Livestock Science 110 (2007) 166 – 173

www.elsevier.com/locate/livsci

Influence of supplementing diet with Oleic and Linoleic acid on the

freezing ability and sex-sorting parameters of ram semen
S.P. de Graaf ⁎, K. Peake, W.M.C. Maxwell, J.K. O'Brien, G. Evans
Centre for Advanced Technologies in Animal Genetics and Reproduction (ReproGen), Faculty of Veterinary Science,
The University of Sydney, NSW 2006, Australia
Received 22 August 2006; received in revised form 3 October 2006; accepted 1 November 2006

Abstract

In an effort to improve the cryosurvival of both non-sorted and sex-sorted ram spermatozoa the effect of supplementing the ram
diet with Oleic and Linoleic acid, in the form of extra virgin olive oil and sunflower oil, respectively, was assessed. Rams (n = 4/
group) were fed either (i) a standard maintenance diet (Control), (ii) maintenance diet + 5% (w/w) sunflower oil (Linoleic), or (iii)
maintenance diet + 5% (w/w) extra virgin olive oil (Oleic) for a period of 6 weeks. The effect of these diets on the post-thaw
(incubated 37 °C, 6 h) motility characteristics (as measured by CASA) of non-sorted, frozen-thawed ram spermatozoa were
assessed every 2 weeks. The sex-sorted, frozen–thawed spermatozoa were assessed at the end of the 6 week trial period in the same
manner. Linoleic and Oleic diets had a negative impact (P b 0.05) on the total sperm motility, viability and acrosome integrity after
a 6 week period of dietary supplementation. Furthermore, the average path velocity and straight line velocity of spermatozoa from
Oleic-fed rams was less when compared to samples originating from rams fed linoleic acid or the control diets after both 2 and
6 weeks post-diet modification. Curvilinear velocity of oleic spermatozoa 2 weeks post diet modification were inferior for Oleic—
(P b 0.05) compared with Linoleic—but not control-fed rams. Spermatozoa from rams fed Oleic diets exhibited lower (P b 0.05)
linearity than spermatozoa from rams fed Linoleic acid (2, 4 and 6 weeks) or the control diets (6 weeks). Diet did not significantly
affect any motility characteristic or the viability/acrosome integrity of sex-sorted spermatozoa. Nutritional supplementation with the
mono-unsaturated fatty acid, Oleic acid, or the polyunsaturated fatty acid, Linoleic acid, did not improve the cryosurvival of ram
spermatozoa — whether or not it had been processed for sex-sorting by flow cytometry. However, these results provide insight into
the relationship between nutrition and male reproductive characteristics and further research to elucidate the mechanisms by which
diet manipulation affects sperm membranes and subsequent sperm quality is warranted.
© 2006 Elsevier B.V. All rights reserved.

Keywords: Cryopreservation; Sex-sorting; Spermatozoa; Sheep; Unsaturated fatty acids; Diet

1. Introduction bated when cryopreservation is combined with sex-

Cryopreservation is known to elicit a deleterious
effect on the motility, viability and membrane status of
spermatozoa (Watson, 1995). These effects are exacer-
⁎ Corresponding author. Tel.: +61 2 9351 5832; fax: +61 2 9351 3957.
E-mail address: simong@vetsci.usyd.edu.au (S.P. de Graaf).
1871-1413/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.livsci.2006.11.001
separation by means of flow cytometry (Hollinshead
et al., 2003) — a technique which has produced pre-
sexed offspring in a number of species (Garner, 2006).
Cryosurvival of sex-sorted spermatozoa is of added
importance due to the time taken to sex-sort sufficient
spermatozoa for an insemination dose and the inverse
relationship between post-thaw sperm survival and total
 S.P. de Graaf et al. / Livestock Science 110 (2007) 166–173
sperm dose required for AI. These factors contribute
significantly to the price premium associated with sexed
spermatozoa (Seidel, 2003). For these reasons, methods
to improve the cryopreservation of both non-sorted and
sex-sorted spermatozoa have been sought, with reason-
able success being achieved (Schenk et al., 1999;
Hollinshead et al., 2003). However, further improve-
ment is needed to reduce the total number of sex-sorted
spermatozoa required for in vitro fertilisation (IVF) or
AI and subsequently improve the commercial potential
of sex-preselection technology.
Recently, a greater understanding of the role of
polyunsaturated fatty acids, and lipids in general, in
sperm function has led to a desire to affect the lipid
content of sperm membranes (White, 1993; He et al.,
2001). While this usually involves the addition of
substances to spermatozoa at some stage during the
cryopreservation process, the idea of modifying sperm
membranes prior to ejaculation within the male, perhaps
via dietary intake, represents an attractive alternative for
successful membrane modification (Perez-Pe et al., 2001;
Purdy and Graham, 2004). It has long been known that
diet plays a significant role in reproductive physiology. In
rams, an increased plane of nutrition increases testicular
size and sperm production (Oldham et al., 1978),
primarily due to heightened digestible energy intake
(Murray et al., 1990). Modification of certain dietary
components, such as water soluble vitamins, has also
improved the rate of spermatogenesis in boars (Audet
et al., 2004). An early report in the ram suggested the
feeding of soya bean oil, high in Linoleic acid, improved
post-thaw motility (Milovanov and Golubj, 1973). This
finding is supported by enhanced semen quality in poultry
(Kelso et al., 1997; Cerolini et al., 2003) and rabbits
(Castellini et al., 2005) fed a diet high in alpha-linolenic
acid. Supplementation of boar diets with polyunsaturated
fatty acids and anti-oxidants has also aided sperm survival
during chilled transport (Strzezek et al., 2004). As for
frozen semen, an improvement in post-thaw quality has
been reported for spermatozoa from stallions fed a diet
high in docosahexaenoic acid (Brinsko et al., 2005), a
polyunsaturated fatty acid prevalent in the semen of most
species (Parks and Lynch, 1992) and known to readily
increase with dietary supplementation (Blesbois et al.,
1997; Conquer et al., 2000). Transfer of unsaturated fatty
acids from the diet to the semen of ruminants is a
somewhat more difficult prospect due to hydrogenation of
lipids by rumen microorganisms (Garton, 1969). Further,
lipid content of the ruminant diet generally should not
exceed 4 to 5% due to the resultant reduction in efficiency
of rumen microorganisms in their ability to breakdown
cellulose (Brooks et al., 1954; White et al., 1958).
167
Encouragingly, recent research has shown the feeding of
extra Linoleic acid to cause a significant increase in the
concentration of this fatty acid in the blood plasma of
goats, thereby suggesting unsaturated fatty acids to have
the capacity to resist biohydrogenation in the rumen
(Yeom et al., 2003, 2005). It is therefore feasible that the
beneficial effects of Linoleic and Oleic acid on stored ram
spermatozoa may be facilitated via manipulation of the
diet (Perez-Pe et al., 2001). This has certainly been
advocated after anecdotal evidence of improved bull
(I. Drummond, pers. comm.) and boar (D. Rath, pers.
comm.) semen freezability in animals fed diets high in
extra virgin olive oil (Oleic acid), particularly when
followed by sex-separation using flow cytometry.
The potential benefit to the quality of frozen–thawed
spermatozoa, whether sexed or non-sexed, and the ease
with which this may be achieved justifies scientific
investigation of the link between dietary oil supplemen-
tation and sperm cryopreservation. Thus, the present
study aims to determine the effect of supplementing the
dietary intake of rams with Oleic and Linoleic acid on
the freezing ability and sex-sorting parameters of ram
semen.
2. Materials and methods
Procedures herein were approved by The University
of Sydney’s Animal Ethics Committee.
2.1. Experimental design
Twelve mature, mixed breed rams of European
descent were housed in individual pens at The Uni-
versity of Sydney farms (Camden, NSW) and were
allocated to one of the three diets: (Treatment 1) a
standard ram maintenance diet (Control), (Treatment 2)
Control + Linoleic acid in the form of sunflower oil
(Linoleic), and (Treatment 3) Control + Oleic acid in the
form of extra virgin olive oil (Oleic). Rams were
allocated to the treatment groups based on principal
component analysis of post-thaw sperm quality (n = 3
ejaculates), prior to commencement of the trial (while all
rams were fed the control diet) in order to balance both
high and low quality semen producing rams between
treatments. With commencement of the experimental
period, ejaculates were collected from each ram once
every 2 weeks for a total of 6 weeks and frozen for later
post-thaw analysis. At the end of the 6 week experi-
mental period further ejaculates (n = 2) were collected
from each ram, sex-sorted and frozen. All thawed
samples were analysed for motility, viability and
acrosome integrity during incubation (0, 3, 6 h; 37 °C).
168 S.P. de Graaf et al. / Livestock Science 110 (2007) 166–173
2.2. Composition of the diets
The control diet was based on a standard mainte-
nance diet for rams containing levels of metabolisable
energy appropriate for each animal based on body
weight (Jordan et al., 1985). The diet was provided in
the form of oats and lupin grains, lucerne and oats chaff.
The actual diet intake per animal was 1 to 1.3 kg/kg
liveweight/day — depending on ram bodyweight. Oleic
and Linoleic treatment diets were formulated by the
addition of 5% (w/w) extra virgin olive (Coobamindra
Olive and Citrus Farm, Kotingal, NSW, Australia) and
sunflower (Woolworths, Yennora, NSW, Australia) oils,
respectively, to the standard control diet. All rams were
exposed to the control diet 4 weeks prior to the intro-
duction of the Oleic or Linoleic diets to the treatment
animals. Throughout the trial period each ram was
monitored closely to ensure complete consumption of
allocated feed occurred.
2.3. Semen collection, freezing and thawing
Ejaculates were collected by artificial vagina from the
rams during the breeding season. Semen not destined for
sex-sorting was diluted 1:4 (semen:diluent, v/v), using a
tris-citrate-glucose diluent containing 15% egg yolk and
5% glycerol (v/v) and frozen and thawed (37 °C, 2 min)
using the pellet method (Evans and Maxwell, 1987).
Thawed non-sorted samples were diluted with tris-
citrate-fructose (TRIS; Evans and Maxwell, 1987) to
standardise concentration with that of sex-sorted sam-
ples (20 × 106 spermatozoa/ml). Non-sorted and sex-
sorted sperm samples were both diluted 1:1 [v/v; 500 μl
semen: 500 μl with Androhep (Minitube Australia,
Smythes Creek, Australia) post-thaw and incubated
(37 °C, 6 h).
2.4. Preparation and flow cytometric sorting of
spermatozoa
Freshly ejaculated samples were diluted to a concen-
tration of 400 × 106 spermatozoa/ml with TRIS, containing
267 μM Hoechst stain (H33342; Sigma-Aldrich, Sydney,
Australia), incubated at 34 °C and inverted every 15 min.
After 1 h, samples were diluted 1:1 (sperm sample:diluent,
v/v) with TRIS containing 4% (v/v) egg yolk and 0.002%
(w/v) food dye (Warner Jenkinson Company, Inc., St
Louis, USA), and filtered (35 μm, Falcon 2235; Becton
Dickinson, MO, USA). Spermatozoa were processed
without separation of X and Y bearing spermatozoa using
a modified high speed flow cytometer (SX MoFlo®; Dako
Colorado Inc., Fort Collins, CO, USA) operating at 40 psi
with a TRIS sheath fluid running a diode pumped solid
state pulse laser (125 mW; Vanguard 350 HMD-355,
Spectra Physics, Mountain View, CA, USA). Sorting gates
were set to select the entire viable, oriented population to
enable maximum sorting rates (∼5000–11,500 spermato-
zoa/s). As previously described, 6-million sorted sperma-
tozoa were collected into each 10 ml tube containing
0.25 ml of Androhep supplemented with 20% egg yolk
(v/v; pH 7.4) with an additional 0.25 ml of egg yolk
supplemented Androhep added after the initial collection
of 2-million spermatozoa (Hollinshead et al., 2004).
Sorted spermatozoa were then centrifuged at 750 ×g for
7.5 min at 21 °C, the supernatant discarded and the re-
maining sperm pellet resuspended 1:4 (pellet:diluent, v/v)
with tris-citrate-glucose cryoprotective medium prior to
freezing by the aforementioned method.
2.5. Evaluation of spermatozoa
2.5.1. Motility characteristics
Total motility, velocity and other kinematic character-
istics of sperm movement were recorded by means of
computer assisted sperm assessment (CASA; HTM-
IVOS v. 12; Hamilton-Thorne, Beverly, USA). Semen
samples (5.5 μl, 10 × 106 spermatozoa/ml) were placed on
a pre-warmed slide (37 °C; Cell Vu, Millennium Sciences
Corp., NY, USA) and enclosed by a cover slip
(22 × 22 mm) before immediate transfer to the CASA
machine (pre-calibrated with factory ram settings and
utilising an image sampling frequency of 60 Hz). Motility
characteristics were determined by assessment of at least
three randomly selected microscopic fields (200–300
spermatozoa per sample).
2.5.2. Viability and acrosome integrity
The membrane impermeable DNA supravital stain
Ethidium homodimer 1 (Eth-D1; Molecular probes,
Eugene, OR, USA), and fluorescein-conjugated peanut
agglutinin (FITC-PNA; Sigma-Aldrich), were used to
simultaneously assess sperm viability and acrosome in-
tegrity (Szasz et al., 2000). Briefly, 12 μl aliquots of
sperm suspension were combined with 10 μl of Eth-D1
(4 μM working stock in Androhep), pre-warmed to 37 °C
and incubated for 30 s before introduction of 4 μl of
FITC-PNA [(10 μg/ml working stock in phosphate
buffered saline (PBS; Sigma-Aldrich)] and fixation with
4 μl of PBS containing 0.5% glutaraldehyde (Sigma-
Aldrich). Samples were observed under phase contrast
(400× magnification; 200 cells/sample) using an Olym-
pus BHS fluorescence microscope comprising a 520–
550 nm band pass filter, supplementary 515 nm exciter
filter, 565 nm dichroic mirror and additional 610 nm
 S.P. de Graaf et al. / Livestock Science 110 (2007) 166–173 169

Fig. 1. Motility characteristics determined by CASA for a) total motility, b) linearity [LIN], c), straight line velocity [VSL; - - - - ] and curvilinear
velocity [VCL;——— ], and d) average path velocity [VAP; ———] for non-sorted spermatozoa originating from rams fed a maintenance diet
▪
[Control; ], or the same diet supplemented with Linoleic [▴] or Oleic [●] acid.

barrier filter. Acrosome non-intact or damaged sperma-
tozoa displayed bright green or patchy green fluores-
cence, respectively, while cells that did not stain positive
for FITC-PNA in this region were regarded as acrosome
intact. Spermatozoa exhibiting red fluorescence were
considered non-viable, and thus plasma membrane dam-
aged, while those regarded as viable excluded the mem-
brane impermeable Eth-D1 dye. Hence, spermatozoa
were classified as viable, acrosome intact; viable, acro-
some non-intact; non-viable, acrosome intact; and non-
viable, acrosome non-intact.
2.5.3. Sorting data
The percentage orientation (spermatozoa with their
flat surface of the head facing the laser beam), rate of
coincidences at maximum sorting rate (related to sample
wastage), and the rate of sorting were recorded via
telemetry from the SX MoFlo® and associated SUM-
MIT sort software (Dako Colorado Inc.).
2.6. Statistical analysis
Statistical analyses were conducted using GENSTAT
(Version 8.1, VSN International, Hemel Hempstead,
UK). Sperm motility characteristics were analysed using
a REML variance component mixed model analysis and
sorting data were analysed by general analysis of vari-
ance, utilising arc-sin transformations to attain normal-
ity where necessary (Petrie and Watson, 2006). Dual
staining for sperm viability and acrosome integrity were
analysed by Poisson regression. Principle component
analysis for balanced allocation of ram to diet type was
undertaken using MINITAB 14 (Minitab Ltd., Pennsyl-
vania, USA).
170 S.P. de Graaf et al. / Livestock Science 110 (2007) 166–173

3. Results Table 1
Motility characteristics determined by CASA for sorted spermatozoa
originating from rams fed a normal maintenance diet (Control), or the
3.1. Effect of diet on the freezing ability of non-sorted same diet supplemented with Linoleic or Oleic acid
ram spermatozoa
Motility Diet
Characteristic
Motility parameters following the post-thaw incuba- Control Linoleic Oleic
tion of non-sorted spermatozoa from rams fed a control, TM (%) 67.4 ± 5.2 54.8 ± 5.9 60.5 ± 6.0
Linoleic or Oleic diet for a 6 week period are depicted in VAP (μms− 1) 91.9 ± 6.6 80.5 ± 6.5 81.3 ± 8.0
VSL (μms− 1) 82.1 ± 5.7 71.4 ± 5.7 72.6 ± 7.1
Fig. 1. No significant interactions were observed for any
VCL (μms− 1) 139 ± 9.2 126.5 ± 9.3 125.6 ± 11.6
motility characteristic between treatments and incuba- ALH (μm) 5.1 ± 0.4 4.7 ± 0.4 4.8 ± 0.4
tion time. Therefore, data were pooled over incubation BCF (Hz) 37.8 ± 0.9 36.9 ± 1.8 35.3 ± 2.3
time (0, 3 and 6 h) to increase the power of comparison STR (%) 87.3 ± 1.3 83.4 ± 3.9 80.1 ± 5.1
between treatments (n = 24/treatment). LIN (%) 58.3 ± 1.9 54.5 ± 2.9 53.0 ± 3.6
Six weeks after supplementation, diets containing
either Linoleic or Oleic acid decreased (P b 0.05) the post-
thaw motility (TM) of ram spermatozoa, when compared Motility parameters were similar for spermatozoa
to the samples from rams fed the control diet (Fig. 1a). A derived from rams fed the control, Linoleic or Oleic
corresponding decrease (P b 0.05) after 6 weeks in the supplemented diets (Table 1). Similarly, the number of
percentage of viable and acrosome intact spermatozoa viable, acrosome intact spermatozoa post-thaw did not
was recorded for the Oleic (41.2 ± 5.1%) and Linoleic differ between the control (59.7 ± 4.8%), Linoleic (48.9 ±
(36.5 ± 2.9%) treatments, compared to the controls (56.2 ± 5.1%) or Oleic acid (56.3 ± 5.1%) treatment groups.
3.6%). Diet supplementation with Oleic acid decreased
(P b 0.05) the average path velocity (VAP; Fig. 1d) and 4. Discussion
straight line velocity (VSL; Fig. 1c) of spermatozoa,
compared to samples originating from rams fed Linoleic Supplementation of ram diets with either Linoleic or
acid or the control diet at 2 and 6 weeks following its Oleic acid failed to improve the cryosurvival of either
introduction to the feed. In addition, curvilinear velocity non-sorted or sex-sorted ram spermatozoa. In fact,
(VCL) of Oleic-treated spermatozoa 2 weeks post diet contrary to the anticipated improvement in post-thaw
modification were inferior (P b 0.05) to the Linoleic- motility and viability of spermatozoa, the supplements
treated group, but not the control samples (Fig. 1c). proved deleterious after 6 weeks of feeding. Such results
Spermatozoa from rams fed Oleic acid diets exhibited cannot be adequately explained by an inhibitory effect
lower (P b 0.05) linearity (LIN) than spermatozoa from on spermatogenesis as the constituents of both olive and
rams fed Linoleic acid (2, 4 and 6 weeks) or the control sunflower oil do not contain substances known to
diet (6 weeks; Fig. 1b). No differences in amplitude of disrupt spermatogenesis or produce a cumulative toxic
lateral head displacement (ALH), beat cross frequency effect. Similarly, contamination of feed by microorgan-
(BCF) or straightness (STR) were observed between isms or oxidation of supplemented oils is unlikely to
treatments at 2, 4 or 6 weeks post diet modification. have occurred, as fresh diets were formulated every 2 to
3 days.
3.2. Effect of diet on the sex-sorting efficiency and The negative effect of the unsaturated fatty acids in
subsequent freezing ability of sorted ram spermatozoa this trial also cannot be attributed to pre-existing dif-
ferences in each treatment group. Prior to the introduction
Flow cytometric analysis showed no differences in of the treated feeds, principal component analysis of
the percentage of correctly oriented spermatozoa, CASA parameters was used to balance rams across each
plasma membrane intact spermatozoa, coincidence rate treatment so as to obtain groups that shared the same
or sort rate, between samples originating from rams fed motility characteristics. Nevertheless, these results are in
a control, Linoleic or Oleic acid supplemented diet. As contrast to the main body of literature concerning dietary
with the non-sorted spermatozoa, no significant inter- oil manipulation.
actions were observed for any motility characteristic The vast majority of research on the supplementation
between treatment and incubation time. Therefore, data of unsaturated fatty acids for improved sperm production
were pooled over incubation time (0, 3 and 6 h) to or cryosurvival has resulted in positive or at least neutral
increase the power of comparison between treatments findings, with improvements noted in roosters (Blesbois
(n = 24/treatment). et al., 1997; Surai et al., 2000), turkeys (Blesbois et al.,
 S.P. de Graaf et al. / Livestock Science 110 (2007) 166–173
2004), trout (Labbe et al., 1995), boars (Rooke et al.,
2001) and stallions (Brinsko et al., 2005) fed diets high in
n-3 long chain polyunsaturated fatty acids (usually 22:6).
However, direct comparison of the results obtained in
these species and ruminants, such as sheep, is inappro-
priate due to the widely differing manner in which
unsaturated fatty acids are dealt within the respective
gastrointestinal systems. While monogastrics such as the
pig, chicken or human are capable of directly absorbing
unsaturated fatty acids in the gastrointestinal tract, when
fed to ruminants such as sheep and cattle these are subject
to breakdown by ruminant microorganisms. This process,
known as biohydrogenation, effectively alters unsaturat-
ed fatty acids into saturated fatty acids which are then
utilised by the ruminant in a similar manner to
monogastrics (Garton, 1969). While there is some
evidence to suggest that unprotected unsaturated fatty
acids may be able to at least partially survive biohy-
drogenation, it is entirely possible that this did not occur
in the current study (Yeom et al., 2003, 2005). Both
Linoleic (18:2) and Oleic (18:1) acids may have been
converted to stearic acid (18:0), a saturated fatty acid, and
subsequently absorbed by the animals. It follows that the
alteration of the experimentally active constituents of the
diet in this manner may have caused a subsequent
increase in the amount of saturated fatty acids within the
sperm membrane and/or a decrease in the percentage of
polyunsaturated fatty acids. It has been hypothesised that
a low polyunsaturated to saturated fatty acid ratio may
cause poor resistance to temperature change as a result of
reduced membrane fluidity at low temperatures (Watson,
1981). The poorer velocity characteristics and linearity of
Oleic acid treated spermatozoa, in comparison to Linoleic
acid samples may be attributed to only partial hydroge-
nation of Linoleic acid resulting in a decreased level of
stearic acid, and its putative negative effects, produced by
this diet. This hypothesis would account for the reduced
quality of spermatozoa originating from rams fed
supplemented diets in the present study, but it does not
support the previous results of Milovanov and Golubj
(1973) or Davidenko et al. (1990). These authors found
that rams fed diets high in soya bean (high in both
Linoleic and Oleic acid) and sunflower oil, respectively,
produced semen with superior post-thaw quality and
fertility of spermatozoa. However, in these studies the
authors claim to have protected the unsaturated fatty
acids from hydrogenation in the rumen by homogenisa-
tion in a 1% solution of egg yolk and an antioxidant
(Trivitamin), increasing the proportion bypassing the
rumen and thus facilitating their absorption and incorpo-
ration in sperm membranes. In retrospect, the unsaturated
oils in the present study could have been protected, but
171
the main aim was to test the recent anecdotal reports on
the beneficial effects of specific, unprotected oils in the
diet of cattle (I. Drummond, pers. comm.). Nevertheless,
any further experimentation involving addition of
unsaturated oils to ruminant diets would benefit from
the inclusion of a protected diet as an additional control.
An alternative explanation for the negative impact of
unsaturated dietary oils in the present study is increased
sperm damage due to lipid peroxidation. This assumes
that the oils survived passage through the rumen in
sufficient quantities to elicit the desired change in
membrane lipid profile within the spermatozoa. In this
case, the theorised beneficial aspects of increased
unsaturated to saturated fatty acid ratio on resistance to
temperature change may have been counteracted by an
increased propensity of lipid membranes high in
unsaturated fatty acids to undergo peroxidation (Jones
et al., 1979). Lipid peroxidation is known to negatively
impact on sperm motility, fertility and acrosome integrity
(Aitken et al., 1993; Sharma and Agarwal, 1996). This
theory is not without precedent as rabbits fed diets high
in polyunsaturated fatty acids were found to have sperm
producing higher levels of reactive oxygen species and
an increased susceptibility to oxidative stress resulting in
poorer cell viability and acrosome integrity (Castellini
et al., 2003). This issue was only rectified by the
inclusion of high levels of anti-oxidants, such as Vitamin
C and E within the diet (Castellini et al., 2003, 2005). It
may be that the studies of Milovanov and Golubj (1973)
and Davidenko et al. (1990) were successful in part due
to the effect of additional anti-oxidant supplementation.
As the levels of incorporation of fatty acids in sperm
membranes could not be assessed in the present study, no
definite conclusion can be drawn on the means by which
the deleterious impact of unsaturated fatty acids was
conferred. Future research to quantify the nature of
sperm membrane changes is warranted to elucidate the
impact of dietary Oleic and Linoleic acids on the func-
tion of spermatozoa in ruminants.
No significant differences in the quality of sex-sorted,
frozen-thawed spermatozoa between dietary treatments
were obtained in the present study. Nevertheless, the
negative trend observed when combined with the
findings using non-sorted spermatozoa suggest Oleic
and Linoleic acid may also be detrimental in this system.
Membrane-damaged spermatozoa are removed from the
population during flow cytometric sorting, by inclusion
of food dye, which may explain the less pronounced
negative effect of the supplemented diets on the quality
of spermatozoa after sex-sorting (Johnson and Welch,
1999; Garner, 2006). Without published results for
comparison, little explanation can be offered for
172 S.P. de Graaf et al. / Livestock Science 110 (2007) 166–173
differences between the findings of this study and reports
in cattle that extra virgin olive oil has a beneficial effect
on the freezing of sex-sorted bull spermatozoa. One
possible explanation is a potential difference in the
ability of rumen microorganisms of cattle and sheep to
hydrogenate unsaturated fatty acids, or alternatively an
interaction between components of the diet of bulls not
present in the ration used in the present study. The
markedly different lipid profiles of spermatozoa from
bulls and rams may also play a role (Watson, 1981).
As for the vehicle by which the lipid profile is altered, a
clue lies in the time that functional changes in sper-
matozoa occurred after the diet was modified. Changes in
velocity and linearity began after only 2 weeks, ruling out
an effect on early stages of the spermatogenic cycle, as
such alterations would have taken considerably longer to
manifest (40 day spermatogenic cycle in rams) (Evans and
Maxwell, 1987). These results suggest that putative
alterations in the lipid profiles, and hence sperm function,
occurred during epididymal transit, where spermatozoa
are known to undergo a number of morphological, phy-
siological and biochemical changes as they are exposed to
the ever changing luminal fluid micro-environment
(Fournier-Delpech and Thibault, 1993). Lipid content is
but one aspect of the modifications which occurs during
epididymal maturation (Poulos et al., 1975). So any
change in the luminal fluid brought about by dietary
manipulation could alter the lipid make-up of the fluid and
hence the maturing spermatozoa.
5. Conclusion
To conclude, the results of this study indicate that
dietary supplements of Oleic and Linoleic acids are
deleterious to the quality of non-sorted, frozen-thawed
ram spermatozoa and do not confer a beneficial effect to
sex-sorted, frozen–thawed ram spermatozoa. While
these results proved disappointing for improving the
cryosurvival and yield of sex-sorted spermatozoa, they
do provide some insight into the relationship between
nutrition and male reproductive characteristics. Further
research to elucidate the mechanisms by which diet
manipulation affects sperm membranes and subsequent
sperm quality is warranted.
Acknowledgements
This work was supported by XY, Inc. (Fort Collins,
CO, USA). S.P. de Graaf was supported by The Australian
Sheep CRC with a postgraduate research scholarship. Mr.
B. Biffin and Mr. K. Tribe are thanked for on farm
assistance. The authors wish to thank Dr. M. Ruckholdt
for operation of the MoFlo® SX cell sorter, Dr. P.
Thomson for statistical advice, Ms. S. Underwood and
Mr. C. Peake for technical support and Dr. R. Newman for
his contributions to the authors' understanding of mem-
brane physiology.
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