A comparison of BoviPure® and Percoll® on bull sperm separation protocols for IVF
M. Samardzijaa, , , M. Karadjolea, M. Matkovicb, M. Cergolja, I. Getza, T. Dobranica, A. Tomaskovica, J. Petricb, J. Surinab, J. Grizelja and T. Karadjolea
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 82.33×106spz/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 25.50×106 and 27.67×106spz/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 (P0.05) between the evaluated parameters before and after sperm processing for both separation protocols. We found no significant differences (P0.05) 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 (P0.05) 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 (P0.05). 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
Article Outline
1. Introduction
2. Material and methods
2.1. General approach
2.2. BoviPure® gradient
2.3. Percoll® gradient
2.4. Sperm quality parameters asssessment
2.5. Collection of cumulus–oocyte complexes (COC) and in vitro maturation (IVM)
2.6. In vitro fertilization and culture (IVF and IVC)
2.7. Statistical analyses
3. Results
4. Discussion
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 (Zavos, 1992), and also at the same time initiates the capacitation of the sperm (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 (15–30nm 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 (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 (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 (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 (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 (Söderquist et al., 1991 and 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. Correa and Zavos, 1994 and Correa and Zavos, 1996 and 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 (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 (Yanagimachi, 1994). The normal acrosome reaction of spermatozoa is an essential requirement for mammalian fertilization (Henkel et al., 1993). In addition, flourescein-conjugated plant lectins have been shown to be useful in selective acrosomal staining of human (Cross et al., 1986), stallion (Farlin et al., 1992), ram (Sukardi et al., 1997) and bovine spermatozoa (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, Göthenborg, 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 400μL of thawed semen were gently placed into a warm test tube and diluted with BoviPure® buffer in 1:1 ratio. The amount of 800μL of the prepared semen was gently loaded onto the top of the gradient and centrifugated for 20min at 300×g. 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 500×g. This final pellet was resuspended in 150μL of mIVF, and the final concentration was adjusted to 1×106spz/mL.
2.3. Percoll® gradient
Percoll® gradient was made according to 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 400μL of thawed semen was layered and then tubes were centrifugated for 15min at 700×g. The pellets were resuspended in the same amount of HEPES-TALP media and centrifugated for 7.5min at 300×g. Afterwards the pellets were resuspended in mIVF and the final concentration was adjusted to 1×106spz/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 Jeyendran et al. (1984) with the exception that osmolarity was adjusted to 100mOsm/kg as described for frozen-thawed bovine spermatozoa by Correa and Zavos (1994). The assay was performed mixing 50μL of semen with 1mL of hypoosmotic solution and incubating at 37°C 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 Januskauskas et al. (1999). Aliquots of 50μL thawed semen were diluted in 150μL of mTALP containing 3μL PI and 2μL 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 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 Januskauskas et al. (1999) was used. Aliquots (15μL) 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 200×g for 5min twice, preventing that excess of dye stain live spermatozoa after permeabilization with ethanol. We kept smear for 15min at −20°C and then eliminated the ethanol. Twenty microliters of FITC-labeled pisum sativum agglutinin (FITC-PSA) solution (100μg/mL) in PBS were spread over each smear and incubated in moist chamber at 37°C for 7min. They were subsequently agitated in destilled water to remove unbound dye, air dried and mounted with 15μL 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 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 100μg streptomycin/mL) at 37°C. 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. Cumulus–oocyte complexes were matured in vitro in TCM 199 bicarbonate medium supplemented with 10% FCS, FSH/LH (Pergonal® 75/75 I.U./mL, Serono), 1μg/mL estradiol-17β and 100μM cysteamine. Oocytes were incubated in 50μL droplets of maturation media under mineral oil at 39°C 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 3μg/mL BSA-FAF and transfered in 40μL droplets of IVF medium under mineral oil. The IVF medium was modified Tyrode's bicarbonate buffered solution supplemented with 10μg/mL heparin, 0.5μg/mL hypotaurine, 0.5μg/mL epinephrine and 6mg/mL BSA. The sperm suspension was then added at a volume of 10μL to the droplets with oocytes. After sperm-oocytes co-incubation at 39°C 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 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 39°C in 5% CO2, 7% O2 in 88% N2, according to 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 (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 (50±3.65%) and after sperm processing were (66.67±4.01%) for BoviPure® and (64.17±3.75%) 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 (P0.05) between them. However, there were no significant differences (P0.05) in the motility values between the sperm preparation methods. The sperm concentration before processing was (82.33±1.73×106spz/mL) and after sperm processing was (25.50±1.73×106spz/mL) for BoviPure® and for Percoll® (27.67±1.52×106spz/mL). Comparing the results of sperm concentration before processing with the results after sperm processing it was found out that there were significant differences (P0.01) between them. However, there were no significant differences (P0.05) for the concentration values between the sperm preparation methods.
The HOS test (Fig. 1) results before the processing were 39.05±2.97% and after the sperm processing with BoviPure® and Percoll® were 53.84±1.79, and 56.58±1.95%, respectively. Using the SYBR-14/PI test (Fig. 2) the obtained results before the processing were 42.97±2.22% and after processing 70.85±2.49 and 68.76±3.47% of the spermatozoa with the intact plasma membrane, respectively. The EthD-1/FITC-PSA test (Fig. 3) of live spermatozoa with the intact acrosomes were 46.90±4.64% before the sperm processing and after the processing were 74.12±1.34 and 67.46±5.17%, respectively. The significant differences were found out (P0.001) 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 Table 1. Investigating the methods of sperm preparation for the IVF (BoviPure® and Percoll®) it was found out that there were significant differences (P0.01) between the evaluation parameters before and after the sperm processing for the both separation protocols. We found no significant differences (P0.05) regarding sperm evaluation parameters between the protocols.
Fig. 1.Inactive sperms (white arrows) and different swelling patterns of the active sperms (red arrows) after HOS test (400×).
Fig. 2.Live (green) and dead (red) sperms after SYBR-14/PI test (400×).
Fig. 3.Live and dead sperms with intact acrosoma (red arrow) after EthD-1/FITC-PSA test (1000×).
Table 1.
Sperm parameters results (means±S.E.M.)
Sperm separation protocol / Progressive motility (%) / Concentration (106Ml) / HOS % active / SYBR-14/PI % live / EthD-1/FITC-PSA % live with intact acrosomeInitial (n=6) / 50.00±3.65 / 82.33±1.73* / 39.05±2.97 / 42.97±2.22 / 46.90±4.64
BoviPure (n=6) / 66.67±4.01* / 25.50±1.73 / 53.78±1.79** / 70.85±2.49** / 74.16±1.34**
Percoll (n=6) / 64.17±3.75* / 27.67±1.52 / 56.58±1.95** / 68.76±3.47** / 67.46±5.18*
*P0.05.
**P0.001.
The IVF and IVC results are shown in 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 75.80±1.77% for BoviPure® and 61.58±2.87% for Percoll®. The percentage of morulas and blastocysts on the 7th day of the culture were 28.21±2.42% for BoviPure® and 20.83±2.45% for Percoll®. The results of hatched blastocysts on the 10th day of the culture were 14.48±1.58% for BoviPure® and 10.58±2.32% for Percoll®. All mentioned percentages refer to the starting material. The IVF and IVC results were compared and significant differences (P0.05) 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 (P0.05).
Fig. 4.Cleavage, blastocysts and hatched blastocysts rates for BoviPure® and Percoll® (mean±S.E.M.). *P0.05.
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 (Mendes et al., 2003).
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 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 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, Göthenborg, 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®. 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 (P0.05). On the other hand, there were no significant differences (P0.05) in concentration results between the methods. 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.