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Theriogenology 76 (2011) 252–260

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Evaluation of an animal protein-free semen extender for

cryopreservation of epididymal sperm from North
American bison (Bison bison)
S. Krishnakumara, D.P. Whitesideb,c, B. Elkinb,d, J.C. Thundathila,*
a
Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, T2N4N1, Canada
b
Department of Ecosystem and Public Health, Faculty of Veterinary Medicine, University of Calgary, Calgary, AB, T2N4N1, Canada
c
Animal Health Centre, Calgary Zoo, Calgary, AB, T2E8K2, Canada
d
Wildlife Division, Government of Northwest Territories, Environment and Natural Resources, Yellowknife, NT, X1A3S8, Canada
Received 17 June 2010; received in revised form 17 December 2010; accepted 3 February 2011

Abstract
The objective was to evaluate the suitability of an animal protein-free semen extender for cryopreservation of epididymal sperm from
the two subspecies of North American bison: plains (Bison bison bison) and wood (Bison bison athabascae) bison. Both cauda
epididymides (from six plains and five wood bison) were minced and incubated in Sp-TALPH buffer for approximately 2 h at 37 °C
to release actively motile sperm. Sperm suspensions were filtered, centrifuged and the sperm pellet from each bull was divided into two
fractions and diluted either in egg yolk containing extender, Triladyl, or in an animal protein-free extender, Andromed, and equilibrated
for 20 min at 37 °C. Thereafter, samples were chilled and cryopreserved. Frozen-thawed sperm were evaluated for motility (computer
assisted sperm analysis), viability (SYBR 14 and propidium iodide), acrosome integrity (FITC conjugated PSA), cryocapacitation
(tyrosine phosphorylation of sperm proteins as a biomarker), and fertilizing ability (in a heterologous IVF system). There was no
significant difference for progressive motility, viability, and acrosome integrity between the two extenders for plains bison (36.8 ⫾ 9.0,
60.5 ⫾ 17.4, and 77.3 ⫾ 4.6%; overall mean ⫾ SD) as well as for wood bison (11.7 ⫾ 8.1, 13.7 ⫾ 5.6, and 73.4 ⫾ 4.2%). Levels of
tyrosine phosphorylation did not differ for sperm preserved in the two extenders for both subspecies, although an inter-bull variability
in the response to tyrosine phosphorylation between extenders was suggested for plains bison. Fertilization percent did not differ
significantly between extenders for plains bison (84.16 ⫾ 9.92%, overall mean ⫾ SD) and for wood bison (59.53 ⫾ 19.99%). In
conclusion, in the absence of significant difference between extenders in post-thaw sperm characteristics, we inferred that Andromed
(animal protein-free) was suitable for cryopreservation of epididymal sperm from North American bison.
© 2011 Elsevier Inc. All rights reserved.

Keywords: American bison; Epididymal sperm; Cryopreservation; Semen extenders; Sperm analysis; Heterologous IVF

1. Introduction
There are two subspecies of the North American
bison: wood (Bison bison athabascae) and plains (Bi-
* Corresponding author. Tel.: ⫹1 403 220 8244; fax: ⫹1 403 210
3939.
E-mail address: jthundat@ucalgary.ca (J.C. Thundathil).
son bison bison) bison, with wood bison being classi-
fied as ‘threatened’ in Canada [1]. Organizations in-
volved in wood bison conservation (e.g., the national
Wood Bison Recovery Team) have supported research
into the development and assessment of appropriate
reproductive technologies to salvage bison genetics.
Sperm collection, cryopreservation and use for IVF or
AI are powerful technologies for conserving popula-

0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.theriogenology.2011.02.001
 S. Krishnakumar et al. / Theriogenology 76 (2011) 252–260
tions of wild animals. Sperm cryopreservation facili-
tates movement of male genetics between populations
and across borders, without the need for physically
moving animals. Furthermore, it can be used for pre-
serving the genetics of a male after death and is suitable
for preserving the genetics of diseased animals, since
the threat of disease transmission through this technol-
ogy is very low [2].
Our laboratory is involved with developing repro-
ductive technologies for salvaging the genetics of wood
bison. Using plains bison as a model, we previously
reported an efficient method for recovery and cryo-
preservation of epididymal sperm [3] in Triladyl, an
egg yolk containing extender. Egg yolk or milk has
conventionally been added to semen extenders. The
role of these components in protecting sperm has not
fully been understood, although several mechanisms
have been proposed [4,5]. Low density lipoproteins
in egg yolk and casein micelles in skim milk interact
with and sequester bovine seminal plasma proteins
which have been demonstrated to induce cholesterol
and phospholipid efflux from sperm membranes,
leading to membrane destabilization [5]. However,
addition of egg yolk and milk contribute to variabil-
ity among batches of extender, and increase the risk
of disease transmission [6]. The use of an extender
free of animal proteins decreases the risk of trans-
mission of pathogens and viruses and would be pre-
ferred if found to efficiently preserve post-thaw
sperm function. Andromed is a commercially avail-
able semen extender containing phospholipids de-
rived from soybean extract. Sperm from European
bison (Bison bonasus) and African buffalo (Syncerus
caffer) have been successfully cryopreserved using
egg yolk-free semen extender [7,8]. In the European
bison, significantly more viable sperm with intact
acrosomes were present in Triladyl, an egg yolk
based semen extender, compared to an extender con-
taining lipids from plant extract. However there was
no significant difference in the fertilizing ability of
sperm between the two extenders. For the North
American bison, based on preliminary results, we
concluded that Andromed, a synthetic semen ex-
tender, could be used for cryopreserving epididymal
sperm from plains bison [3]. However, the fertilizing
ability of such sperm has not been evaluated. The
objective of the present study was to compare post-
thaw characteristics and in vitro fertilizing ability of
sperm cryopreserved in Andromed versus Triladyl,
from both subspecies of the North American bison.
253
2. Materials and methods
Unless otherwise stated, all chemicals were pur-
chased from Sigma-Aldrich (Oakville, ON, Canada).
2.1. Recovery and cryopreservation of epididymal
sperm
Testes from plains bison (n ⫽ 6 bulls) were col-
lected at a local abattoir, whereas testes of wood bison
(n ⫽ 5 bulls) were collected from free-ranging animals,
following an annual harvest for disease monitoring,
conducted by the Wildlife Division, Government of
Northwest Territories. Bison epididymal sperm were
recovered and cryopreserved according to the proce-
dure previously described [3], with minor modifica-
tions. Briefly, testes were recovered within 30 min after
slaughter. Paired cauda epididymides from each bull
were minced and transferred into tubes containing mod-
ified Tyrode’s Hepes-buffered medium (Sp-TALPH;
[9]) at 37 °C. Samples were held in Sp-TALPH me-
dium at 37 °C for 2–3 h to allow actively motile sperm
to swim out of the epididymides. Thereafter, samples
with ⬎ 50% motility (visual assessment using 200 ⫻
magnification) were filtered through sterile gauze and
suspensions obtained were centrifuged (500 ⫻ g) for 5
min. The sperm pellet was divided into two fractions;
one fraction was extended in ⬃1 mL of pre-warmed
animal protein-free semen extender (Andromed; Mini-
tube, Ingersoll, ON, Canada), and the other fraction in
⬃1 mL of pre-warmed egg yolk containing extender
(Triladyl; Minitube). Concentration of each fraction
was determined using a hemocytometer, and adjusted
to approximately 200 ⫻ 106/mL with the respective
extender. Extended sperm were incubated in a water
bath at 37 °C for 20 min. Tubes of extended sperm were
then transferred to a beaker containing water at 37 °C
and the beaker was placed in a fridge at 4 °C for at least
4 h for chilling and equilibration. Preparations with ⬎
50% motility (visual assessment using 200 ⫻ magnifi-
cation) were manually loaded into 0.5 mL straws and
sealed with glass beads (Minitube). Filled and sealed
straws were held in liquid nitrogen vapours for 15 min
(approximately 2 cm above the surface of the liquid
nitrogen in a styrofoam box) before plunging into liquid
nitrogen. Cryopreserved sperm extended in Andromed
or Triladyl were used for subsequent experiments.
2.2. Evaluation of post-thaw sperm characteristics
2.2.1. Motility
Sperm motility characteristics were evaluated for
sperm cryopreserved in Andromed or Triladyl, using
254 S. Krishnakumar et al. / Theriogenology 76 (2011) 252–260
computer assisted sperm analysis (Sperm Vision 3.5
software; Minitube). Frozen sperm were thawed in a
water bath for 1 min at 37 °C and diluted in warm
extender to a concentration of 30 ⫻ 106/mL. An aliquot
(4 ␮L) was loaded into the chamber of a pre-warmed
microscopic slide (depth 20 ␮m; Leja Products, Nieuw-
Vennep, The Netherlands), and analyzed under 200 ⫻
magnification at 37 °C for total and progressive motil-
ity and motion characteristics of average path velocity
(VAP), curve line velocity (VCL), straight line velocity
(VSL), and amplitude of lateral head displacement
(ALH). Sperm were considered immotile if average
orientation change of sperm head (AOC) was ⬍ 5
degrees and locally motile if distance straight line
(DSL) was ⬍ 4.5 ␮m. Frame rate used was 60 frames/s
and seven fields per sample were analyzed.
2.2.2. Viability
Sperm viability was determined using a combination
of SYBR 14 and propidium iodide (PI; Live Dead
Sperm Viability Kit, Molecular probes, Invitrogen Can-
ada, ON, Canada). Post-thaw, sperm were diluted to a
concentration of 50 ⫻ 106/mL in Sp-TALPH and in-
cubated at room temperature for 30 min with SYBR 14
(4 ␮M) and PI (9.6 ␮M). Gluteraldehyde (0.006% final
concentration) was added as fixative. An aliquot of 10
␮L was pipetted onto a clean glass slide and a cover
slip was mounted on it. A minimum of 200 cells per
sample were counted and classified as viable or non-
viable under epifluorescence (20 ⫻ objective, Leica
Microsystems GmbH Wetzlar, Germany).Viable cells
with intact plasma membranes stained green with
Fig 1. Epididymal sperm from plains bison stained with FITC-PSA to determine acrosomal integrity. (a) Sperm with an intact acrosome. (b) Sperm
with a damaged/reacting acrosome.
SYBR 14, whereas non-viable cells stained red with
propidium iodide.
2.2.3. Acrosomal integrity
Acrosomal integrity of sperm was evaluated using
FITC-conjugated P. sativum agglutinin (FITC-PSA), as
previously described [9]. Briefly, sperm samples were
diluted in Sp-TALPH to a concentration of 50 ⫻ 106/
mL. Aliquots (20 ␮L) were used to prepare smears on
microscopic slides. After drying, slides were immersed
in 100% ethanol maintained at ⫺20 °C, for 2 min.
Smears were allowed to air-dry, after which they were
covered with 50 ␮L of FITC-PSA and left in a dark
humid chamber for 30 min at room temperature. Slides
were rinsed in Sp-TALPH, allowed to air dry, and
stored in a box at ⫺20 °C until evaluated. For evalua-
tion, cover slips were mounted with DABCO and slides
were examined using a 100 ⫻ oil immersion objective
and epifluorescent microscope (Leica Microsystems
GmbH Wetzlar, Germany). A minimum of 200 sperm
per slide were evaluated for acrosomal integrity. Sperm
were classified as either acrosome intact or acrosome
reacted/damaged (Fig. 1).
2.2.4. Cryocapacitation as determined by levels of
tyrosine phosphorylation
To compare tyrosine phosphorylation levels of
sperm proteins, SDS-PAGE and immunoblotting were
done as previously described [10], with minor modifi-
cations. Frozen-thawed sperm from each treatment
group were washed two times with Sp-TALPH medium
to remove as much of the extender as possible. Sperm
 S. Krishnakumar et al. / Theriogenology 76 (2011) 252–260
were then resuspended to a concentration of 100 ⫻
106/mL in Sp-TALPH, and boiled with Laemmli’s
sample buffer (containing 5 mM Na2VO3) for 5 min at
100 °C. To exclude protein bands contributed by the
extenders, Andromed and Triladyl were run as controls.
For this, extenders were diluted (1:20 in Sp-TALPH)
and treated similarly to sperm. Boiled sperm samples
and extenders were then centrifuged (16,000 ⫻ g, 5
min, room temperature) and solubilized proteins in the
supernatant were separated on 10% SDS-polyacryl-
amide gels. Protein bands were electrotransferred to
nitrocellulose membranes in transfer buffer (Tris gly-
cine buffer, pH 8.5 containing 20% methanol) at a
constant voltage (100V) for 1 h. Non-specific binding
sites were blocked with skim milk (3% w/v), and mem-
branes were immunoblotted with monoclonal anti-
mouse anti-phosphotyrosine antibody (Millipore Cor-
poration, Billerica, MA, USA; Clone 4G10; 1:5000)
overnight at 4 °C. Immunoreactive bands were detected
using enhanced chemiluminescence and quantified us-
ing scanning densitometry. Membranes were reprobed
with monoclonal anti ␤-tubulin antibody (1:5000) as
loading control.
2.2.5. In vitro fertilizing ability
Fertilizing ability of plains and wood bison sperm
cryopreserved in either Andromed or Triladyl was eval-
uated in a heterologous in vitro system, using bovine
oocytes. Oocytes were recovered by aspirating visible
follicles (3– 8 mm) from abattoir-derived bovine ova-
ries. Oocytes were matured for 22 h at 39 °C and 5%
CO2, in TCM199 medium supplemented with 25 ␮M
sodium pyruvate, 25 ␮g/mL gentamicin, 0.5 ␮g/mL
FSH, 5 ␮g/mL LH, 2 ␮g/mL estradiol-17␤, and 10%
fetal calf serum (Hyclone, Logan, UT, USA). Mature
oocytes were transferred to drops of fertilization me-
dium, FERT-TALP [11], supplemented with 6 mg/mL
fatty acid-free bovine serum albumin, 100 units/ml pen-
icillin, 100 ␮g/mL streptomycin, 25 ␮M sodium pyru-
vate, 20 ␮M penicillamine, 10 ␮M hipotaurine, 1 ␮M
epinephrine, and 5 ␮g/mL heparin. Bovine oocytes
were randomly allocated to either of two treatment
groups. Bison sperm (cryopreserved in Andromed or
Triladyl) were processed by swim-up technique [12] for
in vitro fertilization. Straws were thawed in a water
bath for 1 min at 37 °C. Sperm were washed by cen-
trifugation (500 ⫻ g, 5 min) and the pellet resuspended
in fertilization medium. The washing step was repeated
to ensure removal of extender. The final sperm pellet
was then overlaid with 200 ␮L of fertilization medium
and incubated at 39 °C and 5% CO2 for 15 to 20 min.
Thereafter, the upper two-thirds of the medium, which
255
contained actively motile sperm, were used for insem-
inating bovine oocytes. Using a hemocytometer, sperm
concentration in the post-swim up medium was deter-
mined and adjusted to 1 ⫻ 106 sperm/mL of fertiliza-
tion medium. An aliquot of 10 ␮L of sperm suspension
was added to each 20 ␮L drop of fertilization medium
containing 10 bovine oocytes. Oocytes were co-incu-
bated with sperm for 18 h. For each trial, a group of in
vitro matured oocytes were incubated in fertilization
medium for 18 h without sperm as a parthenogenetic
control. Following 18 h of IVF, presumptive zygotes
and control oocytes were denuded of their cumulus
cells by vortexing, and cultured for 8 d in 40 ␮L drops
(20 presumptive zygotes per drop) of synthetic oviduct
fluid (SOF; 107.7 mM NaCl, 7.16 mM KCl, 1.19 mM
KH2PO4, 25.06 mM NaHCO3, 0.3 mM sodium pyru-
vate, 2.5 mM sodium lactate 60% syrup, 1 mM glu-
tamine, 8 mg/mL bovine serum albumin, 1 ⫻ BME
essential amino acids, and 1X MEM non-essential
amino-acids). Fertilizing ability of sperm cryopre-
served in Andromed or Triladyl was based on cleavage
percentage recorded 48 h after fertilization minus the
percentage of parthenogenetically activated oocytes for
that trial. Furthermore, embryos were evaluated every
48 h thereafter to record development to the blastocyst
stage. All hybrid embryos were destroyed at the end of
the culture period.
2.3. Statistical analysis
Analyses were done using STATA (Intercooled
Stata 10, College Station, TX, USA). For comparison
of extenders, percentage post-thaw motility, viability,
and acrosome integrity of sperm were arc-sine trans-
formed and analyzed using a paired Student’s t-test.
Sperm from six plains bison and five wood bison bulls
were used in the fertilization trials (three trials per bull)
and analyzed using Chi-square test. For all analyses,
P ⬍ 0.05 was considered statistically significant.
3. Results
3.1. Sperm motility, viability and acrosome integrity
The initial motility (mean ⫾ SD) of the six plains
bison used in the study was 71.67 ⫾ 4.08% and the five
wood bison bulls was 62 ⫾ 2.74%. There was higher
total motility and lower VCL at post-thaw, for sperm
preserved in Triladyl compared to Andromed (P ⬍
0.05) for both plains and wood bison. However, there
were no significant differences between extenders for
post-thaw progressive motility and kinematic parame-
256 S. Krishnakumar et al. / Theriogenology 76 (2011) 252–260

Table 1
Mean (⫾ SD) motility measurements for post-thaw sperm cryopreserved in Andromed and Triladyl extenders from plains bison (n ⫽ 6) and
wood bison (n ⫽ 5).
Parameter Plains bison Wood bison
Andromed Triladyl Andromed Triladyl
Motility
Total motility (%) 45.39 ⫾ 9.6a 53.55 ⫾ 6.48b 15.74 ⫾ 9.62a 23.60 ⫾ 11.72b
Progressive motility (%) 34.50 ⫾ 9.86 39.03 ⫾ 8.17 10.89 ⫾ 7.04 12.45 ⫾ 9.20
VAP (␮m/s) 43.93 ⫾ 3.60 42.35 ⫾ 3.76 39.24 ⫾ 4.34 37.48 ⫾ 4.43
VCL (␮m/s) 79.71 ⫾ 6.86a 70.15 ⫾ 6.44b 78.89 ⫾ 10.53a 61.06 ⫾ 12.96b
VSL (␮m/s) 33.48 ⫾ 2.88 33.22 ⫾ 3.34 33.86 ⫾ 5.05 31.49 ⫾ 3.24
ALH (␮m) 3.73 ⫾ 0.37 3.64 ⫾ 0.26 3.79 ⫾ 0.30 3.56 ⫾ 0.57
Viability
Viable sperm (%) 59.26 ⫾ 14.26 61.64 ⫾ 20.50 12.6 ⫾ 6.10 14.86 ⫾ 4.99
Acrosomal integrity
Intact acrosome (%) 76.56 ⫾ 4.89 78.12 ⫾ 4.20 71.13 ⫾ 4.7 75.6 ⫾ 3.69
a,b
Within a row, for each subspecies, means without a common superscript differed (P ⬍ 0.05).

ters of VAP, VSL, and ALH for both subspecies. There
was also no significant difference between extenders
for percentage viability and acrosome integrity for both
subspecies (Table 1).
3.2. Cryocapacitation as determined by levels of
tyrosine phosphorylation
Tyrosine phosphorylation of sperm proteins was
studied using immunoblotting. The two extenders, An-
dromed and Triladyl, were diluted and run in parallel
lanes on the same gel as the sperm samples (Fig. 2). No
protein bands were detected for Andromed extender
following Ponceau staining of the membrane (data not
shown). For Triladyl extender several immunoreactive
bands were observed. Sperm-specific immunoreactive
bands were observed at approximately 75, 45, and 37
kDa (p75, p45, and p37), excluding immunoreactive
bands contributed by Triladyl extender alone. The pixel
intensity of the three protein bands considered were not
significantly different for sperm cryopreserved in An-
dromed or Triladyl, for both plains and wood bison
(Fig. 2a and 2b). However, a variation was observed
between plains bison bulls in levels of tyrosine phos-
phorylation of these proteins. Although this variability
was not found to be significant, it resulted in high
standard deviation values for plains bison. Although
p37 (Bull1), p37 and p45 (Bull2), and p45 and p75
(Bull3) had similar pixel intensity values for sperm
extended in Andromed and Triladyl, p75 and p45
(Bull1), and p37 (Bull3) had greater pixel intensity
values for sperm extended in Triladyl. Conversely, p75
(Bull2) had greater pixel intensity values for sperm
extended in Andromed. This inter-bull difference was
not observed in wood bison sperm.
3.3. Fertilizing ability in a heterologous system
A total of 1478 bovine oocytes were used to evaluate
in vitro fertilizing ability of plains and wood bison
sperm cryopreserved in the two semen extenders. Fer-
tilization percentage (based on oocytes cleaved on Day
2; fertilization ⫽ Day 0) and subsequent development
of hybrid embryos until blastocyst stage was recorded
(Fig 3). There was no significant difference in fertiliza-
tion percentage and blastocyst yield for sperm cryopre-
served in Andromed and Triladyl for both subspecies
(Table 2).
4. Discussion
The present study investigated the efficiency of an
animal protein-free semen extender, Andromed, for
preserving epididymal sperm function in the two sub-
species of the American bison. For epididymal sperm
from plains and wood bison cryopreserved either in
Andromed or Triladyl, there was no significant differ-
ence between extenders for percentage progressive mo-
tility, viability, or acrosome integrity. Furthermore,
there was no significant difference in cryocapacitation
levels (based on tyrosine phosphorylation of sperm
proteins) and in vitro fertilization rate (based on cleav-
age) for sperm cryopreserved in the two extenders for
both subspecies.
Sperm motility, viability, integrity of the acrosome,
plasma membrane and DNA, and mitochondrial status
are conventionally used as indicators of sperm quality
and fertilizing ability. In the current study, progressive
motility at post-thaw was not significantly different
between the extenders for the two subspecies of the
 S. Krishnakumar et al. / Theriogenology 76 (2011) 252–260 257

Fig 2. Representative image of tyrosine phosphorylated sperm protein profile from (a) plains and (b) wood bison. Lanes 1– 6: protein extract
prepared from sperm extended in Andromed (A) or Triladyl (T) from three bulls; Lane 7: Andromed extender only; Lane 8: Triladyl extender only.
Based on densitometry analysis (mean pixel ⫾ SD, from replicates replicates), there were no differences (P ⬎ 0.05) between extenders for p75,
p45 and p37 for both subspecies.

American bison. Motility of epididymal and ejaculated total motility when cryopreserved in Triladyl compared
sperm from American bison has previously been re- to Andromed [15]. Our results were consistent with
ported [3,15]. Post-thaw ejaculated sperm had higher these studies. However, percent progressive motility,

Fig 3. In vitro produced hybrid embryos [(a) 2-cell, (b) 4-cell, (c) 8-cell stage, (d) morula and (e) hatched blastocyst] following heterologous IVF
using bovine oocytes and wood bison sperm.
258 S. Krishnakumar et al. / Theriogenology 76 (2011) 252–260
Table 2
In vitro fertilization (Mean ⫾ SD) of sperm from plains (n ⫽ 6)
and wood (n ⫽ 5) bison cryopreserved in Andromed and Triladyl
extender.
Subspecies Extender No. Cleavage (%) Blastocyst
oocytes (%)
Plains Andromed 439 82.00 ⫾ 7.52 16.62 ⫾ 10.86
bison Triladyl 431 86.31 ⫾ 12.32 21.57 ⫾ 11.02
Wood Andromed 300 58.67 ⫾ 18.64 22.33 ⫾ 13.53
bison Triladyl 308 60.39 ⫾ 21.33 19.16 ⫾ 17.35
No difference (P ⬎ 0.05) between the two extenders for each sub-
species for fertilization and blastocyst percentage.
which excludes locally motile sperm, was not signifi-
cantly different between the extenders in the present
study. Since progressively motile sperm contribute to
fertilizing ability, we expect that this end point has
more relevance for assessing sperm quality. Further-
more, the present findings were consistent with our
previous study that reported no significant difference
between Andromed and Triladyl extenders for post-
thaw viability and acrosomal integrity for epididymal
sperm from plains bison [3]. In contrast to these obser-
vations, however, there were higher intact membranes,
acrosomes, and viable cells, for European bison sperm
diluted in Triladyl compared to a synthetic semen ex-
tender [7]. Perhaps these differences could be attributed
to the sperm source, since ejaculated sperm from Eu-
ropean bison were used in the latter study.
For the other CASA parameters, VCL was signifi-
cantly higher for sperm cryopreserved in Andromed,
while there was no significant difference in sperm mo-
tion parameters of VAP, VSL and ALH between the
extenders. A higher VCL and ALH are indicative of
hyperactivated sperm [29]. A significantly higher VCL
and higher (although not significant) ALH for An-
dromed-extended sperm in the current study might sug-
gest a tendency toward hyperactivation for bison sperm
cryopreserved in the absence of animal source proteins.
Higher velocities (VAP, VSL and VCL) for bovine
sperm were detected following cryopreservation in se-
men extender containing soybean extract, compared to
an extender containing egg yolk [13]. Another study
using bovine sperm reported higher VSL in soy-bean
lecithin extract based extender and higher ALH in egg
yolk containing extender [14] and suggested that these
differences could be due to a difference in the density
and viscosities of the two extenders under study.
As noted, progressive motility in the present study
was not different between the extenders for both plains
and wood bison. However, overall post-thaw motility
of wood bison epididymal sperm was low compared to
plains bison sperm. Effect of reproductive seasonality
on motility of ejaculated wood bison sperm has previ-
ously been documented [15]. In the present study, wood
bison sperm was collected in March, outside the phys-
iological breeding season and had a lower initial mo-
tility compared to plains bison; this could have ac-
counted for the subsequent poor post-thaw motility.
However, the effect of reproductive seasonality on ep-
ididymal sperm has yet to be documented.
Mean values for VAP, VCL and VSL recorded in
this study appeared low compared to values previously
reported for bison [15] and cattle [16,17]. This however
did not adversely affect the in vitro fertilization rate.
Distinct subpopulations of sperm, varying in morpho-
logical and functional characteristics, exist within an
ejaculate in several species of animals: cattle [18],
horses [19], pigs [20], Iberian red deer [21]), and hu-
mans [22]. During IVF, good quality sperm are selected
by swim-up or Percoll gradient centrifugation. In the
present study, swim up method selected sperm sub-
populations with high motility parameters, resulting in
high fertilization rates. Therefore, it can be concluded
that eliminating animal source proteins from the semen
extender does not compromise in vitro fertilizing abil-
ity. However, the effects of low sperm motion velocity
parameters on fertility, especially following AI, remain
to be investigated.
Cryopreservation has been shown to induce capaci-
tation-like changes in sperm, with changes in sperm
motility patterns, plasma membrane stability and orga-
nization, intracellular calcium levels and tyrosine phos-
phorylation of sperm proteins [30]. This precocious
capacitation or cryocapacitation is detrimental to the
fertilizing ability of sperm. Tyrosine phosphorylation
of sperm proteins has been identified as a reliable bio-
chemical marker for bovine sperm cryocapacitation
[23] and in the current study, levels of tyrosine phos-
phorylation of bison sperm proteins were compared
following cryopreservation in Andromed or Triladyl as
an indirect measure of extender efficiency. There was
no difference between the two extenders in the levels of
the three tyrosine phosphorylated sperm protein bands
considered, indicating similar cryoprotective abilities
of Andromed and Triladyl. However, variability be-
tween plains bison bulls in tyrosine phosphorylation
status indicated a different response of individual bulls
to extender type. Although this variability was not
found to be statistically significant, it was an interesting
observation and a possible direction for further inves-
tigation, since it could explain differences in cryocap-
acitation levels leading to possible variability in in vivo
 S. Krishnakumar et al. / Theriogenology 76 (2011) 252–260
fertility, previously seen in bovine bulls following
sperm cryopreservation in different extenders [26]. Dif-
ferences in sperm cryosurvival among individual males
within a species have previously been reported and are
suggested to have a genetic basis [20].
To our knowledge, this is the first report evaluating
the fertilizing ability of sperm from the American bison
cryopreserved in an extender free of animal proteins. In
the current study, eliminating animal source proteins
did not compromise in vitro fertilizing ability at post-
thaw. Similar studies have been conducted in domestic
cattle, with varying results. There was no significant
difference between extenders in the in vitro or in vivo
fertilizing ability, nor in 90 d non-return rates [6,24] for
bovine sperm, whereas in another study, higher non-
return rates were reported for the synthetic semen ex-
tender [25]. Contrary to these findings, a decrease in
non-return rates for bovine sperm cryopreserved in egg
yolk-free extender was reported [26]. For the European
bison, ejaculated sperm cryopreserved in a synthetic
semen extender performed significantly better (than
sperm cryopreserved in Triladyl) in a sperm penetration
assay into bovine oocytes [7]. For the American bison
the fertilization rate for sperm preserved in Triladyl was
not significantly different compared to sperm preserved
in Andromed. However, since all previous studies men-
tioned used ejaculated sperm, those results cannot be
directly compared with the current study.
Finally, an additional advantage of Andromed over
Triladyl is that since the former does not contain pro-
teins or any large particles, it does not interfere with or
confound sperm protein studies, flow cytometry, and
motility evaluation using CASA. For the immunoblot-
ting experiments in the present study, two wash steps
for post-thaw sperm were incorporated to remove as
much of the egg yolk particles as possible. In spite of
the additional wash steps, tyrosine phosphorylated
bands contributed by the extender were detected in
Triladyl extended sperm samples. Egg yolk particles
have scatter properties similar to sperm and have been
known to take up fluorescent stains [27]. Furthermore,
it has been suggested that properties of egg yolk con-
taining extender such as density, viscosity and presence
of large particles, interfere with CASA evaluation of
motility parameters [13,28].
In conclusion, the results of the current study dem-
onstrated that Andromed can be used as a semen ex-
tender for the cryopreservation of epididymal sperm
from plains and wood bison. Andromed has the advan-
tage of being free of animal source proteins and it
preserved post-thaw sperm quality and in vitro fertiliz-
259
ing ability as efficiently as Triladyl, a conventional
semen extender. However, further studies are required
using epididymal sperm from wood bison retrieved
during the physiological breeding season, to fully doc-
ument the efficiency of Andromed for cryopreservation
of wood bison epididymal sperm.
Acknowledgments
Funding was provided by the Advancing Canadian
Agriculture and Agri-Food Program, through the pro-
vincial councils of Alberta, Northwest Territories, Sas-
katchewan, Manitoba, Yukon and British Columbia
(Project #AB0122CO), the Government of Northwest
Territories, and Calgary Zoo. We acknowledge Sun-
terra Meat Plant, Innisfail, AB for facilitating the col-
lection of plains bison tissue, and Alta Genetics, Cal-
gary, AB, for generously providing reconstituted
Triladyl extender. We also thank Douglas Nickel for
his help in procuring samples.
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