Conservation of fresh ram spermatozoa at 5°C
in the presence of seminal plasma
A. Morrier1, F. Castonguay1,2, and J. L. Bailey1,3
Centre de Recherche en Biologie de la Reproduction,
1Département des sciences animales,
Université Laval, Québec City, Québec, Canada G1K 7P4;
2Dairy and Swine Research and Development
Centre, Agriculture and Agri-Food Canada, Lennoxville, Québec, Canada J1M 1Z3.
Received 2 August 2002, accepted 3 February 2003.
Morrier, A., Castonguay, F. and Bailey, J. L. 2003. Conservation of fresh ram spermatozoa at 5°C in the presence of seminal
. Can. J. Anim. Sci. 83
: 221–227. Seminal plasma aids sperm transport and contains factors beneficial for sperm function.
In artificial insemination, however, diluting the semen reduces the concentration of seminal plasma. To test the hypothesis that
supplemental seminal plasma in extended ram semen improves conservation at 5°C, we added various concentrations of seminal
plasma to semen during storage, and investigated subsequent sperm function in vitro. Semen was divided into three aliquots,
extended in a commercial diluent (Triladyl) supplemented with 0, 10 or 25% (vol:vol) ovine seminal plasma and cooled to 5°C.
After 8 and 24 h at 5°C, sperm were suspended in a modified synthetic oviduct fluid (SOF-m) at 39°C to mimic the female geni-
tal tract at insemination. Sperm aliquots were assessed for motility and chlortetracycline fluorescence after 0, 4 and 8 h in the SOF-
m. No significant differences were observed due to seminal plasma supplementation during conservation at 5°C or incubation in
SOF-m at 39°C. However, decreased sperm motility and fewer non-capacitated sperm were observed concomitant with an aug-
mentation of capacitated and acrosome-reacted cells during incubation in SOF-m. Therefore, the hypothesis that diluent supple-
mentation with homologous seminal plasma improves ram sperm conservation or subsequent sperm function was not supported.
: Ovine, ram, sperm, motility, viability, chlortetracycline fluorescence, artificial insemination, SOF
Morrier, A., Castonguay, F. et Bailey, J. L. 2003. Conservation des spermatozoïdes frais de bélier à 5 °C en présence de plas-
. Can. J. Anim. Sci. 83
: 221–227. Le plasma séminal facilite le transport du sperme et renferme des facteurs qui lui
sont bénéfiques. Lors de l'insémination artificielle cependant, en diluant la semence, on réduit aussi la concentration du plasma.
Pour vérifier l'hypothèse qu'un supplément de plasma séminal améliorerait la conservation du sperme de bélier allongé à 5 °C, les
auteurs ont ajouté à ce dernier une quantité variable de plasma séminal pendant le stockage et examiné subséquemment la motil-
ité des spermatozoïdes in vitro
. Le sperme a été divisé en trois parties, allongé avec un diluant commercial (Triladyl) auquel on a
ajouté 0, 10 ou 25 % (v:v) de plasma séminal d'ovin puis a été refroidi à 5 °C. Après avoir passé 8 ou 24 heures à cette tempéra-
ture, le sperme a été mis en suspension dans un fluide synthétique modifié imitant celui des trompes de Fallope (SOF-m) à 39 °C,
de manière à reproduire les conditions dans le tractus génital de la femelle à l'insémination. Les auteurs ont évalué la motilité des
spermatozoïdes et la fluorescence à la chlortétracycline des aliquotes de sperme laissés 0, 4 et 8 heures dans le SOF-m. Aucune
variation sensible n'a été relevée à la suite de l'addition de plasma séminal lors de la conservation du sperme à 5 °C ou son incu-
bation dans le SOF-m à 39 °C. Néanmoins, la motilité réduite des spermatozoïdes et le plus petit nombre de spermatozoïdes non
capacités sont cohérents avec la hausse du nombre de cellules capacitées et de cellules avec réaction acrosomique pendant l'incu-
bation dans le SOF-m. On en déduit que la dilution du sperme de bélier avec du plasma séminal homologue n'améliore pas sa con-
servation ni le fonctionnement subséquent des spermatozoïdes.
: Ovins, bélier, sperme, motilité, viabilité, fluorescence à la chlortétracycline,
insémination artificielle, fluide synthétique modifié des trompes de Fallope
Seminal plasma facilitates sperm transport in the female
of epididymal compounds (Setchell et al. 1993) or seminal
genital tract [reviewed by Yanagimachi (1994)] and may
plasma proteins (Barrios et al. 2000).
regulate capacitation by contributing decapacitation factors
The specific effects of seminal plasma on sperm during
to the sperm (Maxwell and Johnson 1999). However, artifi-
semen conservation are controversial, but many recent
cial insemination implies the dilution of semen, which
reports indicate a positive influence of seminal plasma com-
removes certain proteins adsorbed to the sperm surface, and
ponents on sperm quality. The inclusion of seminal plasma
reduces the concentration of natural antioxidants and othercompounds present in seminal plasma. In fact, it has beensuggested that better diluents for freezing and storage of
, for acrosome-reacted sperm with dull
fluorescence over the head; B
, capacitated sperm for those
semen would be formulated following the systematic study
with fluorescence only along the acrosome; CTC
, non-capacitated sperm for those with uniform
3To whom correspondence should be addressed (e-mail:
head fluorescence; SOF-m
, synthetic oviduct fluid; VAP
medium path velocity
CANADIAN JOURNAL OF ANIMAL SCIENCE
in the media improved the viability and membrane integrity
tion. Therefore, each male was used once for this experi-
of boar (Maxwell et al. 1998) and ram sperm (Maxwell et al.
= 12). Volume was determined and morphology and
1997) subjected to flow cytometric sorting. Seminal plasma
subjective motility were visually assessed by a trained
can suppress premature capacitation and acrosome reactions
andrologist, using phase contrast microscopy (400×) at
(Cross 1993), due to the presence of membrane-stabilizing
room temperature. Samples with less than 75% motile
decapacitation factors (Maxwell and Johnson 1999).
sperm were rejected. Sperm concentration was assessed
Furthermore, its proteins are thought to prevent or restore the
with a calibrated spectrophotometer (Spectronic 20D,
membrane integrity of cold-shocked ram sperm (Barrios et
Bausch and Lomb, Markham, ON).
al. 2000; Pérez-Pé et al. 2001). Although removal of seminalplasma before freezing increased the viability of ram sperm,
Semen Preparation and Conservation
plasma membrane integrity, motility and heterogeneity were
Immediately after collection, the semen was divided into
enhanced when either whole seminal plasma or a specific
three aliquots. Each aliquot was diluted with Triladyl
fraction were added to the semen diluent (Ollero et al.
(Minitube Canada, Woodstock, ON), a commercial diluent
1997a,b, 1998). Moreover, Pérez-Pé et al. (2002) reported
prepared with 20% egg yolk (from commercial eggs kept
that seminal plasma prevented the appearance of a capacita-
at 4°C until use), as per manufacturer's directions. Triladyl
tion-associated tyrosine phosphoprotein in ram sperm during
is composed of Tris [Tris(hydroxymethyl)aminomethane],
cold-shock, reinforcing the theory that seminal plasma com-
citric acid, fructose and 7% glycerol containing tylosin,
ponents stabilize the sperm plasma membrane.
gentamycin, lincomycin and spectinomycin as antibiotics.
Maxwell et al. (1999) demonstrated that the addition of
Each aliquot was supplemented with either 0, 10 or 25%
seminal plasma to frozen-thawed semen prior to artificial
(vol:vol) seminal plasma. Semen was extended at room
insemination improved sperm motility, with more uncapac-
temperature with Triladyl ± seminal plasma (prewarmed to
itated and fewer acrosome-reacted sperm compared to
30°C) to reach a final concentration of 1.6 × 109 sperm mL–1
unsupplemented controls. Furthermore, it was found that the
(Baril et al. 1993) as normally conducted at CIOQ. Diluted
addition of seminal plasma increased pregnancy rates of
semen was placed into straws of 250 µL (IMV, l'Aigle,
ewes after cervical insemination with frozen-thawed ram
France), sealed with polyvinyl acid, progressively cooled
sperm. Therefore, the hypothesis of the present study was
to 5°C over 3 h in a transport box (CIOQ), and subse-
that the addition of homologous seminal plasma to a com-
quently maintained at this temperature. This specially
mercial semen diluent improves ram sperm quality during
designed box contains the exact proportion of ice and
conservation at 5°C. Motility and physiological status
water at 30°C in order to reach 5°C in 3 h (approximately
assessed by the chlortetracycline (CTC) fluorescence assay
0.14°C min–1); thereafter, the temperature can be main-
were used as indicators of sperm quality.
tained at 5°C for 36 h. The same transport box was used forall replicates.
MATERIALS AND METHODS
At 8 and 24 h post-collection, one sealed straw of semen
was cut open (with clean scissors) and diluted (about 32-
Chemicals and Animal Care Guidelines
fold; to 50 × 106 sperm mL–1) in synthetic oviductal fluid
All experiments were conducted as authorized by the uni-
(SOF-m; Morrier et al. 2002). The SOF-m was composed
versity animal care committee and were conducted in accor-
of 108 mM NaCl, 7.2 mM KCl, 1.2 mM KH PO ,
dance with the guidelines of the Canadian Council on
25 mM NaHCO , 5 mM CaCl , 0.5 mM MgCl , 1.5 mM
Animal Care (1993). All products used in this experiment
glucose, 3.3 mM sodium lactate, 0.33 mM sodium pyruvate,
were purchased from Sigma Chemical Co. (St. Louis, MO)
1 mM glutamine, 20 µM penicillamine, 10 µM hypotaurine,
unless otherwise specified.
and 50 mg L–1 gentamycin. This solution was supplement-ed with 20% heat-inactivated estrus sheep serum (the
Seminal Plasma Preparation
same lot for all replicates) and adjusted to pH 7.36,
One ejaculate from each of 10 rams (two Charollais, two
290 mOsm kg–1.
Polled-Dorset, one Romanov, three Hampshire, and two
Sperm quality (motility and CTC assay) was assessed
Texel) was pooled and centrifuged (12 000 × g
, 15 min,
immediately following dilution in SOF-m ("Time t0"). The
22°C). Ejaculates with less than 75% sperm motility were
samples diluted in SOF-m were incubated in a humidified
rejected. The supernatants were collected and held at –20°C
atmosphere of 5% CO in air at 39°C. After 4 and 8 h in the
until use (Catt et al. 1997).
SOF-m, sperm quality was re-assessed. Figure 1 shows aschematic description of this protocol.
Six mature Polled-Dorset, one Hampshire, one Texel, one
Sperm Motility Analysis
Charollais, one Arcott Canadian, and two Arcott Rideau
For the assessment of sperm motility, three 2-µL samples of
rams aged from 2 to 7 yr were housed at the Centre
sperm in SOF-m ( 50 × 106 cells mL–1) were deposited into
d'Insémination Ovine du Québec (CIOQ, La Pocatière,
a 10-µm Makler counting chamber (Sefi-Medical
QC). Rams were under light regime, alternating from long
Instruments, Haifa, Israel) placed on the motility analyser
(16 h of light) to short days (8 h of light) monthly. Semen
(Hamilton Thorn Research, Beverly, MA) as described by
was collected twice consecutively with an artificial vagina
Morrier et al. (2002). At least 200 cells from five random
and the two ejaculates were then pooled for experimenta-
fields were recorded. The settings used were the following:
MORRIER ET AL. — RAM SPERM AND SEMINAL PLASMA
dure, followed by the Fisher's protected least significant dif-ferences (LSD) test when there were significant variableeffects. The statistical model used for sperm parameters(percentage motile, percentage progressively motile, andpercentage CTC pattern F, B and AR sperm) included theeffect of ram, the volume of seminal plasma added (treat-ment), duration of conservation in the diluent at 5°C, andtime of incubation in SOF-m at 39°C. The two-way interac-tions among these variables were also tested and the resid-ual error was the error term. Significance was judged at theprobability level of P
< 0.05. SAS Institute, Inc. (1990) soft-ware was used for the analyses.
The initial concentrations of the ejaculates and total motili-ty were 3.67 × 109 ± 1.31 sperm mL–1 and 88 ± 4% (means± SD), respectively, with less than 25% morphologicallyabnormal sperm for each ejaculate. The presence of seminalplasma in the Triladyl diluent did not affect the percentageof total motile sperm (P
= 0.5321; Fig. 2) at any of the timepoints studied. However, the percentage of total motilesperm increased very slightly during conservation in diluentat 5°C (83% after 8 h versus 86% after 24 h, with a least sig-nificant difference of 2.4%; P
< 0.05). The percentage oftotal motile sperm decreased during subsequent incubation
Outline of experimental protocol.
in SOF-m at 39°C (P
< 0.05) at all times (0 to 4 to 8 h).
The percentage of progressively motile sperm was not
frames acquired, 20; frame rate, 20 Hz; minimum contrast,
affected by the seminal plasma treatments at any of the time
10; minimum size, 5; lo/hi size gates, 0.2 to 2.0; lo/hi inten-
points studied (P
= 0.8932; Fig. 3). However, the conserva-
sity gates, 0.2 to 1.8; non motile head size, 5; non motile
tion in Triladyl diluent at 5°C increased the percentage of
brightness, 63; medium path velocity (VAP) value, 95; low
progressively motile sperm (41% after 8 h versus 44% after
VAP value, 20; slow cells motile, no; and threshold (STR),
24 h when the data of all SOF time points are combined,
60. Only progressive (proportion of cells moving with a cer-
with a least significant difference of 1.7%; P
< 0.05). The
tain straightness) and total motilities (or motile sperm; pro-
percentage of progressively motile sperm was lower during
portion of cells moving at or above a minimum speed) were
incubation in SOF-m at 39°C (P
< 0.05) after 8 h of conser-
determined for this study.
vation at 5°C. The percentage of progressively motile spermactually increased after 4 h of incubation in SOF-m at 39°C,
Chlortetracycline Fluorescence Assay
then decreased again when the semen was conserved for 24
The CTC fluorescence assay used was conducted as
h in Triladyl (46, 49 and 36% for times 0, 4 and 8 h in SOF-
described previously (Morrier et al. 2002). In brief, the CTC
m, respectively; all times were significantly different with a
solution was 5 mM cysteine and 750 µM CTC diluted in a
least significant difference of 2.3%).
buffer of 20 mM Tris base and 130 mM NaCl (pH 7.8). For
The percentage of sperm displaying the non-capacitated
the assay, 15 µL CTC solutions were mixed with 10 µL
CTC pattern (pattern F) was not affected by the addition of
sperm diluted in SOF-m, and fixed with 0.5 µL 12.5%
seminal plasma to the Triladyl diluent at any of the time
(vol:vol) glutaraldehyde solution prepared in 2.5 M Tris
points studied (P
= 0.5701, Fig. 4). However, the percent-
base. Ten µL of this suspension were placed on a micro-
ages of F pattern sperm decreased during conservation in
scope slide, covered with a coverslip and evaluated by fluo-
Triladyl diluent at 5°C and incubation in SOF-m at 39°C (P
rescent microscopy. Slides were prepared in duplicate and
< 0.05). The inclusion of seminal plasma did not affect the
200 sperm slide–1 were scored at 400× magnification. The
frequency of AR-pattern sperm (P
= 0.6027) either.
CTC patterns were scored as described by Gillan et al.
Contrary to the F pattern, however, the percentage of AR
(1997) as follows: "F" or non-capacitated sperm for those
pattern sperm increased with time in either the Triladyl at
with uniform head fluorescence; "B" or capacitated sperm
5°C or SOF-m at 39°C. The percentage of B pattern sperm
for those with fluorescence only along the acrosome; and
was increased due to conservation Triladyl at 5°C (P
"AR" for acrosome-reacted sperm with dull fluorescence
and duration of incubation in SOF-m (P
over the head.
In the light of reports by Maxwell et al. (1997, 1999) on the
The normality of the data was confirmed. Analysis of vari-
benefits of seminal plasma on sperm function, our study uti-
ance was performed using the General linear models proce-
lized a different, more practical approach: the addition of
CANADIAN JOURNAL OF ANIMAL SCIENCE
Percentages of motile sperm (means ± SE) from ram semen
Percentages of progressively motile ram sperm (means ±
conserved in a liquid state at 5°C for (a) 8 or (b) 24 h in Triladyl
SE) conserved in liquid state at 5°C for (a) 8 or (b) 24 h in Triladyl
diluent (TDIL = 8 and TDIL = 24, respectively) containing 0, 10
diluent ( TDIL = 8 and TDIL = 24, respectively) containing 0, 10%
or 25% (vol:vol) seminal plasma, and after incubation in SOF-m at
or 25% (vol:vol) seminal plasma and after dilution in SOF-m at
39°C, 5% CO for 0, 4 and 8 h (n
= 12). Seminal plasma did not
39°C, 5% CO for 0, 4 and 8 h (n
= 12). Seminal plasma did not
affect the percentage of motile sperm (P
= 0.5321). Sperm motili-
affect the percentage of progressively motile sperm (P
ty increased very slightly during 8 and 24 h of storage in Triladyl
The percentage of progressively motile sperm differed due to con-
diluent at 5°C (P
< 0.05). Within time of conservation in Triladyl
servation in the Triladyl diluent at 5°C only at 4 h of incubation in
(TDIL = 8 or TDIL = 24), motility declined (P
< 0.05) with each
< 0.05) (TDIL = 8 compared to TDIL = 24). Within
successive duration (0, 4 and 8 h) of incubation in SOF-m at 39°C.
time of conservation in Triladyl (TDIL = 8 or TDIL = 24), motili-ty declined (P
< 0.05) with each successive duration (0, 4 and 8 h)of incubation in SOF-m at 39°C.
seminal plasma during dilution for artificial insemination,avoiding semen manipulation post-conservation. We testedthe hypothesis that supplementation of ram semen with
plasma is detrimental to sperm motility (de Lamirande and
homologous seminal plasma during chilled conservation
Gagnon 1984) and viability (García and Graham 1987).
would protect sperm function. It is commonly recommend-
Ollero et al. (1997b) suggested that those controversial results
ed to use cooled ram semen for insemination within 8 h of
could be explained by the presence of two components in ram
collection (Baril et al. 1993), due to a marked decline in
seminal plasma. A higher molecular weight compound, pre-
sperm fertilizing capacity within 24 h (Evans and Maxwell
sent in the >10 kDa seminal plasma fraction (Ollero et al.
1987; Maxwell and Salamon 1993). Therefore, we evaluated
1997a) would protect ram sperm; however, a low molecular
sperm parameters after 8 h (which is within the recom-
weight factor would prevent the adsorption of beneficial sem-
mended time frame for artificial insemination) and after
inal plasma proteins to the sperm after ejaculation.
24 h (when the first marked drop in sperm function is evident).
Several studies have reported a beneficial effect of semi-
Contrary to our hypothesis that seminal plasma improves
nal plasma on sperm undergoing flow-cytometric process-
ram sperm conservation at 5°C, sperm motility, viability and
ing (Ashworth 1994; Catt et al. 1997; Maxwell et al. 1997).
distribution of CTC-patterns were not improved with the
However, these studies were performed at high dilution
inclusion of 10 or 25% seminal plasma. Although several
rates (< 1 × 107 sperm mL–1). At such high dilution levels,
studies have provided observations that seminal plasma
the cells die rapidly, a phenomenon called the "dilution
added to semen would be helpful to sperm (Barrios et al.
effect" [Mann and Lutwak-Mann (1981) in Maxwell and
2000; Graham 1994), others have demonstrated that seminal
Johnson (1999)], which particularly affects ram sperm
MORRIER ET AL. — RAM SPERM AND SEMINAL PLASMA
Percentages of sperm (means ± SE) displaying CTC patterns F, B and AR during conservation in a liquid state at 5°C for (a) 8 or (b)
24 h in Triladyl diluent (TDIL = 8 and TDIL = 24, respectively) containing 0, 10% or 25% (vol:vol) seminal plasma, and after dilution in
SOF-m at 39°C, 5% CO for 0, 4 and 8 h (n
= 12). The presence of seminal plasma did not affect CTC pattern distribution (P
> 0.05). The
overall CTC pattern distribution differed within time of incubation in SOF-m at 39°C, due to duration of conservation in Triladyl diluent at5°C (TDIL = 8 versus TDIL = 24; P
< 0.05). *For each CTC pattern, the proportion of sperm differs within time of conservation in Triladyldiluent at 5°C (TDIL = 8 or TDIL = 24), due to incubation in SOF-m at 39°C at all times (0, 4 and 8 h; P
[Mann (1984) in Maxwell and Johnson (1999)]. However,
In our study, sperm were diluted in a medium rich in pro-
in our study, 1.6 × 109 sperm mL–1 were used (a compara-
teins (Triladyl is prepared with 20% egg-yolk). The egg
tively low dilution rate). Furthermore, Harrison et al. (1982)
yolk (contained in the diluent) and the estrous ewe serum
suggested that using a protein-depleted medium results in
(present in the SOF-m) probably play the same role as serum
two factors that can explain the dilution effect: (1) poor
albumin, thus possibly explaining why the seminal plasma
stimulation of motility and (2) the tendency of live sperm to
addition did not improve the physiology of ram sperm.
adhere to container surfaces. According to Harrison et al.
Maxwell et al. (1999) reported an improvement in fertili-
(1982), serum albumin protects against the dilution effect by
ty rates and sperm motility, an increase in the percentage of
stimulating sperm motility and preventing sperm adhesion.
uncapacitated sperm, and a concomitant decrease in capaci-
CANADIAN JOURNAL OF ANIMAL SCIENCE
tated and acrosome-reacted sperm when seminal plasma
Baril, G., Chemineau, P., Cognie, Y., Guérin, Y., Leboeuf, B.,
was added to frozen-thawed ram sperm washed of the
Orgeur, P. and Vallet, J.-C. 1993.
Manuel de formation pour l'in-
semen extender prior to cervical insemination of ewes (com-
sémination artificielle chez les ovins et les caprins. FAO, Rome,
pared to a control without seminal plasma). Although that
study was interesting, their protocol is impractical for large-
Barrios, B., Pérez-Pé, R., Gallego, M., Tato, A., Osada, J.,
Mu i˘no-Blanco, T. and Cebrian-Pérez, J. A. 2000.
scale application under field conditions. As well, the benefi-
ma proteins revert the cold-shock damage on ram sperm mem-
cial effect of seminal plasma was observed relative to sperm
brane. Biol. Reprod. 63
extended only in Dulbecco's phosphate-buffered solution
Bedford, S. J., Jasko, D. J., Graham, J. K., Amann, R. P.,
(DPBS). This a protein-free medium; a decrease in the pro-
Squires, E. L. and Pickett, B. W. 1995.
Effect of seminal exten-
portion of agglutinated cells when seminal plasma was
ders containing egg yolk and glycerol on motion characteristics
added to the diluent can also explain the beneficial effect
and fertility of stallion spermatozoa. Theriogenology 43
observed. Furthermore, it was not established whether the
Canadian Council on Animal Care 1993.
Guide to the care and
beneficial results observed were specific to seminal plasma
use of experimental animals. Volume 1. E. D. Olfert, B. M. Cross,
proteins, since no other protein sources were tested. In the
and A. A. McWilliams, eds. CCAC, Ottawa, ON.
present experiment, egg yolk (which is rich in proteins),
Catt, S. L., O'Brien, J. K., Maxwell, W. M. C. and Evans, G.
could have masked the effect of seminal plasma. Bedford
Assessment of ram and boar spermatozoa during cell-sorting
by flow cytometry. Reprod. Dom. Anim. 32
et al. (1995) proposed a possible negative interaction
Cross, N. L. 1993.
Multiple effects of seminal plasma on the acro-
between egg yolk and seminal plasma; the mechanism is
some reaction of human sperm. Mol. Reprod. Dev. 35
unknown, but may involve lipid peroxidation and release of
de Lamirande, E. and Gagnon, C. 1984
. Origin of a motility
free radicals, which would harm sperm and reduce their
inhibitor within the male reproductive tract. J. Androl. 5
function (Bedford et al. 1995). In our experiment, it is pos-
Evans, G. and Maxwell, W. M. C. 1987.
sible that the beneficial effects of seminal plasma were hid-
insemination of sheep and goats. Butterworths, Sydney, Australia.
den by the presence of egg yolk. Perhaps a positive effect of
seminal plasma would be evident using a less-protein-rich
García, M. A. and Graham, E. F. 1987.
Factors affecting the
semen diluent, such as a simpler Tris-based extender.
removal of low-molecular weight fractions (LMWF) from egg-
Alternatively, pre-incubating freshly collected semen with
yolk and seminal plasma in extended semen by dialysis: effect of
post-thaw sperm survival. Cryobiology 24
the supplemental seminal plasma prior to extension might
Gillan, L., Evans, G. and Maxwell, W. M. C. 1997.
reduce competition between seminal plasma proteins and
status and fertility of frozen –thawed ram spermatozoa. Reprod.
diluent proteins for the sperm plasma membrane, thereby
Fertil. Dev. 9
avoiding any interference by the proteins in the egg yolk-
Graham, J. K. 1994.
Effect of seminal plasma on the motility of
the epididymal and ejaculated spermatozoa of the ram and bull
In conclusion, the effects of seminal plasma on sperm
during the cryopreservation process. Theriogenology 41
function and fertility remain controversial. Seminal plasma
seems to act in duality: some seminal plasma proteins are
Harrison, R. A. P., Dott, H. M. and Foster, G. C. 1982.
beneficial to sperm membranes, while others are detrimen-
serum albumin, sperm motility, and the ‘dilution effect'. J. Exp.
tal. Further investigation is necessary to understand this
complex mixture. In theory, a better understanding of semi-
Maxwell, W. M. C., Welch, G. R. and Johnson, L. A. 1997.
Viability and membrane integrity of spermatozoa after dilution and
nal plasma proteins could improve the formulation of better
flow cytometric sorting in the presence or absence of seminal plas-
diluents for artificial insemination. Specific protein frac-
ma. Reprod. Fertil. Dev. 8
tions or even individual proteins from seminal plasma could
Maxwell, W. M. C., Long, C. R., Johnson, L. A., Dobrinsky, J.
be screened for positive (or detrimental) effects on sperm
R. and Welch, G. R. 1998.
The relationship between membrane
function and semen preservation. However, the present
status and fertility of boar spermatozoa after flow cytometric sort-
work does not indicate that supplementing fresh ram semen
ing in the presence or absence of seminal plasma. Reprod. Fertil.
(diluted in egg yolk) with additional seminal plasma
improves sperm conservation at 5°C.
Maxwell, W. M. C., Evans, G., Mortimer, S. T., Gillan, L.,
Gellatly, E. S. and McPhie, C. A. 1999.
Normal fertility in ewes
after cervical insemination with frozen-thawed spermatozoa sup-
We thank the Centre d'expertise en production ovine du
plemented with seminal plasma. Reprod. Fertil. Dev. 11
and Centre d'insémination ovine du Québec
Maxwell, W. M. C. and Johnson, L. A. 1999.
Physiology of sper-
viding the semen, Jean Paulin for technical assistance and to
matozoa at high dilution rates: the influence of seminal plasma.
Dr. Jean-Paul Laforest for critically reading the manuscript.
This work was supported by le Conseil des recherches en
Maxwell, W. M. C. and Salamon, S. 1993.
Liquid storage of ram
pêche et en agroalimentaire du Québec
semen: A review. Reprod. Fertil. Dev. 5
: 613–638.Morrier, A., Castonguay, F. and Bailey, J.L. 2002.
Ashworth, P. J .C., Harrison, R. A. P., Miller, N. G. A.,
addition and conservation of fresh and cryopreserved ram sperma-
Plummer, J. M. and Watson, P. F. 1994.
Survival of ram sper-
tozoa. Can. J. Anim. Sci. 82
matozoa at high dilution: protective effect of simple constituents of
Ollero, M., Cebrian-Pérez, J. A. and Muiño-Blanco, T. 1997a.
culture media as compared with seminal plasma. Reprod. Fertil.
Improvement of cryopreserved ram sperm heterogeneity and via-
bility by addition of seminal plasma. J. Androl. 6
MORRIER ET AL. — RAM SPERM AND SEMINAL PLASMA
Ollero, M., García-López, N., Pérez-Pé, R., Cebrián-Pérez, J.
SAS Institute, Inc. 1990.
SAS/STAT user's guide: Statistics.
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12690 • The Journal of Neuroscience, September 17, 2014 • 34(38):12690 –12700 Serotonin Affects Movement Gain Control in the Spinal Cord Kunlin Wei,1 Joshua I. Glaser,2,3,4,5 Linna Deng,1 Christopher K. Thompson,5,6 Ian H. Stevenson,2,3,4,5 Qining Wang,1Thomas George Hornby,2,3,4,5,6 Charles J. Heckman,2,3,4,5 and Konrad P. Kording2,3,4,51Department of Psychology, Peking University, Beijing, China 100871, Departments of 2Physical Medicine and Rehabilitation, 3Physiology, and 4AppliedMathematics, Northwestern University, Chicago, Illinois 60611, 5Rehabilitation Institute of Chicago, Chicago, Illinois 60611, and 6Department ofKinesiology and Nutrition, University of Illinois at Chicago, Chicago, Illinois 60607