/mL). Comparing the results of sperm concentration before processing with the results after sperm processing it was found out that there were significant differences (P<0.01) between them. However, there were no significant differences (P>0.05) for the concentration values between the sperm preparation methods. The HOS test ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "fig1" Fig. 1) results before the processing were 39.052.97% and after the sperm processing with BoviPure and Percoll were 53.841.79, and 56.581.95%, respectively. Using the SYBR-14/PI test ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "fig2" Fig. 2) the obtained results before the processing were 42.972.22% and after processing 70.852.49 and 68.763.47% of the spermatozoa with the intact plasma membrane, respectively. The EthD-1/FITC-PSA test ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "fig3" Fig. 3) of live spermatozoa with the intact acrosomes were 46.904.64% before the sperm processing and after the processing were 74.121.34 and 67.465.17%, respectively. The significant differences were found out (P<0.001) between the sperm evaluation parameters before and after the processing with both methods for HOS, SYBR-14/PI and EthD-1/FITC-PSA tests. The results were shown in  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "tbl1" Table 1. Investigating the methods of sperm preparation for the IVF (BoviPure and Percoll) it was found out that there were significant differences (P<0.01) between the evaluation parameters before and after the sperm processing for the both separation protocols. We found no significant differences (P>0.05) regarding sperm evaluation parameters between the protocols.  INCLUDEPICTURE "http://www.sciencedirect.com/scidirimg/sci_dir/line.gif" \* MERGEFORMATINET   INCLUDEPICTURE "http://www.sciencedirect.com/cache/MiamiImageURL/B6T43-4GBWJDD-1-5/0?wchp=dGLbVlb-zSkzV" \* MERGEFORMATINET  Fig. 1.Inactive sperms (white arrows) and different swelling patterns of the active sperms (red arrows) after HOS test (400).  INCLUDEPICTURE "http://www.sciencedirect.com/cache/MiamiImageURL/B6T43-4GBWJDD-1-7/0?wchp=dGLbVlb-zSkzV" \* MERGEFORMATINET  Fig. 2.Live (green) and dead (red) sperms after SYBR-14/PI test (400).  INCLUDEPICTURE "http://www.sciencedirect.com/scidirimg/sci_dir/line.gif" \* MERGEFORMATINET   INCLUDEPICTURE "http://www.sciencedirect.com/cache/MiamiImageURL/B6T43-4GBWJDD-1-1/0?wchp=dGLbVlb-zSkzV" \* MERGEFORMATINET  Fig. 3.Live and dead sperms with intact acrosoma (red arrow) after EthD-1/FITC-PSA test (1000). Table 1. Sperm parameters results (meansS.E.M.) Sperm separation protocolProgressive motility (%)Concentration (106Ml)HOS % activeSYBR-14/PI % liveEthD-1/FITC-PSA % live with intact acrosomeInitial (n=6)50.003.6582.331.73 HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "tbl1fn1" *39.052.9742.972.2246.904.64BoviPure (n=6)66.674.01 HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "tbl1fn1" *25.501.7353.781.79 HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "tbl1fn2" **70.852.49 HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "tbl1fn2" **74.161.34 HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "tbl1fn2" **Percoll (n=6)64.173.75 HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "tbl1fn1" *27.671.5256.581.95 HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "tbl1fn2" **68.763.47 HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "tbl1fn2" **67.465.18 HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "tbl1fn1" **P<0.05. **P<0.001.  INCLUDEPICTURE "http://www.sciencedirect.com/scidirimg/sci_dir/line.gif" \* MERGEFORMATINET  The IVF and IVC results are shown in  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "fig4" Fig. 4. A total of 492 oocytes (246 for BoviPure and 246 for Percoll) were matured and fertilized in vitro and cultured in SOFaaBSA in six replications. The oocytes cleavage results were 75.801.77% for BoviPure and 61.582.87% for Percoll. The percentage of morulas and blastocysts on the 7th day of the culture were 28.212.42% for BoviPure and 20.832.45% for Percoll. The results of hatched blastocysts on the 10th day of the culture were 14.481.58% for BoviPure and 10.582.32% for Percoll. All mentioned percentages refer to the starting material. The IVF and IVC results were compared and significant differences (P<0.05) between the methods in cleavage (D2) and morulas and blastocysts (D7) rates were revealed. However, the number of hatched blastocysts (D10) did not differ significantly between sperm separation protocols (P>0.05).  INCLUDEPICTURE "http://www.sciencedirect.com/scidirimg/sci_dir/line.gif" \* MERGEFORMATINET   INCLUDEPICTURE "http://www.sciencedirect.com/cache/MiamiImageURL/B6T43-4GBWJDD-1-3/0?wchp=dGLbVlb-zSkzV" \* MERGEFORMATINET  Fig. 4.Cleavage, blastocysts and hatched blastocysts rates for BoviPure and Percoll (meanS.E.M.). *P<0.05.  INCLUDEPICTURE "http://www.sciencedirect.com/scidirimg/sci_dir/line.gif" \* MERGEFORMATINET  4. Discussion Until recently, gradient of Percoll was the most widely used method in the preparation of bull sperm for the purpose of in vitro fertilization ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib18" Mendes et al., 2003).  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib10" Getz (1999) compared the swim up method versus Percoll for the in vitro fertilization of ova and found that the Percoll method is simple, fast and resulted with a higher percentage of progressively motile sperm cells (65%) versus the swim up method (50%). Our results agree with  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib27" Tanghe et al. (2002) who investigated the Percoll method and obtained motility of 68% for the first group that consisted of three bulls, but weaker motility (52%) for the second group that consisted of three other bulls. Our results are also congruent with those obtained by  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib25" Sonderlund and Lundin (2000). They investigated the use of silane-coated silica particles for density gradient centrifugation in IVF and established no difference in the sperm motility between Percoll and PureSperm (Nidacon Laboratories AB, Gthenborg, Sweden) gradient. Puresperm is an iso-osmotic salt solution containing colloidal silica particles coated with silane, and it differs very little from BoviPure protocol. The above-mentioned authors confirmed PureSperm as an acceptable alternative to Percoll.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib1" Centola et al. (1998) in the human sperm established there was no difference in the percent motility and motile count between Percoll and PureSperm gradient. They also concluded that PureSperm appears to be as effective as Percoll for the recovery of good progressively motile sperm for use in IUI or other assisted reproductive techniques. Comparing the results of initial concentration of the sperm cells with the results of the final concentration we found out that there are significant differences between them (P<0.05). On the other hand, there were no significant differences (P<0.05) in concentration results between the methods.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib2" Chen and Bongso (1999) evaluated the sperm separation efficiency of PureSperm and Percoll gradients. They found out that Percoll was superior to PureSperm in concentration, percentage motility and percentage of live spermatozoa. Comparing the initial sperm results of HOS (39.05%), SYBR-14/PI (42.97%) and EthD-1/FITC-PSA (46.90%) with results of these tests after both sperm separation protocols, we found out a significant difference (P<0.001). These results indicate that both protocols, Percoll and BoviPure, improve the quality of the separated sperm in regard to higher viability and acrosome integrity. Our results for the HOS test performed after Percoll yielded 56.58% of sperm cells with functional active plasma membrane.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib3" Correa and Zavos (1996) yielded 4146% and  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib17" Matkovi et al. (2002) obtained 53% of sperm cells with functionally active plasma membrane using the Percoll method.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib24" Somfai et al. (2002) evaluated the viability and acrosome integrity of frozen-thawed bull sperm before and after Percoll gradient. They found out the significantly increased proportion of live spermatozoa with intact acrosomes, before the treatment (45.80%) and after Percoll separation (88.20%). It should be mentioned that the force of centrifugation might affect sperm motility and membrane integrity in bulls ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib29" Verberckmoes et al., 2000) and also in rams ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib11" Gil et al., 1999). Because of that and because of the different volume of gradients and volume of spermatozoa loaded on gradients, the sperm parameters results in those two protocols should be interpreted with caution. We also compared the results of IVF and IVC for the both sperm separation protocols. We did not find a high, significant correlation between the sperm parameters with the IVF results (r<0.5; P<0.05). The cleavage (D2) and blastocysts (D7) rate were significantly higher (P<0.05) for the BoviPure group compared to the Percoll group: 75.80 and 28.21%; 61.58 and 20.83%, respectively. However, the number of hatched blastocysts (D10) did not differ significantly between the sperm separation protocols (P>0.05). These results are not in agreement with  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib18" Mendes et al. (2003) who found no differences in cleavage rates and embryo production between Percoll and Puresperm protocols. Puresperm appears to be comparable to Percoll for the separation of spermatozoa in human studies ( HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib1" Centola et al., 1998 and  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib25" Sonderlund and Lundin, 2000).  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bib22" Sieren and Youngs (2001) used BoviPure protocol and obtained 77.20% of the cleaved ova and 21.60% of the blastocysts. The above mentioned authors evaluated the effect of coincubation of oocytes with frozen/thawed bull sperm using the BoviPure method and compared the same effect with a modified Brackett-Oliphant medium as a control group in the in vitro procedures of obtaining the cow embryos. The percentage of cleaved ova was determined 48h after the insemination, while after 8 days of in vitro culture, the percentage of formed blastocysts was determined too. The preparation of the bull sperm by the BoviPure method did not show significantly better (P>0.05) effects on the percentage of cleavage of ova 48h after the insemination (77.2%) and on the percentage of formed blastocysts after 8 days of cultivation in vitro (21.60%) in comparison with the control group (71.90 and 17.10%). Those results demonstrated that the preparation of the bull sperm by BoviPure method didnt significantly improve the ability of obtaining the bovine embryos in in vitro procedures, which is not congruent with our results. Although sperm evaluation parameters did not differ between our two protocols we observed that cleavage rates and embryo production appeared to be superior following BoviPure compared to Percoll separation. Our findings suggest that BoviPure is a good and acceptable alternative to Percoll for bull sperm separation for the fertilization in vitro.  INCLUDEPICTURE "http://www.sciencedirect.com/scidirimg/sci_dir/line.gif" \* MERGEFORMATINET  References  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bbib1" Centola et al., 1998 G.M. Centola, R. Herko, E. Andolina and S. Weisensel, Comparison of sperm separation methods: effect on recovery, motility, motion parameters, and hyperactivation, Fertil. Steril. 70 (1998), pp. 11731175.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T6K-3V8RRF1-13&_user=3875467&_coverDate=12%2F31%2F1998&_fmt=summary&_orig=browse&_cdi=5033&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=02f91914432d192dbc6017fff1a18221&ref=full" SummaryPlus |  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T6K-3V8RRF1-13&_user=3875467&_coverDate=12%2F31%2F1998&_fmt=full&_orig=browse&_cdi=5033&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=868b28e2e23e78601dd72c1747e611fa&ref=full" Full Text + Links |  HYPERLINK "http://www.sciencedirect.com/science?_ob=MiamiImageURL&_imagekey=B6T6K-3V8RRF1-13-1&_cdi=5033&_user=3875467&_check=y&_orig=browse&_coverDate=12%2F31%2F1998&view=c&wchp=dGLbVlb-zSkzV&md5=39a9a5b1ea80baeda4cadebd8f9bd73d&ie=/sdarticle.pdf" PDF (50 K) |  HYPERLINK "http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=655&_targetURL=http%3A%2F%2Fwww.scopus.com%2Fscopus%2Finward%2Frecord.url%3Feid%3D2-s1.0-49BW-YDY0-TWC1-C1GK-00000-00%26partnerID%3D10%26rel%3DR3.0.0&_acct=C000050661&_version=1&_userid=3875467&md5=0a574724880559f73c27615bb5e7865b" \t "outwardLink" Abstract + References in Scopus |  HYPERLINK "http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=656&_targetURL=http%3A%2F%2Fwww.scopus.com%2Fscopus%2Finward%2Fcitedby.url%3Feid%3D2-s1.0-49BW-YDY0-TWC1-C1GK-00000-00%26partnerID%3D10%26rel%3DR3.0.0&_acct=C000050661&_version=1&_userid=3875467&md5=42d0c0d880d6a1752e4d91036535496b" \t "outwardLink" Cited By in Scopus  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bbib2" Chen and Bongso, 1999 M.J. Chen and A. Bongso, Comparative evaluation of two density gradient preparations for sperm separation for medically assisted conception, Hum. Reprod. 14 (1999), pp. 759764.  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Correa and P.M. Zavos, Preparation and recovery of frozen-thawed bovine spermatozoa via various sperm selection techniques employed in assisted reproductive technologies, Theriogenology 46 (1996), pp. 12251232.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCM-3W2V7CG-13&_user=3875467&_coverDate=11%2F01%2F1996&_fmt=summary&_orig=browse&_cdi=5174&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=52269ba352cda94acb95aaf3c239018e&ref=full" Abstract |  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCM-3W2V7CG-13&_user=3875467&_coverDate=11%2F01%2F1996&_fmt=full&_orig=browse&_cdi=5174&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=aab09f215d79ec3f0789e1d3b91e429f&ref=full" Full Text + Links |  HYPERLINK "http://www.sciencedirect.com/science?_ob=MiamiImageURL&_imagekey=B6TCM-3W2V7CG-13-1&_cdi=5174&_user=3875467&_check=y&_orig=browse&_coverDate=11%2F01%2F1996&view=c&wchp=dGLbVlb-zSkzV&md5=825ac11ebd853f40caa8775acb50068f&ie=/sdarticle.pdf" PDF (581 K) |  HYPERLINK "http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=655&_targetURL=http%3A%2F%2Fwww.scopus.com%2Fscopus%2Finward%2Frecord.url%3Feid%3D2-s1.0-49G4-GYV0-TWC1-C036-00000-00%26partnerID%3D10%26rel%3DR3.0.0&_acct=C000050661&_version=1&_userid=3875467&md5=6a1e0cf86478e687b1307fe4a7d2dc9c" \t "outwardLink" Abstract + References in Scopus |  HYPERLINK "http://www.sciencedirect.com/science?_ob=RedirectURL&_method=outwardLink&_partnerName=656&_targetURL=http%3A%2F%2Fwww.scopus.com%2Fscopus%2Finward%2Fcitedby.url%3Feid%3D2-s1.0-49G4-GYV0-TWC1-C036-00000-00%26partnerID%3D10%26rel%3DR3.0.0&_acct=C000050661&_version=1&_userid=3875467&md5=1732b58e965c1f5f907e6cbf63fcc9b2" \t "outwardLink" Cited By in Scopus  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bbib4" Correa and Zavos, 1994 J.R. Correa and P.M. Zavos, The hypoosmotic swelling test: its employment as an assay to evaluate the functional integrity of the frozen-thawed bovine sperm membrane, Theriogenology 42 (1994), pp. 351360.  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCM-49NH84R-79&_user=3875467&_coverDate=08%2F01%2F1994&_fmt=summary&_orig=browse&_cdi=5174&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=07c20e37a0bdf077dbc3b196e361dfb6&ref=full" Abstract |  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCM-49NH84R-79&_user=3875467&_coverDate=08%2F01%2F1994&_fmt=full&_orig=browse&_cdi=5174&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=08ceb8acc61ad874f91263c8273f26a8&ref=full" Full Text + Links |  HYPERLINK "http://www.sciencedirect.com/science?_ob=MiamiImageURL&_imagekey=B6TCM-49NH84R-79-1&_cdi=5174&_user=3875467&_check=y&_orig=browse&_coverDate=08%2F01%2F1994&view=c&wchp=dGLbVlb-zSkzV&md5=050a8d1de3f1050083fc920e9c306402&ie=/sdarticle.pdf" PDF (737 K)  HYPERLINK "http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T43-4GBWJDD-1&_user=3875467&_handle=V-WA-A-W-AAB-MsSAYWW-UUA-U-AABCUBWYDA-AABWZABZDA-VYYBDDVEE-AAB-U&_fmt=full&_coverDate=06%2F08%2F2005&_rdoc=93&_orig=browse&_srch=%23toc%234963%239999%23999999999%2399999%21&_cdi=4963&view=c&_acct=C000050661&_version=1&_urlVersion=0&_userid=3875467&md5=f1ca4be49d89da1d9b62ca0f9db970eb&artImgPref=F" \l "bbib5" Cross et al., 1986 N.L. Cross, P. Morales, J.W. Overstreet and F.W. Hanson, Two simple methods for detecting acrosome-reacted human sperm, Gamete Res. 15 (1986), pp. 213226.  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