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Animal Reproduction Science 111 (2009) 141148

Motility, acrosome integrity, membraneintegrity and oocyte cleavage rate of spermseparated by swim-up or Percoll gradient

method from frozenthawed buffalo semen

A. Mehmood a,

, M. Anwar a, S.M. Saqlan Naqvi ba Animal Sciences Institute, National Agricultural Research Centre, Islamabad 45500, Pakistan

b Department of Biochemistry, University of Arid Agriculture, Rawalpindi, Pakistan

Received 21 May 2007; received in revised form 31 January 2008; accepted 18 February 2008

Available online 10 March 2008

Abstract

Frozenthawed semen of five buffalo bulls was used to compare efficacy of swim-up and Percoll gra-

dient methods for separating viable spermatozoa. Sperm separated by the two methods were also tested

to differentiate buffalo bulls on the basis of in vitro fertilization (IVF) rates. Recovery of motile sperm

(%), increase in membrane integrity (%) and acrosome integrity (%) were compared after two sperm sep-

aration methods in experiment I, and in vitro fertilization rate (cleavage rate and cleavage index) was

compared in experiment II. Swim-up separated sperm showed a higher motility (P < 0.05), while per-

cent recovery of motile sperm was higher with Percoll separation (P < 0.05). Membrane integrity (%) of

sperm separated with swim-up was significantly higher (P < 0.05) as compared to sperm separated with

Percoll gradient. Swim-up separated sperm gave a higher cleavage rate and cleavage index ( P < 0.001).

Sperm separated by swim-up showed significant difference among the bulls in cleavage rate and cleav-

age index (P < 0.05), while the Percoll gradient method did not. It has been concluded that separation

of sperm from frozenthawed buffalo semen by swim-up method can be more expedient for IVF inbuffalo.

2008 Elsevier B.V. All rights reserved.

Keywords: Sperm separation; Swim-up; Percoll; Fertility; IVF; Buffalo

Corresponding author. Tel.: +92 51 9255363.

E-mail address: mehmood [email protected] (A. Mehmood).

0378-4320/$ see front matter 2008 Elsevier B.V. All rights reserved.

doi:10.1016/j.anireprosci.2008.02.011
mailto:[email protected]://dx.doi.org/10.1016/j.anireprosci.2008.02.011http://dx.doi.org/10.1016/j.anireprosci.2008.02.011mailto:[email protected]


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142 A. Mehmood et al. / Animal Reproduction Science 111 (2009) 141148

1. Introduction

Interest in the in vitro embryo production (IVEP) techniques in water buffalo (Bubalus bubalis)

has increased over the years (Nandi et al., 2002; Gasparrini et al., 2008). However, a low IVF

rate is one of the major factors limiting the commercial use of IVEP in this species (Nandi et al.,2002). Separation of highly motile sperm with intact membrane and acrosome is an important

step to improve IVF rate. Swim-up and Percoll gradient methods are mostly used for IVF. Per-

coll centrifugation, an easily performed procedure, results in a final pellet with large number of

spermatozoa including dead and abnormal cells, while swim-up procedure that uses the migrating

ability of sperm cells, provides highly motile spermatozoa but yields only 1020% of the total

initial sperm population in cattle (Parrish et al., 1995). Swim-up and Percoll separation techniques

have been compared to harvest viable sperm in bovine (Parrish et al., 1995; Somfai et al., 2002).

Although successful IVF in buffalo was carried out by sperm separated from frozenthawed

semen with swim-up (Nandi et al., 1998) or Percoll gradient technique (Totey et al., 1996), no

comparative study is available.Integrity of the sperm plasma membrane is essential for cell survival and fertilizing ability.

Moreover for a successful fertilization, a spermatozoon must maintain an intact acrosome up to

the time it binds to zona pellucida of the oocyte and undergoes the acrosome reaction to release

acrosome enzymes (Graham and Moce, 2005). Acrosome integrity was evaluated by Coomassie

Blue G-250 staining method, which has been shown to be a reliable method for the assessment

of acrosomal status in a variety of species including cattle (Larson and Miller, 1999) and buffalo

(Mehmood et al., 2007). Functional integrity of the sperm by hypoosmotic swelling test was

standardized for buffalo bulls (Rasul et al., 2000).

Individual bulls differ in their ability to fertilize oocytes in vitro as sperms have to depict

motility, membrane and acrosome integrity, and the ability to penetrate oocytes (Ward et al.,

2003). Harvesting viable population by sperm separation methods could also be informative to

predict in vitro or in vivo fertilizing ability of bulls (Zhang et al., 1998). Combination of three

sperm attributes i.e. the proportion of motile, acrosome intact and HOST-responsive sperm was

identified as significant predictor of the in vitro fertilizing potential of bull (Brito et al., 2003).

Present study was conducted to compare efficacy of swim-up and Percoll gradient methods

for separating viable spermatozoa. Sperm viability was evaluated by their motility, membrane

integrity, acrosome integrity and ability to fertilize oocytes in vitro. Furthermore, sperm separated

by the two methods were tested to differentiate buffalo bulls on the basis of IVF rates.

2. Material and methods

2.1. Semen source

Frozen semen (Tris, citric acid, egg yolk and glycerol) of five buffalo bulls was procured from

Semen Production Unit Karaniwala, Punjab, Pakistan. The bulls used were AI bulls, maintained

under standard management and examined routinely for all andrological parameters.

2.2. Experiment I

Semen of three different ejaculates collected and cryopreserved (0.5 ml straws) on three differ-

ent days was pooled for each bull separately. Pooled semen from the same bull was separated by

swim-up and Percoll gradient methods on the same day, replicated three times. Same amount of


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A. Mehmood et al. / Animal Reproduction Science 111 (2009) 141148 143

semen (0.5 ml) was processed for swim-up and Percoll gradient method. Sperm were evaluated

on the basis of progressive motility, recovery of motile sperm (%), increase in membrane integrity

(%) and acrosome integrity (%).

2.2.1. Swim-up

Sperm were separated by swim-up procedure as described by Lu et al. (1987). Briefly, 0.5 ml

(volume initial) of frozen thawed semen was layered in duplicate (0.25 ml each) under 1 ml of

modified Tyrodes medium with lactate and pyruvate (TALP) and incubated for 30 min at 39C

in humidified atmosphere of 5% CO2. The top 0.80 ml of medium from each tube was removed,

pooled and centrifuged at 1000 g for 10 min. The pellet was washed by centrifuging in fresh

TALP and final pellet was reconstituted in 0.5 ml of TALP (volumefinal).

2.2.2. Percoll gradient

Percoll gradient method was adopted from Parrish et al. (1995). Percoll (SigmaAldrichChemie GmbH, Germany) was mixed 9:1 (v/v) with a concentrated solution of TALP contain-

ing 31 mM KCl, 800 mM NaCl, 3 mM NaH2PO4 and 100 mM Hepes. The pH was adjusted to

7.3 with 1 N NaOH. Then CaCl2 2.0 mM, MgCl2 0.4 mM, lactic acid 21.6 mM and NaHCO325 mM were added to prepare 90% Percoll solution. The 90% Percoll was mixed with TALP (1:1,

v/v) to prepare 45% Percoll solution. Percoll density gradient consisted of 0.5 ml (volumeinitial)

frozenthawed semen layered over 2 ml of a 45% Percoll and 2 ml of a 90% Percoll in a 15 ml

conical plastic test tube. The gradient was centrifuged at 700 g for 15 min. After centrifugation,

the supernatant above the sperm fraction was carefully removed. Bottom fraction (0.5 ml) was

washed in 5 ml of TALP by centrifugation and final pellet was reconstituted in 0.5 ml of TALP

(volumefinal).

2.2.3. Sperm evaluation

Progressive motility, concentration, membrane and acrosome integrity of frozenthawed sper-

matozoa were assessed before and after sperm separation methods. Visual motility was evaluated

subjectively using a phase-contrast microscope (400). Sperm concentration was estimated using

a haemocytometer. Acrosome integrity (AcI) of sperm was determined by staining with Coomassie

Blue as described earlier for buffalo (Mehmood et al., 2007). Membrane integrity (MI) of sperm

was assessed using the hypoosmotic swelling (HOS) test validated for optimum conditions in

buffalo (Rasul et al., 2000). Briefly, HOS solution (200 mOsm/kg) containing sodium citrate(0.735%, w/v) and fructose (1.351%, w/v) was maintained at 37 C for 5 min before use. The

osmotic pressure of the final solution was adjusted to 100 mOsm/kg by diluting HOS with dis-

tilled deionised water (1:1). Each semen sample (25l) was mixed with HOS solution (250l)

and incubated at 37 C for 10 min. After incubation, two hundred sperm were counted for their

intact membrane (characterized by coiled tail) under a phase-contrast microscope (400). Percent

recovery of motile sperm, increase in MI (%) and AcI (%) was calculated as:

Recovery of motile sperm (%)=(Concentrationfinal volumefinal motilityfinal)

(Concentrationinitial volumeinitial motilityinitial)100

Increase in MI (%) =MI after treatmentMI before treatment

MI before treatment 100


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Increase in AcI (%) =AcI after treatmentAcI before treatment

AcI before treatment

2.3. Experiment II

Frozenthawed semen of five buffalo bulls were same as used in experiment I. Spermatozoa

were separated by swim-up and Percoll gradient method (experiment I). In vitro fertilization

rate (cleavage rate and cleavage index) was used to evaluate the sperm separation meth-

ods.

Method of COC recovery, in vitro maturation and in vitro fertilization was the same as reported

earlier for buffalo (Mehmood et al., 2007). Briefly, cumulus oocyte complexes (COCs) were

recovered from slaughter house buffalo ovaries by aspiration and in vitro matured in M 199 with

10% oestrus buffalo serum. Sperm pellet separated with swim-up and Percoll gradient methods

(experiment I) was diluted 1:1 with 100g/ml heparin solution in TALP and incubated for 30 min

for capacitation. Sperm dose used for IVF was 12 106/ml. After 48 h of co-incubation, eggs

were washed with medium 199 by vigorous pipetting and examined under stereomicroscope to

evaluate cleavage rate (number of oocytes cleaved 100/total COCs incubated) and cleavage

index (number of oocytes > two-cell stage 100/number of oocytes cleaved). The experiment

was repeated three times (replicates).

2.4. Statistical analysis

A 2 (separation methods) 5 (bulls) factorial experiment with completely randomized design

(CRD) was used and effect of separation method and bulls was observed. The experiment was

repeated three times. Data are expressed as mean S.D. in experiment I. Spermatological param-

eters of swim-up and Percoll gradient separated spermatozoa were analyzed by two-way ANOVA

and comparisons were made with LSD. In experiment II, cleavage rate and cleavage index were

compared using Chi-square (Minitab 12.22, 1996).

3. Results

3.1. Sperm recovery and viability

Post-thaw sperm concentration and motility did not differ (P > 0.05) among semen from

five buffalo bulls. There was no bull effect (P > 0.05) on sperm recovery, membrane integrity

and acrosome integrity after sperm separation with swim-up and Percoll gradient methods.

Therefore, data on these parameters have been pooled. Data on concentration, motility and

recovery of motile sperm after separation with swim-up and Percoll gradient method are pre-

sented in Table 1. Overall motility was significantly higher with swim-up than the Percollgradient method (P < 0.05). However, number of spermatozoa recovered and percent recov-

ery of motile sperm was significantly higher with Percoll gradient as compared to swim-up

(P < 0.05).


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Table 1

Sperm recovery from frozenthawed buffalo bull semen separated with swim-up and Percoll gradient methods

Sample Concentration (106 /ml) Motility (%) Volume layered (ml) Recovery of motile sperm* (%)

Frozenthawed 139.0 13.9 38.5 4.9

Swim-up 2.8 1.5a 69.1 8.0a 0.5 4.1 2.8a

Percoll gradient 4.7 1.5b 63.1 9.0b 0.5 6.0 3.0b

Values are meanS.D. of five bulls and three replicates (n = 15). a,bValues in a column with different superscript differ

(P < 0.05).* Volumeinitial (0.5 ml) = volumefinal (0.5 ml), therefore not used in the calculation.

Table 2

Increase in membrane integrity (increase MI %) and acrosome integrity (increase AcI %) of spermatozoa separated from

frozenthawed buffalo bull semen with swim-up and Percoll gradient methods

Method Increase MI (%) Increase AcI (%)

Swim-up 77.3 8.9a 73.0 7.4

Percoll 70.5 7.6b 70.6 11.1

Data are cumulative values of five bulls and three replicates (n = 15). a,bValues in a column with different superscript differ

(P < 0.05).

Increase in membrane integrity and acrosome integrity of frozenthawed buffalo bull semen

separated by swim-up or Percoll gradient is shown in Table 2. The increase in membrane integrity

of swim-up sperm was significantly greater (P < 0.05) compared with Percoll gradient separated

sperm. Increase in acrosome integrity of the sperm did not differ between two separation methods

(P > 0.05).

3.2. In vitro fertilization

Fertilizing ability of spermatozoa separated by the swim-up and Percoll gradient methods is

shown in Table 3. Overall cleavage rate of the oocytes inseminated with swim-up (66.8%) sep-

arated sperm was significantly greater (P < 0.001) than with Percoll gradient (55.6%) separated

spermatozoa. Similarly the cleavage index was significantly higher (P < 0.001) with sperm sep-

Table 3

Cleavage rate and cleavage index after in vitro fertilization by sperm from five buffalo bulls separated by swim-up andPercoll gradient methods

Bull ID no. Cleavage rate (frequency) Cleavage index (frequency)

Swim-up Percoll gradient Swim-up Percoll gradient

1 71.6ab (48/67) 61.5 (40/65) 56.3ab (27/48) 40.0 (16/40)

2 64.6b (42/65) 50.8 (33/65) 38.1b (16/42) 30.3 (10/33)

3 53.7c (36/67) 56.9 (37/65) 33.3b (12/36) 24.3 (9/37)

4 82.1a (55/67) 56.9 (37/65) 65.5a (36/55) 40.5 (15/37)

5 61.5b (40/65) 51.6 (33/64) 40.0b (16/40) 30.3 (10/33)

Overall

66.8x

(221/331) 55.6y

(180/324) 48.4p

(107/221) 33.3q

(60/180)

abcValues in a column with different superscript differ (P < 0.001). x,y and p,q Values in a row within cleavage rate and

cleavage index differ (P < 0.001). Data are cumulative values of five bulls and three replicates (n = 15).


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arated by swim-up method (48.4%) as compared to Percoll gradient (33.3%). Bull effect was

significant (P < 0.001) for the cleavage rate and cleavage index of spermatozoa separated with

swim-up method. Highest cleavage rate (82.1%) and cleavage index (65.5%) was observed in Bull

ID no. 4. Whereas, Bull ID no. 3 showed lowest cleavage rate (53.7%) and cleavage index (33.3%).

The differences among the bulls in cleavage rate and cleavage index of oocytes inseminated withsperm separated with Percoll gradient method were non significant (P > 0.05).

4. Discussion

Buffalo embryos have been produced in vitro using sperm separated with swim-up (Nandi et

al., 1998) and Percoll gradient (Totey et al., 1996) methods from frozenthawed semen. However,

no direct comparison has been reported to harvest viable spermatozoa. Present study compared

the quality of sperm separated from frozenthawed buffalo semen by swim-up or Percoll gradient

method. There was no significant difference in recovery of motile sperm, membrane integrity

and acrosome integrity among semen samples of five buffalo bulls separated by both methods inthe present study and is in conformity with earlier study in cattle (Brito et al., 2003). Therefore,

cumulative data on these parameters were used to compare swim-up and Percoll gradient. Sperm

separated by the two methods were tested to differentiate buffalo bulls on the basis of IVF rates.

The recovery of motile spermatozoa (%) was significantly higher (P < 0.05) with Percoll gra-

dient separation. This is because of higher concentration of spermatozoa recovered with Percoll

gradient as compared to swim-up method and is in consistent with the earlier findings in human

and cattle. However, overall motility was significantly higher with swim-up method. Motility

enhances the ability of sperm to penetrate the zona pellucida of the oocyte (Suarez and Ho, 2003).

Less in number but more viable sperm were recovered after swim-up separation in comparison

with Percoll gradient in human (Englert et al., 1992). In cattle more sperm were recovered after

Percoll gradient separation than swim-up, however penetration rate was higher with swim-up

separated sperm (Parrish et al., 1995). Observations of the present study with buffalo sperm sup-

port the notion that swim-up method is based on the separation of sperm with respect to function

i.e. motility (Parrish et al., 1995) whereas Percoll gradient selects sperm with respect to their

density (Le Lannou and Blanchard, 1988) and is not a physiological mean of separating viable

spermatozoa.

Swim-up method rendered a significantly greater number of sperm with intact membrane

compared with Percoll gradient whereas acrosome integrity of the sperm did not differ between

two separation methods. Parrish et al. (1995) reported greater number of viable sperm after swim-up than Percoll gradient in cattle. Palomo et al. (1999) found significantly high motility, membrane

integrity and acrosome integrity after separating freshly ejaculated goat sperm with swim-up as

compared to Ficoll and Percoll gradient. However, Somfai et al. (2002) observed higher viable

sperm with intact acrosome after Percoll separation than that after swim-up of frozenthawed

bull sperm. Nonsignificant difference in acrosome intact sperm in the present study might be due

to incubation time, as lingering time period used in swim-up method increases the likelihood of

acrosome-reacted sperm (Correa and Zavos, 1996).

Cleavage rate after in vitro fertilization is the best available endpoint for expressing fertility

when obtaining data to validate a potential diagnosis assay for sperm viability (Ward et al.,

2003). Thus in vitro fertility of spermatozoa separated with swim-up and Percoll gradient methodfrom frozenthawed semen of five buffalo bulls was evaluated on the basis of cleavage rate in

the present study. Significantly higher cleavage rate and cleavage index were observed when

oocytes were inseminated with swim-up separated sperm than with Percoll gradient ones. Swim-


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up separated bovine spermatozoa have also been shown to penetrate significantly greater number

of oocytes as compared to Percoll gradient ones (Parrish et al., 1995). This might be due to the

significantly higher motility of spermatozoa separated by swim-up as indicated in experiment

I. Motility is an important parameter in fertility that is why sperm motility enhancers such as

pencillaminehypotaurineepinephrine syrup (Lu et al., 1987) and caffeine (Pomeroy et al., 1988)have been employed in IVF procedures to improve IVF rate.

The results showed significant difference among the buffalo bulls on the basis of IVF rate by

swim-up separated sperm. The cleavage rate of individual bulls was categorized into three groups

with almost similar trend in the cleavage index. There were no differences among the bulls in

cleavage rate and cleavage index of oocytes inseminated with Percoll gradient separated sperm.

When development of embryos fertilized by either swim-up or Percoll separated spermatozoa

was compared for semen from five cattle bulls, a difference in cleavage rate was found in favor

of swim-up separated spermatozoa (Parrish et al., 1995). However, the authors reported that this

disadvantage of the Percoll procedure was overcome by increasing sperm concentration from 1

to 5 106/ml sperm during IVF. Comparatively lower sperm concentration for IVF was used inthe present study as IVF was reported to be the best predictor of field fertility at lower sperm

concentration in cattle (Ward et al., 2003) and sheep (OMeara et al., 2005). Hillery et al. (1990)

showed that the yield of embryos after IVF for high in vivo fertility bulls (non return rate = 78%)

was almost double (32% vs. 18%) than that for the low fertility bulls (non return rate = 66%).

Therefore, the bull difference in cleavage rate by swim-up separated sperm showed the perspective

of using IVF technique for in vivo fertility test in buffalo bulls. However, relationship of IVF by

swim-up separated spermatozoa to in vivo fertility of buffalo bulls should be undertaken before

its recommendation for routine buffalo bull fertility test.

It is concluded that swim-up and Percoll gradient are effective to recover viable sperm from

frozenthawed buffalo semen. Although Percoll gradient separated greater number, swim-up

separated sperm had significantly higher motility and showed greater IVF rate (cleavage rate and

cleavage index). Moreover swim-up separated sperm were able to differentiate buffalo bulls on

the basis of IVF rate.

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