Effects of bovine spermatozoa preparation on embryonic development in vitro

Reproductive Biology and Endocrinology BioMed Central Research Open Access Effects of bovine spermatozoa preparation on embryonic development in vitro Marko Samardzija*, Martina Karadjole, Iva Getz, Zdenko Makek, Marijan Cergolj and Tomislav Dobranic Address: Clinic for Obstetrics and Reproduction, Faculty of Veterinary Medicine, University of Zagreb, Heinzelova 55, Zagreb, Croatia Email: Marko Samardzija* - ; Martina Karadjole - ; Iva Getz - ; Zdenko Makek - ; Marijan Cergolj - ; Tomislav Dobranic - * Corresponding author Published: 13 November 2006 Reproductive Biology and Endocrinology 2006, 4:58 doi:10.1186/1477-7827-4-58 Received: 06 September 2006 Accepted: 13 November 2006 This article is available from: http://www.rbej.com/content/4/1/58 © 2006 Samardzija et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract The aim of our research was to examine the ability of density gradient preparation BoviPure® and swim up method on bull sperm separation and in vitro embryo production (IVP) systems. Frozen/ thawed semen from six Simmental bulls was pooled and treated using both methods. The sperm motility, concentration, membrane activity, membrane integrity and acrosomal status were evaluated and compared before and after sperm processing using BoviPure® and swim up methods. We also evaluated and compared cleavage rates, embryo yield and quality between the methods. There were significant differences (P < 0.05) between the sperm characteristics before and after BoviPure®, but not after swim up method. However, there were significant differences for sperm results among those two mentioned methods. A total of 641 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA. The percentage of cleavage (Day 2) and the percentage of hatched embryos (Day 9) were similar for both methods. However, embryo production rate (Day 7) was significantly higher using BoviPure® method (P < 0.05). Also, total cell number and embryo differential staining (inner cell mass and trophectoderm cells) of Day 7 morulas and blastocysts showed that BoviPure® treated sperm displayed higher quality embryos compared to swim up method (P < 0.05). Our results indicate that BoviPure® method has an enhanced capacity in sperm selection for in vitro embryo production when compared with swim up method. So, we concluded that BoviPure® could be considered as a better alternative to swim up method for separating bull spermatozoa from frozen/thawed semen for IVP of bovine embryos. Background Mammal spermatozoa have very expressive heterogeny in morphology, motility and nuclear stability. During copulation, cervical mucus represents a barrier which allows only migration of progressively motile spermatozoa with normal morphology and high nuclear stability [1]. Frozen bull spermatozoa after thawing have lower percentage of progressive motility (30–70%), but percentage of mor- phologically normal spermatozoa in thawed ejaculate is equal to fresh semen [2]. Sperm separation procedures are able to significantly improve the sperm quality with higher rate of progressive motility and morphologically normal spermatozoa. In the in vitro production of embryos, sperm separation methods have very important role. Such selection of spermatozoa separates motile sperm from nonmotile, removes seminal plasma, cryoPage 1 of 7 (page number not for citation purposes) Reproductive Biology and Endocrinology 2006, 4:58 protective and infectious agents, other background materials and debris [3,4] and also in the same time initiates the capacitation of sperm [5]. The morphological selection of spermatozoa in the prepared population varies, mostly with tail and midpiece defects being primarly excluded. Many sperm separation methods have been developed to improve sperm quality based on high rate of progressive motility and morphologically normal spermatozoa. Some of the most important sperm separation methods are: selective fractionation of subpopulations (density-gradient centrifugation) and self-migration techniques swimup [1]. The efficacy of sperm preparation methods could be evaluated using different sperm parameters such as sperm motility, morphology, concentration, viability, membrane activity, acrosomal status, reactive oxygen species (ROS) formation, chromatin maturity and integrity, protamination degree and IVP rates [6-9]. BoviPure® is a commercial medium for the density-gradient centrifugation of bull spermatozoa. It is an iso-osmotic salt solution containing colloidal silica particles coated with silane specifically formulated for use with bull sperm. At this time, very few studies have been conducted to evaluate BoviPure® for in vitro production of bovine embryos [9,10]. In contrast, swim up method is routinely used for many years in in vitro procedures of bovine embryos [6,11-13]. Comparing swim up method and Percoll gradient Parrish et al. [2] obtained similar sperm results for both methods, although a lower concentration resulted for swim up method. However, the fact that cleavage rate was significantly higher for swim up method compared to Percoll compensated a lower sperm concentration results. With swim up method we can safely separate spermatozoa based on their motility and morphology [1]. Research that compared these two methods (BoviPure and swim up) of bull sperm preparation for in vitro production of bovine embryos was not done until today. The present study was designed to compare the efficiency of two sperm separation methods evaluating sperm quality parameters and subsequent development and quality of bovine IVP embryos. Methods General approach For the purpose of our research a group of six Simmental bulls with proven fertility was chosen. Frozen-thawed sperm of all the six bulls was pooled and then the sperm parameters were estimated. Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise stated. BoviPure® gradient Sperm preparation for in vitro fertilization (IVF) on BoviPure® gradient was accomplished according to pro- http://www.rbej.com/content/4/1/58 ducer's directions (Nidacon International AB, Göthenborg, Sweden). BoviPure® works at room temperature. In a 10 mL centrifuge tube 2 mL of BoviPure® Bottom Layer Medium was placed and then carefully layered with 2 mL of BoviPure® Top Layer Medium. Aliquots of 400 µL of thawed semen were gently placed into a warm test tube and diluted with BoviPure® Extender in 1:1 ratio. The amount of 800 µL of the prepared semen was gently loaded onto the top of the gradient and centrifugated for 20 min at 300 × g. After centrifugation, the fluid above the sperm pellet was carefully removed. The pellet was resuspended with 5 mL of BoviPure® Wash and centrifuga (...truncated)