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
a
Clinic for Obstetrics and Reproduction, Faculty of Veterinary Medicine, University of
Zagreb, Heinzelova 55, 10000 Zagreb, Croatia
b
Centre 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 × 106 spz/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 × 106 spz/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
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 (15–30 nm 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 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®
buffer 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 centrifugated for 10 min at 500 × g. This final pellet was resuspended in
150 μL of mIVF, and the final concentration was adjusted to 1 × 106 spz/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 1.5 mL of 45% Percoll® solution on the 1.5 mL of
90% solution in 15 mL Falcon® tubes. On the top of the gradient 400 μL of thawed semen
was layered and then tubes were centrifugated for 15 min at 700 × g. The pellets were
resuspended in the same amount of HEPES-TALP media and centrifugated for 7.5 min at
300 × g. Afterwards the pellets were resuspended in mIVF and the final concentration was
adjusted to 1 × 106 spz/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
100 mOsm/kg as described for frozen-thawed bovine spermatozoa by Correa and Zavos
(1994). The assay was performed mixing 50 μL of semen with 1 mL of hypoosmotic solution
and incubating at 37 °C for 60 min. 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 50 μL thawed semen were diluted in 150 μL of mTALP containing
3 μL PI and 2 μL 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-14stained 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 (15 μL) of ethidium homodimer (EthD-1) counter-stained semen were
smeared onto microscope slides, air dried, fixed and permeabilized with 96% ethanol for 30 s.
The unbounded dye of EthD-1 was removed using centrifugation at 200 × g for 5 min twice,
preventing that excess of dye stain live spermatozoa after permeabilization with ethanol. We
kept smear for 15 min at −20 °C and then eliminated the ethanol. Twenty microliters of FITClabeled pisum sativum agglutinin (FITC-PSA) solution (100 μg/mL) in PBS were spread over
each smear and incubated in moist chamber at 37 °C for 7 min. They were subsequently
agitated in destilled water to remove unbound dye, air dried and mounted with 15 μL of antifade 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 2 h after slaughter and transported to the laboratory in
physiological saline (0.9%) with antibiotics (100 I.U. penicillin and 100 μg streptomycin/mL)
at 37 °C. Immature bovine oocytes were aspirated from 2 to 8 mm 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 15 mM 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), 1 μg/mL estradiol-17β and 100 μM cysteamine. Oocytes were incubated in 50 μL
droplets of maturation media under mineral oil at 39 °C and with 5% CO2 for 24 h.
2.6. In vitro fertilization and culture (IVF and IVC)
The expanded COCs were washed in TALP-HEPES medium supplemented with 3 μg/mL
BSA-FAF and transfered in 40 μL droplets of IVF medium under mineral oil. The IVF
medium was modified Tyrode's bicarbonate buffered solution supplemented with 10 μg/mL
heparin, 0.5 μg/mL hypotaurine, 0.5 μg/mL epinephrine and 6 mg/mL BSA. The sperm
suspension was then added at a volume of 10 μL to the droplets with oocytes. After spermoocytes co-incubation at 39 °C and with 5% CO2 for 18–24 h, 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
8 mg/mL BSA, according to Edwards et al. (1997) was used. Fertilized oocytes were cultured
in vitro in SOF medium without glucose for 48 h and then transfered in SOF with 1.5 mM
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 (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 (82.33 ± 1.73 × 106 spz/mL) and
after sperm processing was (25.50 ± 1.73 × 106 spz/mL) for BoviPure® and for Percoll®
(27.67 ± 1.52 × 106 spz/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 (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 (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 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.
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
(106 Ml)
HOS %
active
SYBR-14/PI
% live
EthD-1/FITC-PSA
% live with intact
acrosome
Initial (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*
*
P < 0.05.
P < 0.001.
**
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 (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).
Fig. 4. Cleavage, blastocysts and hatched blastocysts rates for BoviPure® and Percoll® (mean ± S.E.M.).
*
P < 0.05.
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 (P < 0.05). On
the other hand, there were no significant differences (P < 0.05) 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.
Comparing the initial sperm results of HOS (39.05%), SYBR-14/PI (42.97%) and EthD1/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. Correa and Zavos (1996)
yielded 41–46% and Matković et al. (2002) obtained 53% of sperm cells with functionally
active plasma membrane using the Percoll® method. 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 (Verberckmoes et al., 2000) and also in rams (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 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 (Centola et al.,
1998 and Sonderlund and Lundin, 2000). 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 48 h 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 48 h 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 didn’t 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.
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