Considerable research evaluating the effects of bovine sperm preparation methods such as swim up and density gradient centrifugation have been done. Most of them referred to Percoll
® gradient [
22‐
24]. The problem is that some batches of Percoll
® have endotoxic effect so it was discarded for use in assisted reproduction technics in human medicine [
25]. There have been reports that batches of Percoll
® differ in composition and this variation may affect cleavage rates and embryo development [
26]. As a result of Percoll
® endotoxicity many pharmaceutical companies researched for a good quality substitute for Percoll
® like Bovipure
®. Bovipure
® is a sperm separation and purification product formulated specifically for use with bull sperm. Our results indicate that BoviPure
® was more effective at preparing sperm for IVF when compared to swim up (P < 0.05) regarding sperm motility 70.00 % vs. 53.75 %, respectively. Our results of sperm motility for swim up method are congruent to Risopatron et al. [
27]. Mentioned authors compared two sperm separation methods and gained significantly higher percentage of motility for swim up (54.00%) compared to washing method (43.50%). Sperm viability, membrane activity and acrosome status evaluated by HOS, SYBR-14/PI and EthD-1/FITC-PSA tests showed a higher percentage of live and acrosome intact spermatozoa obtained after BoviPure
® method compared to swim up. Somfai et al. [
24] used the dual stain to evaluate the viability and acrosome integrity of frozen-thawed bull sperm before and after Percoll
® and swim up methods. They found significantly increased proportion of live spermatozoa with intact acrosomes for Percoll
® (88.20%), than after the swim up method (69.40%) which is similar to our results obtained after comparing density gradient BoviPure
® and swim up method. Piomboni et al. [
28] found that swim up selection based on sperm motility excludes many sperm with reacted acrosome and broken plasma membrane which was not established in our research. Determining sperm viability for swim up method Risopatron et al. [
27] using dual staining found 63.20% of live sperm with intact membrane, while using washing method they found 53.20% of live sperm with intact membrane. Samardzija et al. [
9] used HOS, SYBR-14/PI and EthD-1/FITC-PSA tests for determination of sperm viability, membrane activity and acrosome status for BoviPure
® and Percoll
® gradients. They found no differences between Percoll
® and BoviPure
® methods in percentage of live spermatozoa with intact acrosomes. It should be mentioned that the force of centrifugation might affect sperm motility and membrane integrity in bulls [
29] and also in rams [
30]. Because of that the sperm parameters results in those two protocols should be interpreted with caution. In the present research we compared the cleavage rates, embryo yields and quality for the both sperm separation methods and found no significant differences between methods in cleavage and hatched blastocysts rates. However, the number of morulas and blastocysts on Day 7 of culture was significantly higher for BoviPure
® method. We did not find high, significant correlations between the sperm parameters and embryo yield, except for acrosome status after EthD-1/FITC-PSA test (r>0.7;P < 0.05). That could be explained with the fact that spermatozoa with intact acrosomes were not capacitated. Non-capacitated spermatozoa showed a significant, although low relation to fertility [
31,
32]. Sieren and Youngs [
10] used BoviPure
® method and obtained 77.20% of the cleaved oocytes 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 production of bovine embryos. The preparation of the bull sperm by the BoviPure
® method did not show significantly better effects on the cleavage rate (77.20%) and on the percentage of blastocysts on Day 8 of in vitro culture (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 did not significantly improve the ability of obtaining the bovine embryos in procedures in vitro. That is not consistent to our results because we established that bovine embryo development appeared to be superior following BoviPure
® compared to swim up method. However, we did not observe differences in cleavage rates and percentage of hatched blastocysts which was similar to results of mentioned authors. Samardzija et al. [
9] examined the effect of BoviPure
® and Percoll
® on bull sperm separation for IVP of bovine embryos. They found no significant differences regarding sperm evaluation parameters between the methods. The cleavage (Day 2) and blastocysts (Day 7) rates 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 (Day 9) did not differ significantly between sperm separation methods. Our previous work indicates that BoviPure
® is acceptable method for sperm separation in bovine IVP which was in accordance to our results. In our research significantly higher blastocyst rates in BoviPure
® group vs swim up group make us suggest that BoviPure
® method allowed a faster cleavage and blastocyst yield than swim up method. Other explanation of different embryo yield could be that density gradient method selects spermatozoa with more compacted chromatin and less nuclear DNA damage than swim up method [
33]. Embryos from BoviPure
® treated group displayed significantly higher total cell number comparing to swim up group. Cesari et al. [
34] compared two bull sperm separation methods and revealed a significantly higher number of inner cell mass cells in Percoll treated group compared to swim up. Similar results were found by Rho et al. [
35] who reported that goat blastocysts obtained from the Percoll treatment group had significantly more cells compared to swim up group (167 ± 5 vs 149 ± 4, respectively). Our study also demonstrated that BoviPure
® method resulted in significantly higher number of inner cell mass cells compared to swim up method which can be correlated with embryo quality. Although differences were found in cell counting, sperm treatment did not affect hatching rates. Our research showed predominance of sperm preparation by BoviPure
® in terms of blastocyst formation, total cell number and allocation of ICM. Lane and Gardner [
36] reported that mouse fetal development after transfer was positively correlated with number of blastocyst cells and with ICM development, but not with number of TE cells or hatching ability. Therefore, it would be advisable to extend the comparisons of these two sperm preparation methods by embryo transfer into recipient cows which will allow for more reliable resultes of subsequent embryo development.