|

Received: 24 February 2017 Accepted: 18 June 2017

DOI: 10.1111/rda.13036

ORIGINAL ARTICLE

Single layer centrifugation of fresh dromedary camel semen

improves sperm quality and in vitro fertilization capacity
compared with simple sperm washing

C Malo1 |EG Crichton1|JM Morrell2 |BS Pukazhenthi3|JA Skidmore1

1
Camel Reproduction Center, Dubai, United
Arab Emirates
2
Clinical Sciences,Swedish University of
Agricultural Sciences (SLU), Uppsala, Sweden
3
Center for Species Survival,Smithsonian
Conservation Biology Institute, Front Royal,
VA, USA
Correspondence
Clara Malo, Camel Reproduction Center,
Dubai, United Arab Emirates.
Email: clara@camelreproductioncenter.ae
Contents
Single layer centrifugation (SLC) through a colloid is a tool for selecting viable
mammalian spermatozoa but has not been used previously for fresh dromedary camel
sperm. Semen from six camels (2 ejaculates/male) was diluted 1:5 (v:v) or 1:10 (v:v) in
a Triscitratefructose buffer for mechanical liquefaction by gentle pipetting.
Following liquefaction, semen was processed either by SLC or by centrifugation with-
out a colloid (control). Total and progressive motilities, CASA kinematics, vitality and
acrosome integrity (eosinnigrosin) and plasma membrane integrity (Hypo-osmotic
swelling test; HOST), and fertilizing ability in a heterologous assay (zona-free goat
oocytes) were evaluated. Both total (p=.003) and progressive motilities (p=.003)
were higher in SLC-processed than in control semen samples, irrespective of dilution.
Positive HOST values increased when using colloid in 1:5 (p=.001) and 1:10 dilution
(p=.010). Colloid-selected sperm had higher penetration rates than controls (p<.001
and p=.02 for 1:5 and 1:10 dilutions, respectively). However, only the SLC sperm at
1:5 dilution showed higher percentages of pronuclear formation (p=.02) than con-
trols. Dilution effect was only significant for total motility before in vitro fertilization,
with higher values for the 1:5 dilution (p=.033). The recovery rates of motile sperm
between dilutions were similar (26.1% vs 35.4%; p=.226). In conclusion, SLC is a
promising tool for selecting functional dromedary camel sperm and warrants more
research.

1| INTRODUCTION
Good quality spermatozoa are important to achieve successful fertil-
ization. However, the high viscosity and poor quality of semen from the
dromedary camel are major challenges to the successful processing of
sperm for this species (Deen, Vyas, & Sahani, 2003). Various products
have been developed to improve and purify mammalian cell popula-
tions, including spermatozoa. One such product, Percoll, composed of
a polyvinylpyrrolidone-coated silica colloid, has been used extensively
in many species to prepare spermatozoa for IVF (e.g. Moore, Squires,
& Graham, 2005; Noguchi etal., 2015); however, it is generally consid-
ered to be impractical to use when preparing sperm samples for artifi-
cial insemination (AI; Morrell & Rodriguez-Martinez, 2010) and there
have been reports of toxicity with some batches of Percoll (Avery &
Grav, 1995). Typically two or more layers of colloid of different den-
sities are prepared and utilized to separate populations of live motile
sperm from seminal plasma, dead cells, debris and extender. Khatir,
Anouassi, and Tibary (2009) used Percoll, in two layers, to select live
sperm from dromedary camel ejaculates in order to perform IVF.
In recent years, Morrell, Rodriguez-Martinez, and Johannisson
(2010) have developed new colloid formulations based on silane-
coated silica particles, designed to address the particular semen char-
acteristics of a number of livestock species and overcome the problems
of scaling-up the technology to recover sufficient sperm for AI. Specific
to this technology is that only a single layer of colloid is needed to
select live spermatozoa. This fact renders it more user-friendly and

Reprod Dom Anim. 2017;17. 2017 Blackwell Verlag GmbH | 1

wileyonlinelibrary.com/journal/rda
 |
2 MALO etal.
less time-consuming than a traditional density gradient. Single layer
centrifugation (SLC) has been successfully used for viable sperm
selection in the stallion (Morrell, Johannisson, Dalin, & Rodriguez-
Martinez, 2009; Morrell, Johannisson, & Rodriguez-Martinez, 2011),
bull (Morrell, Rodriguez-Martinez, & Andersson, 2014; Samardzija
etal., 2006; Thys etal., 2009), boar (Morrell & Wallgren, 2011), dog
(Dorado, Glvez, Morrell, Alcarz, & Hidalgo, 2013), goat (Jimnez-
Rabadn etal., 2012), donkey (Ortiz etal., 2015), brown bear (lvarez-
Rodrguez etal., 2016), cat (Chatdarong, Thuwanut, & Morrell, 2010)
and llama (Santa Cruz etal., 2016; Trasorras etal., 2012). The SLC-
selected populations of sperm have higher motility and membrane in-
tegrity than non-selected sperm samples (Macas-Garca etal., 2009),
and also more morphologically normal and functionally competent
sperm, and fewer sperm with damaged DNA. Recent research indi-
cates that SLC-selected sperm also produce less hydrogen peroxide
than controls (Morrell, Lagerqvist, Humblot, & Johannisson, 2016).
Enhanced fertility characteristics have been reported for SLC sperm,
and their fertility confirmed for some species (stallion, Morrell, Richter
etal., 2014; bull, Thys etal., 2009; boar, Sjunnesson, Morrell, &
Gonzlez, 2013; and llama, Trasorras etal., 2012) despite the removal
of seminal plasma proteins and possibly cholesterol during processing
(Kruse, Dutta, & Morrell, 2011).
While colloid selection has been applied to select good quality
sperm from llama ejaculates treated with enzymes (Santa Cruz etal.,
2016; Trasorras etal., 2012), there is no comparative information
on the value of SLC for sperm selection and function in dromedary
camels. Considering the challenges presented by the low volume and
sperm concentration of the dromedary camel ejaculate, and its ex-
cessive viscosity (Skidmore, Morton, & Billah, 2013), the aim of this
study was to determine the efficacy of SLC selection of sperm of this
species. We hypothesized that sperm of higher functionality might be
obtained from processing ejaculates by SLC, leading to improved in
vitro, and ultimately, in vivo functionality. Therefore, this study was
designed to evaluate the motility and plasma membrane character-
istics of fresh camel sperm and their in vitro sperm function using a
heterologous IVF system, following SLC selection.
2| MATERIALS AND METHODS
Unless otherwise indicated, all chemicals were from Sigma-Aldrich Co.
(St Louis, MO, USA).
The medium used for semen collection was Triscitratefructose
buffer (TCF, pH=6.9) composed of 300mM TRIS, 94.7mM citric acid
and 27.8mM fructose (Evans & Maxwell, 1987). Bovine serum albu-
min (0.05%, wt/v) and EDTA (10mM) were added with 4% egg yolk
and the solution sterilized by filtration (0.2m).
Colloid One (JM Morrell, patent applied for) was used for the ex-
periment. The medium used for the collection and washing of goat
cumulus oocyte complexes (COCs) was phosphate-buffered saline
(PBS) composed of 136.89mM NaCl, 2.68mM KCl, 8.1mM Na2HPO4
and 1.46mM CaCl22H2O, 0.34mM sodium pyruvate, 5.4mM glu-
cose and 70mg/ml kanamycin. The basic medium used for oocyte
maturation was a modified North Caroline State University (NCSU)-23
medium supplemented with 10% (v/v) heat-inactivated foetal calf
serum (FCS) and 0.8mM cysteine (Petters & Wells, 1993). Fertilization
medium was M199 with Earles salts supplemented with 6% FCS (v:v),
0.91mM sodium pyruvate, 3.05mM d-glucose, 2.92mM calcium lac-
tate, 50IU kanamycin and 30g/ml streptomycin sulphate (Crichton,
Malo, Pukazhenthi, Nagy, & Skidmore, 2016).
2.1|Animals
Six dromedary camel males from the Camel Reproduction Centre
(Dubai, UAE) were used in this study. Semen (two ejaculates/animal)
was collected between December and March, using an artificial vagina
(Skidmore etal., 2013), and immediately transported to the laboratory
where it was placed in a 37C water bath. All animal procedures were
carried out according to the guidelines of the Animal Care and Use
Committee of the Camel Reproduction Centre, UAE.
2.2|Semen preparation
Semen aliquots were extended with TCF at two dilution rates (1:5
and 1:10, v:v) and manually liquefied by gentle pipetting (usually
2030min), thus releasing the sperm. Aliquots of liquefied semen
were removed for concentration and assessment of total motility, to
calculate the sperm yield after SLC; the remainder was divided into
two aliquots: sperm for SLC were processed according to Morrell etal.
(2009) with some modifications. Briefly, 2ml of the diluted semen
was layered over 2ml of colloid at room temperature in 15-ml tubes
and centrifuged at 300 g for 20min (SLC). At the same time, a 2ml
aliquot of diluted semen was centrifuged at the same speed (control).
Following centrifugation, the supernatant (semen extender, seminal
plasma and colloids) was discarded and the sperm pellets were resus-
pended in TCF for analysis of concentration, motility, acrosome and
membrane integrity or in fertilization medium for IVF.
2.3|Sperm assessment
To determine motility parameters, sperm samples were placed in a
disposable chamber (Cytonix, Beltsville, MD, USA) and 300 sperma-
tozoa were analysed by means of a computer-assisted semen analysis
(CASA) system (CEROS II; Hamilton Thorne, MA, USA). Specifically,
total motility, progressive motility and kinematic parameters were
evaluated. Sperm kinematics included velocity average path (VAP;
m/s), velocity straight line (VSL; m/s), velocity curvilinear (VCL;
m/s), amplitude of lateral head (ALH, M), straightness (STR, %), lin-
earity (LIN, %) and beat-cross frequency (BCF, Hz).
Acrosome and membrane integrity (vitality) were evaluated using
the eosinnigrosin stain (Bamba, 1988). An aliquot of each semen sam-
ple was diluted 1:1 (v:v) with stain solution (5% eosin, 10% nigrosin in
0.1M citrate solution) and smeared on a glass slide which was then
dried on a warm plate. A total of 100 spermatozoa were assessed per
sample, and the percentage of intact acrosomes and live sperm were
calculated.
MALO etal. |
3

T A B L E 1 Effect of SLC and initial dilution (1:5 and 1:10) on total motility (TM), progressive motility (PM), kinematics (ALH, BCF, LIN, STR
VAP, VCL, VSL), vitality (VIT), acrosome integrity (AI) and membrane functionality (HOST) on fresh dromedary camel sperm

1:5 DILUTION 1:10 DILUTION SLC 1:5 vs 1:10

CONTROL CONTROL
(n=12) SLC (n=12) p-Value (n=12) SLC (n=12) p-Value p-Value

TM 41.705.82 62.357.38 .003* 40.395.35 60.376.70 .003* .375*

PM 12.742.11 26.064.68 .003* 10.872.15 26.964.62 .003** .239**
ALH 8.590.83 9.290.83 .272** 8.321.09 9.300.69 .308** .695**
BCF 23.080.41 22.420.82 .373* 23.580.75 22.870.79 .333* .377*
LIN 34.071.69 34.352.05 .875** 34.602.33 33.541.04 .533* .528*
STR 65.342.28 68.102.01 .108* 65.652.48 67.501.63 .408* .758*
VAP 87.669.32 91.458.12 .875** 83.4511.32 91.277.28 .695** .433**
VCL 176.0519.87 194.0318.30 .182** 171.6224.87 194.3216.47 .583** .814**
VSL 54.944.56 60.185.07 .209** 51.525.77 60.874.73 .308** .209**
VIT 69.174.57 73.174.43 .294* 72.083.98 70.754.93 .711* .022*
AI 71.004.19 77.584.25 .103* 73.753.23 75.175.06 .727* .268*
HOST 30.672.70 51.005.16 .001* 30.832.87 48.584.09 .010** .783**

Values represent data from six males (two ejaculates/male). Data represents meanSE
*Significance of Students t test for paired samples.
**Significance of Wilcoxon test.
Bold values express significant differences.

Sperm membrane functionality was assessed by the hypo-osmotic
swelling test (HOST), as described by Jeyendran, Van der Ven, Perez-
Pelaez, Crabo, and Zaneveld (1984). In brief, the hypo-osmotic solu-
tion was prepared by dissolving 0.73g sodium citrate and 1.35g
fructose in 100ml of distilled water. Hypo-osmotic solution (100l)
was mixed with 30l of semen and incubated at 37C for 1hr. A total
of 100 spermatozoa were assessed per sample for their swelling char-
acterized by coiled tails indicating an intact plasma membrane.
The sperm recoveries of the SLC samples were calculated by divid-
ing the total motile sperm obtained after the SLC by the total motile
sperm prior to centrifugation with SLC and multiplying by 100.
2.4|Heterologous zona-free oocyte
penetration assay
The in vitro spermoocyte interaction ability of SLC and control sperm
was evaluated in a heterologous IVF system using in vitro-matured,
zona pellucida-free goat oocytes according to the method of Crichton
etal. (2016). Briefly, COCs aspirated from goat ovaries were matured
overnight in vitro. Following the removal of their cumulus cells and
zonae pellucidae the next day, denuded oocytes were co-incubated
(39C, 5% CO2 in humidified air) for 1824hr in fertilization medium
with 1106 motile SLC or control sperm/ml retrieved from pellets
following centrifugation. Oocytes were mounted on slides under
vaseline-supported cover slips, fixed for 72hr in 25% (v/v) acetic acid
in ethanol, stained with 1% (v/v) Lacmoid and examined (100) using
bright field microscopy. For each male, sperm from each dilution (1:5
and 1:10) and treatment (SLC and control) were co-incubated with
1315 COCs and the experiment replicated twice. A total of 635
oocytes were thus used to evaluate the ability of SLC and control
sperm from two dilutions to penetrate and de-condense within goat
ooplasm. In addition, numbers of sperm/penetrated oocyte were
counted. Degenerated oocytes were not counted.
2.5|Experimental design
Ejaculates were obtained from six bulls and the experiment repeated
twice. The effect of dilution was tested by adding TCF to aliquots of
semen at 1:5 and 1:10, followed by liquefaction by repeated gentle
pipetting. Aliquots of the liquefied semen were centrifuged using SLC,
while other aliquots were centrifuged without the colloid as controls.
Total motility, progressive motility, kinetics, acrosome integrity, func-
tionality and integrity of the membrane were measured after centrifu-
gation. A total of 635 zona pellucida-free goat oocytes were evaluated
following in vitro co-incubation with sperm from different treatments
(in vitro penetration, pronuclear formation and number of sperm per
oocyte). Treatment means in the sperm evaluation assays were ana-
lysed by Students t test or MannWhitney, depending on whether
or not the data were normally distributed. Oocyte penetration results
were analysed by the chi-squared test.
2.6|Statistical analysis
Percentages were used to describe variables, and meansstandard
errors (SE) were calculated.
For quantitative variables (CASA values, vitality, HOST, acrosome
integrity and number of sperm per oocyte penetrated), the Saphiro
Wilk test was applied to check normal distribution of quantitative
variables. To compare several means, those data that were normally
distributed were analysed by Students t test for paired samples
 |
4 MALO etal.

T A B L E 2 Effect of SLC and initial dilution (1:5 and 1:10) on total motility (TM), progressive motility (PM), and kinematics (ALH, BCF, LIN,
STR VAP, VCL, VSL) on pre-IVF dromedary camel sperm

1:5 Dilution 1:10 Dilution SLC 1:5 vs 1:10

Control (n=12) SLC (n=12) p-Value Control (n=12) SLC (n=12) p-Value p-Value

TM 21.204.63 47.335.53 .003** 20.734.95 42.605.30 .004** .033**

PM 8.102.22 22.363.39 .003** 9.232.48 21.923.29 .005** .657**
ALH 9.020.33 8.630.43 .145* 8.700.43 9.560.48 .056* .826*
BCF 24.350.91 23.140.58 .292* 24.580.71 23.500.60 .334* .578*
LIN 32.480.89 33.240.51 .398* 33.431.06 33.501.05 .961* .807*
STR 63.151.64 68.141.42 .004* 65.711.29 70.771.89 .036* .156*
VAP 87.906.33 96.035.94 .325* 89.885.86 97.435.64 .199* .749*
VCL 181.1611.76 203.789.86 .127* 181.9911.65 210.9412.33 .039* .421*
VSL 54.523.64 65.173.20 .051* 57.983.76 68.353.18 .011* .326***

Values represent data from six males (two ejaculates/male). Data represents meanSE
*Significance of Students t test for paired samples.
**Significance of Wilcoxon test.
Bold values express significant differences.

andthe non-normal data were analysed by Wilcoxon test except in
the case of number of sperm per oocyte penetrated when the Mann
Whitney test was used. Values were expressed as meansSE.
For penetration and pronuclear formation percentages, the as-
sociation between two qualitative variables was checked using the
Pearsons chi-squared test, as comparison of post-centrifugation
sperm parameters for control and SLC sperm in 1:5 and 1:10 dilutions.
PercentagesSE were used to describe qualitative variables.
All analyses were performed using SPSS 19.0 for Windows, and
significance was set at p<.05.
3| RESULTS
Both total and progressive motility were higher in SLC sperm samples
compared to controls at 1:5 and 1:10 (p=.003 for both; Table1). A
higher proportion of membrane-intact sperm were identified by HOST
in SLC samples than in controls in both 1:5 and 1:10 dilution (p=.001;
p=.010 respectively). There were no differences (p>.05) between SLC
and control sperm in sperm kinematics, acrosome integrity or vitality.
No significant differences were found between the two dilutions in all of
the parameters studied. There were no differences (p=.226) in sperm
recovery between dilution rates (1:5, 26.15.2%; 1:10, 35.46.9%).
Motility patterns in pre-IVF samples are shown in Table2. Total
and progressive motilities were higher in the SLC group in both 1:5
(p=.003 both) and 1:10 dilution (p=.004; p=.005, respectively).
Among kinematic values, only STR showed significance in 1:5 and 1:10
dilution (p=.004, p=.036, respectively) between the treatments with
SLC sperm showing higher values than their respective controls. VCL
and VSL values were higher in SLC group than in control for dilution
1:10 (p=.039, p=.011, respectively), but no differences were seen
at 1:5 dilution. In pre-IVF parameters, dilution rate exerted an effect
only at TM (p=.033), reaching higher values for 1:5 than 1:10 dilution.
There were significant differences between SLC (p<.001) and con-
trol (p=.017) sperm for oocyte penetration and pronuclear formation,
with higher values found for SLC sperm (Tables3 and 4). There were no
differences between dilutions in the ability of sperm to perform in IVF.
There were no differences among treatments for the number of
sperm per oocyte penetrated (p=.094; Table5).
4|DISCUSSION
To our knowledge, this is the first attempt to select sperm by SLC
in the dromedary camel. Our results indicated that SLC processing
of semen resulted in an enriched population of motile sperm with
enhanced total and progressive motility, positive HOST and in vitro
penetration rates compared to non-selected sperm (control). These
benefits were common to both sperm dilutions with no difference be-
tween them, including the recovery of motile sperm.
Dromedary camel semen is highly viscous in nature; sperm are ren-
dered motionless in the gel, and liquefaction to release them is slow
(Deen etal., 2003). Various studies have used enzymatic approaches
to liquefaction (Bravo, Skidmore, & Zhao, 2000; El-Bahrawy & El-
Hassanien, 2009; Giuliano etal., 2010; Tibary & Anouassi, 1997) with
recent studies indicating papain as the most effective treatment for
camelid sperm (Kershaw, Evan, Rodney, & Maxwell, 2016; Kershaw-
Young, Stuart, Evans, & Maxwell, 2013). However, the use of enzymes
remains contentious as some researchers have observed a detrimental
impact on sperm quality (Morton, Gibb, Leathy, & Maxwell, 2012). The
present study assessed the possibility of liquefying the samples using
large dilutions and gentle pipetting in extender which together re-
duced the viscosity substantially within a period of 2030min. In gen-
eral, no differences in sperm quality or performance were observed
between the two dilutions, such that either could be used in the future
handling of dromedary camel sperm.
MALO etal. 5|
T A B L E 3 Effect of SLC and initial
SLC Control Total p-Value
dilution (1:5 and 1:10) of fresh dromedary
camel sperm and their ability to penetrate Dilution 1:5 83.404.17 64.306.92 74.104.09 <.001
ZP-free goat oocytes Dilution 1:10 76.305.45 63.906.95 70.404.44 .02
Total 79.903.42 64.304.89 72.303.01 <.001
p-Value .102 .933 .29

Data represent co-incubation of sperm of six males (two ejaculates/male) and 635 zona-free goat oo-
cytes (groups of 1315 oocytes per treatment per ejaculation). PercentagesSE.
Bold values express significant differences.

T A B L E 4 Effect of SLC and initial

Dilution SLC Control Total p-Value
dilution (1:5 and 1:10) on the ability of
fresh dromedary camel sperm to form 1:5 53.007.51 40.107.24 46.705.30 .02
pronuclei in ZP-free goat oocytes 1:10 48.107.52 42.207.35 45.205.28 .292
Total 50.605.32 41.205.16 46.003.74 .017
p-Value .375 .709 .703

Data represent results of co-incubation of sperm of six males (two ejaculates/male) and 635 zona-free
goat oocytes with groups of 1315 oocytes per treatment per ejaculation). PercentagesSE.
Bold values express significant differences.

T A B L E 5 Effect of SLC and initial

Dilution SLC Control Total p-Value
dilution (1:5 and 1:10) on numbers of
sperm/penetrated oocyte in a heterologous 1:5 4.070.338 2.930.243 3.580.222 .057
IVF system. Data represents meanSE 1:10 3.730.276 3.400.273 3.580.195 .576
Total 3.910.221 3.170.182 3.580.149 .094
p-Value .867 .173 .222

Total and progressive motility are essential parameters to eval-
uate the overall quality of spermatozoa (Love, 2011). In our study,
both these parameters were increased in SLC-prepared samples
compared with controls. Similar results have been reported for bulls
(Thys etal., 2009), stallions (Morrell etal., 2009, 2010), donkeys (Ortiz
etal., 2015), dogs (Dorado etal., 2013) and boars (Sjunnesson etal.,
2013). However, in their study of bull sperm Goodla, Morrell, Yusniza,
Stlhamma, and Johannisson (2014) did not find improved motility
following SLC selection, probably because the proportion of motile
sperm in the control samples was high (80%90%).
Vitality was not affected by SLC treatment of camel semen. These
results were comparable to earlier reports in stallions (Gamboa etal.,
2016) and bulls (Goodla etal., 2014). In contrast, previous studies on
stallion (Costa etal., 2012; Morrell etal., 2009), donkey (Ortiz etal.,
2015) and boar (Martinez-Alborcia etal., 2013) sperm reported im-
proved percentages of live sperm after SLC.
Gamboa etal. (2016) reported that SLC selection did not improve
the percentages of membrane-intact (swollen tails) stallion sperm
as measured by HOST. In contrast, we found an increase in positive
HOST reactions in SLC-treated sperm. Similarly, Trasorras etal. (2012)
found increased percentages of positive HOST when using SLC for
selecting fresh llama sperm.
In the present study, acrosome integrity was not different between
SLC and control sperm. Similar findings were reported by Bucci etal.
(2013) who did not find a detrimental effect of SLC treatment on the
acrosome integrity of boar semen. However, in studies performed on
cryopreserved sperm of dogs (Dorado etal., 2013) and stallions (Costa
etal., 2012), SLC-selected sperm showed increased acrosome integ-
rity compared to controls.
In terms of their performance in the heterologous IVF system,
SLC sperm performed better than controls in all parameters mea-
sured. This can be explained by the fact that SLC selects good quality
sperm from the ejaculate. Our finding agrees with previous studies
using colloid-prepared sperm from boars in which sperm treated with
Percoll had higher motility percentages and penetration rates (includ-
ing polyspermy) than non-treated sperm (Coy, Gadea, Rath, & Hunter,
2009; Matas etal., 2003). Similarly in another study of boar sperm,
it was necessary to reduce the sperm dose substantially to reduce
polyspermy (Sjunnesson etal., 2013). In vivo fertility improvement has
been reported for SLC-treated cooled stallion semen (Morrell, Richter
etal., 2014). Further studies are needed to examine the effect of SLC
treatment of camel sperm on conception rates.
A major challenge for the future will be to increase the recovery
rate of camel sperm from SLC. Only 26%33% of sperm present in
the original ejaculate passed through the colloid into the sperm pellet
in the present study, although we do not know whether the sperm
that were retained at the interface were, in fact, normal. Previously,
significant loss of sperm during the centrifugation procedure has been
 |
6 MALO etal.
reported (Morrell & Rodriguez-Martinez, 2010), and sperm recovery
can be lower than for conventional centrifugation (Heutelbeck etal.,
2015) because the immature, immotile or damaged spermatozoa are
not able to pass through the colloid. Scaled-up versions of SLC have
been reported for stallion and boar semen, enabling larger volumes of
semen to be prepared (Morrell, van Wienen, & Wallgren, 2011); these
results are encouraging for future work with camel semen. Different
colloid formulations, centrifugation speeds and semen:colloid ratios,
as well as improved liquefaction of the ejaculate, are areas of future
investigations to improve the system.
In conclusion, this study has shown that dromedary camel sperm
can be released from the viscous ejaculate by dilution (1:5 or 1:10) in
TCF. Furthermore, sperm recovered from the liquefied ejaculate follow-
ing SLC have improved motility, membrane functionality and IVF abil-
ity compared with sperm recovered by simple washing. Increasing the
efficiency of the SLC technique will be the subject of future research.
ACKNOWLE DG E MEN TS
The expertise of semen collectors M. Billah and Abdul Rachman Khan
is gratefully acknowledged. We thank Dr Ignacio de Blas for statistical
help. This study was sponsored by H.H. General Sheikh Mohammed
bin Rashid Al Maktoum, Ruler of Dubai.
D ISCLOSURE S
JM Morrell is the inventor of the colloid and has applied for a patent.
CO NFLI CT OF I NTE RE ST
None of the authors have any conflict of interest to declare.
AUTHOR CONTRI B UTI O N S
CM contributed to the study concept, carried out the measurements,
analysed and interpreted the results and drafted the manuscript.
JMM provided the study concept, made the colloid and contributed
to drafting the manuscript. EGC contributed to the study concept,
measurements and drafting the manuscript. BSP and JAS contributed
to the study concept, data interpretation, and manuscript prepara-
tion. All authors critically revised and approved the final version of
the manuscript.
REFERENCES
lvarez-Rodrguez, M., lvarez, M., Anel-Lpe, L., Lpez-Uruea, E.,
Manrique, P., Borragn, S., Anel, L. (2016). Effect of colloid (Androcoll
Bear, Percoll, and PureSperm) selection on the freezability of brown
bear (Ursus arctos) sperm. Theriogenology, 85, 10971105.
Avery, B., & Grav, T. (1995). Impact of Percoll on bovine spermatozoa used
for in vitro insemination. Theriogenology, 44, 871878.
Bamba, K. (1988). Evaluation of acrosome integrity of boar sperm by bright
field microscopy using an eosin-nigrosin stain. Theriogenology, 29,
12451251.
Bravo, P. W., Skidmore, J. A., & Zhao, X. X. (2000). Reproduction aspects
and storage of semen in Camelidae. Animal Reproduction Science, 62,
173193.
Bucci, D., Spinaci, M., Morrell, J., Vallorani, C., Tamanini, C., Guidetti, R., &
Galeati, G. (2013). Effects of single layer centrifugation with Androcoll-P
on boar sperm. Animal Reproduction Science, 138, 276281.
Chatdarong, K., Thuwanut, P., & Morrell, J. M. (2010). Single-layer cen-
trifugation through colloid selects improved quality of epididymal cat
sperm. Theriogenology, 73, 12841292.
Costa, A., Martins-Bessa, A., Andrade, A., Guimares, T., Rebordo, M.,
Gamboa, S., Rocha, A. (2012). Single layer centrifugation with
Androcoll-E improved progressive motility and percentage of live
spermatozoa with intact acrosome of chilled stallion semen but did
not have an effect on DNA integrity. Open Journal of Animal Science,
2, 159165.
Coy, P., Gadea, J., Rath, D., & Hunter, R. H. (2009). Differing sperm ability
to penetrate the oocyte in vivo and in vitro as revealed using colloidal
preparations. Theriogenology, 72, 11711179.
Crichton, E. G., Malo, C., Pukazhenthi, B. S., Nagy, P., & Skidmore, J. A.
(2016). Evaluation of cholesterol- treated dromedary camel sperm
function by heterologous IVF and AI. Animal Reproduction Science, 174,
2028.
Deen, A., Vyas, S., & Sahani, M. S. (2003). Semen collection, cryopreser-
vation and artificial insemination in the dromedary camel. Animal
Reproduction Science, 77, 223233.
Dorado, J., Glvez, M. J., Morrell, J. M., Alcarz, L., & Hidalgo, M. (2013).
Use of single-layer centrifugation with Androcoll-C to enhance sperm
quality in frozen-thawed dog semen. Theriogenology, 80, 955962.
El-Bahrawy, K. A., & El-Hassanien, E. E. (2009). Effect of different mucolytic
agents on viscosity and physical characteristics of dromedary camel
semen. Alexandria Journal of Agricultural Research, 54, 16.
Evans, G., & Maxwell, W. M. C. (1987). Salamons artificial insemination of
sheep and goats. Sydney: Butterworths.
Gamboa, S., Quaresma, A., Castro, F., Bravo, P., Rebordo, M. R., Oom, M.
M., & Rocha, A. (2016). In vivo fertilizing ability of stallion spermatozoa
processed by single layer centrifugation with Androcoll-E. Saudi Journal
of Biological Science. https://doi.org/10.1016/j.sjbs.2016.01.030
Giuliano, S., Carretero, M., Gambarotta, M., Neild, D., Trasorras, V., Pinto,
M., & Miragaya, M. (2010). Improvement of llama (Lama glama) semi-
nal characteristics using collagenase. Animal Reproduction Science, 118,
98102.
Goodla, L., Morrell, J. M., Yusniza, Y., Stlhamma, H., & Johannisson, A.
(2014). Quality of bull spermatozoa after preparation by single-layer
centrifugation. Journal of Dairy Science, 97, 22042212.
Heutelbeck, A., Oldenhof, H., Rohn, K., Martinsson, G., Morrell, J. M., &
Sieme, H. (2015). Use of density centrifugation for delayed cryopres-
ervation of stallion sperm: Perform sperm selection directly after col-
lection or after storage? Reproduction in Domestic Animals, 50, 7683.
Jeyendran, R. S., Van der Ven, H. H., Perez-Pelaez, M., Crabo, B. G., &
Zaneveld, L. J. (1984). Development of an assay to assess the func-
tional integrity of the human sperm membrane and its relationship to
other semen characteristics. Journal of Reproduction and Fertility, 70,
219228.
Jimnez-Rabadn, P., Morrell, J. M., Johannisson, A., Ramn, M., Garca-
lvarez, O., Maroto-Morales, A., Soler, A. J. (2012). Single layer
centrifugation (SLC) improves sperm quality of cryopreserved Blanca-
Celtibrica buck semen. Animal Reproduction Science, 136, 4754.
Kershaw, C. M., Evan, G., Rodney, R., & Maxwell, W. M. C. (2016). Papain
and its inhibitor E-64 reduce camelid semen viscosity without impair-
ing sperm function and improve post-thaw motility rates. Reproduction
Fertility and Development. https://doi.org/doi.org/10.1071/RD15261
Kershaw-Young, C. M., Stuart, C., Evans, G., & Maxwell, W. M. C. (2013).
The effect of glycosaminoglycan enzymes and proteases on the viscos-
ity of alpaca seminal plasma and sperm function. Animal Reproduction
Science, 138, 261267.
MALO etal.
Khatir, H., Anouassi, A., & Tibary, A. (2009). In vitro and in vivo developmen-
tal competence of dromedary (Camelus dromedarius) oocytes following
in vitro fertilization or parthenogenetic activation. Animal Reproduction
Science, 113, 212219.
Kruse, R., Dutta, P. C., & Morrell, J. M. (2011). Colloid centrifugation re-
moves seminal plasma and cholesterol from boar spermatozoa.
Reproduction Fertility and Development, 23, 858865.
Love, C. (2011). Relationship between sperm motility, morphology and the
fertility of stallions. Theriogenology, 76, 547557.
Macas-Garca, B., Gonzlez Fernndez, L., Morrell, J. M., Ortega Ferrusola,
C., Tapia, J. A., Rodriguez-Martnez, H., & Pea, F. J. (2009). Single-layer
centrifugation through colloid positively modifies the sperm subpopu-
lation structure of frozen-thawed stallion spermatozoa. Reproduction in
Domestic Animals, 44, 523526.
Martinez-Alborcia, M. J., Morrell, J. M., Gil, M. A., Barranco, I., Maside, C.,
Alkmin, D. V., Roca, J. (2013). Suitability and effectiveness of single
layer centrifugation using Androcoll-P in the cryopreservation protocol
for boar spermatozoa. Animal Reproduction Science, 140, 173179.
Matas, C., Coy, P., Romar, R., Marco, M., Gadea, J., & Ruiz, S. (2003). Effect of
sperm preparation method on in vitro fertilization in pigs. Reproduction,
125, 133141.
Moore, A. I., Squires, E. L., & Graham, J. K. (2005). Effect of seminal plasma
on the cryopreservation of equine spermatozoa. Theriogenology, 63,
23722381.
Morrell, J. M., Johannisson, A., Dalin, A. M., & Rodriguez-Martinez, H.
(2009). Morphology and chromatin integrity of stallion spermatozoa
prepared by density gradient and single layer centrifugation through
silica colloids. Reproduction in Domestic Animals, 44, 512517.
Morrell, J. M., Johannisson, A., & Rodriguez-Martinez, H. (2011). Effect
of osmolarity and density of colloid formulations on the outcome of
SLC-selection of stallion spermatozoa. International Scholarly Research
Notices Veterinary Science. https://doi.org/10.5402/2011/128984
Morrell, J.M., Lagerqvist, A., Humblot, P., & Johannisson, A. (2016). Effect of
Single Layer Centrifugation on reactive oxygen species and sperm mi-
tochondrial membrane potential in cooled stallion semen. Reproduction
Fertility and Development. https://doi.org/10.1071/RD15440.
Morrell, J. M., Richter, J., Martinsson, G., Stuhtmann, G., Hoogewijs, M.,
Roels, K., & Dalin, A. M. (2014). Pregnancy rates are higher after arti-
ficial insemination with cooled stallion spermatozoa selected by single
layer centrifugation than with control semen doses. Theriogenology, 82,
11021105.
Morrell, J. M., & Rodriguez-Martinez, H. (2010). Practical applications of
sperm selection techniques as a tool for improving reproductive ef-
ficiency. Veterinary Medicine. https://doi.org/10.4061/2011/894767
Morrell, J. M., Rodriguez-Martinez, H., & Andersson, M. (2014). Colloid
centrifugation selects normal spermatozoa from polymorphic bull ejac-
ulates: A case study. Reproduction in Domestic Animals, 49, 281284.
Morrell, J. M., Rodriguez-Martinez, H., & Johannisson, A. (2010). Single
layer centrifugation of stallion spermatozoa consistently selects the
most robust spermatozoa from the rest of the ejaculate in a large sam-
ple size. Equine Veterinary Journal, 42, 579585.
Morrell, J. M., van Wienen, M., & Wallgren, M. (2011). Single Layer
Centrifugation can be scaled-up further to process up to 150 mL
|
7
semen. International Scholarly Research Notices Veterinary Science.
https://doi.org/10.5402/2011/183412
Morrell, J. M., & Wallgren, M. (2011). Colloid centrifugation of boar semen.
Reproduction in Domestic Animals, 46, 1822.
Morton, K. M., Gibb, Z., Leathy, T., & Maxwell, W. M. C. (2012). Effect of
enzyme treatment and mechanical removal of alpacas (Vicugna pacos)
seminal plasma on sperm functionality integrity. Journal of Camelid
Science, 5, 6281.
Noguchi, M., Yoshioka, K., Hikono, H., Iwagami, G., Suzuki, C., & Kikuchi, K.
(2015). Centrifugation on Percoll density gradient enhances motility,
membrane integrity and in vitro fertilizing ability of frozen-thawed boar
sperm. Zygote, 23, 6875.
Ortiz, I., Dorado, J., Acha, D., Glvez, M. J., Urbano, M., & Hidalgo, M.
(2015). Colloid single-layer centrifugation improves post-thaw don-
key (Equus asinus) sperm quality and is related to ejaculate freezability.
Reproduction, Fertility and Development, 27, 332340.
Petters, R. M., & Wells, K. D. (1993). Culture of pig embryos. Journal of
Reproduction and Fertility, 48, 6173.
Samardzija, M., Karadjole, M., Matkovic, M., Cergolj, M., Getz, I., Dobranic, T.,
Karadjole, T. (2006). A comparison of BoviPure and Percoll on bull sperm
separation protocols for IVF. Animal Reproduction Science, 91, 237247.
Santa Cruz, R., Giuliano, S. M., Gambarotta, M. C., Morrell, J. M., Abraham,
M. C., Miragaya, M. H., & Carretero, M. I. (2016). Comparison of
different methods of sperm selection of llama raw semen. Animal
Reproduction Science, 173, 812.
Sjunnesson, Y. C., Morrell, J. M., & Gonzlez, R. (2013). Single layer
centrifugation-selected boar spermatozoa are capable of fertilization in
vitro. Acta Veterinaria Scandinavica, 55, 2026.
Skidmore, J. A., Morton, K. M., & Billah, M. (2013). Artificial insemination in
dromedary camels. Animal Reproduction Science, 136, 178186.
Thys, M., Vandaele, L., Morrell, J. M., Mestach, J., Van Soom, A., Hoogewijs,
M., & Rodriguez-Martinez, H. (2009). In vitro fertilizing capacity of
frozen-thawed bull spermatozoa selected by single-layer (glycidoxy-
propyltrimethoxysilane) silane-coated silica colloidal centrifugation.
Reproduction in Domestic Animals, 44, 390394.
Tibary, A., & Anouassi, A. (1997). Theriogenology in Camelidae, 1st ed. United
Arab Emirates: Ministry of Agriculture and Information.
Trasorras, V., Giuliano, S., Chaves, G., Neild, D., Agero, A., Carretero, M.,
Miragaya, M. (2012). In vitro embryo production in llamas (Lama
glama) from in vivo matured oocytes with raw semen processed with
Androcoll-E using defined embryo culture media. Reproduction in
Domestic Animals, 47, 562567.
How to cite this article: Malo C, Crichton EG, Morrell JM,
Pukazhenthi BS, Skidmore JA. Single layer centrifugation of
fresh dromedary camel semen improves sperm quality and in
vitro fertilization capacity compared with simple sperm
washing. Reprod Dom Anim. 2017;00:17.
https://doi.org/10.1111/rda.13036