Guimaraes 2012

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Animal Reproduction Science 136 (2012) 85– 89
Contents lists available at SciVerse ScienceDirect
Animal Reproduction Science
jou rn al h om epa ge: www .e lsev ier .com/ locate /an i reprosc i
haracteristics of stallion epididymal spermatozoa at collection and
ffect of two refrigeration protocols on the quality of the
rozen/thawed sperm cells
. Guimarãesa,b,∗, G. Lopesa,b, P. Ferreiraa,b, I. Leal c, A. Rochaa,b
ICBAS – Abel Salazar Biomedical Institute, University of Porto, Portugal
CECA/ICETA – Animal Sciences Centre, University of Porto, Portugal
Private Practitioner, Porto, Portugal
 r t i c l e i n f o
rticle history:
eceived 14 August 2012
eceived in revised form 11 October 2012
ccepted 13 October 2012
vailable online 1 November 2012
eywords:
tallion
pididymal sperm cells
ryopreservation
a b s t r a c t
Cryopreservation of epididymal spermatozoa is a useful tool to preserve genetic material
of valuable stallions after emergency castration or unexpected death. For that, testicles and
epididymides are generally sent refrigerated to the laboratory. Collection of epididymal
spermatozoa is a simple procedure that reduces the volume of the material to be shipped,
and may improve the quality of the chilled epididymal sperm cells. In the present study
we compared the characteristics of frozen/thawed epididymal spermatozoa after refriger-
ation of the epididymis or after direct refrigeration of the extended epididymal sperm cells.
Ejaculated sperm samples were obtained from 10 healthy stallions with at least 15 days of
sexual rest, before routine orchiectomies. Spermatozoa were recovered from the epididy-
mal tail immediately after castration (EPI), after refrigeration of the epididymis for 24 h at
4 ◦C (EPI R) and recovered from epididymal tail immediately after castration and stored
for 24 h at 4 ◦C (EPI RR). Total motility, straight-line velocity, percentage of rapid cells, via-
bility and morphological defects were similar (p > 0.05) among different treatments, and
post-thaw viability was higher (pto
collect semen with an artificial vagina (Missouri type)
lubricated with non-spermicidal gel and filled with warm
water to obtain an inner temperature of 45–50 ◦C. A mare
in estrous was utilized as mount. Ejaculated semen was
immediately evaluated for volume, concentration, pro-
gressive motility (PMO), percentage of live sperm cells
(viability) and percentage of morphological defects (DEF).
Semen was then diluted (1:1, v/v), in a pre-warmed (37 ◦C)
Kenney’s medium and subsequently processed for cryo-
preservation.
2.3. Harvesting of epididymal tail spermatozoa
Bilateral orchiectomy was carried out in a stand-
ing position, under deep sedation (20–40 �g/kg of
detomidine hydrochloride depending upon the stallion
reaction, Domosedan®, Laboratórios Pfizer, Portugal) and
local anaesthesia (600 mg of lidocaine hydrochloride,
Anestesin®, Laboratório Sorológico, Portugal). After cas-
tration, testis and the corresponding epididymis were
immediately taken to an adjacent laboratory and testis and
epididymal tail weights were registered. Epididydeferec-
tomy and flushing of the epididymal tail were performed
immediately after castration (EPI and EPI RR groups) or 24 h
later (EPI R group). For this procedure, the epididymal tail
and vas deferens were isolated from the testis and care-
fully dissected removing all connective tissue and fascias.
The epididymal tail duct was then cut when its diameter
was less than 1 mm. A 21 G needle was inserted into the
open end of vas deferens 1 cm above the epididymal tail and
a flushing with 50 mL of Kenney’s medium pre-warmed
(37 ◦C) was performed into a 50 mL Falcon tube.
Spermatozoa suspensions were evaluated for concen-
tration, progressive motility (PMO), percentage of live
sperm cells (viability) and percentage of morphological
defects (DEF) after flushing fresh (EPI) and refrigerated
(EPI R) epididymis, as well as after flushing spermatozoa
in epididymis refrigerated for 24 h (EPI RR). Samples of all
experimental groups were cryopreserved.
2.4. Cryopreservation and thawing
The ejaculates (AV) and all epididymal spermato-
zoa suspensions obtained (EPI, EPI R and EPI RR) were
centrifuged 10 min at 900 × g and the supernatant was
discarded. Cryopreservation of sperm cells was then per-
formed as described by Monteiro et al. (2011b). Briefly,
the pellet was re-suspended in an egg-yolk based freez-
ing extender (Botucrio®, Botupharma, Botucatu, Brazil) to
a final concentration of 200 million sperm cells per mL. Five
T. Guimarães et al. / Animal Reproduction Science 136 (2012) 85– 89 87
Table 1
Settings utilized for computer-assisted semen analysis (ISAS®).
Characteristic
Number of frames 25/s
Maximum cell size 75 �m
Minimum cell size 4 �m
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Table 2
Mean values (±SEM) of testis weight, cauda epididymis weight and total
number of spermatozoa recovered in EPI and EPI R groups.
EPI EPI R
Testis weight (g) 170.5 ± 14.74 169.9 ± 10.58
Epididymal tail weight (g) 14.14 ± 0.79 13.92 ± 0.68
T
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f
Velocity of rapid cells >90 �m/seg
Straightness 35%
Temperature 37 ◦C
traws (0.5 mL) were filled for each experimental group
nd maintained at 4 ◦C for 20 min. Subsequently, straws
ere placed 6 cm above the liquid nitrogen surface and
ept in nitrogen vapours for 25 min. After this period straws
ere immersed in liquid nitrogen and stored. Straws were
hawed in a water bath, at 37 ◦C for 1 min, and its content
ransferred into an eppendorf tube pre-heated at 37 ◦C.
.5. The evaluation of ejaculated and epididymal tail
permatozoa
Concentration of ejaculated semen (AV) was accessed
sing Spermacue® (Minitub Ibérica S.L., Tarragona, Spain)
nd for ejaculates with a concentration reading in the
permacue® ≤ 150 × 106 spermatozoa/mL, the concentra-
ion was re-calculated using a Neubauer chamber. Concen-
ration of the recovered epididymal tail spermatozoa was
etermined using a Neubauer chamber. Progressive motil-
ty (PMO) was assessed by light microscopy (200×). The
ercentage of live sperm cells (viability) and the percent-
ge of spermatozoa with morphological defects (DEF) were
valuated using light microscopy (1000×) in eosin-nigrosin
nd Diff-QuickTM stained smears, respectively. A total of
00 sperm cells were evaluated for each parameter.
The motility parameters of frozen–thawed samples (AV,
PI, EPI R, EPI RR) were evaluated using a computer assisted
perm analysis system (ISAS®; Proiser, Valencia, Spain).
ive fields per sample were analyzed for total motility (TM),
traight-line velocity (VSL) and percentage of rapid cells
RAP), using the settings described in Table 1. Viability and
EF of frozen–thawed samples were assessed as described
bove.
.6. Statistical analysis
Statistical analysis was performed using the software
rism 5 (Graph Pad S, GraphPad Prism, version 5.00 edition,
an Diego, 2007). An analysis of variance (one way ANOVA)
able 3
ean (±SEM) total number of spermatozoa collected (total no. SPZ) and of perc
ith abnormal morphology (DEF), after spermatozoa collection in the different g
Total no. SPZ (×106) PMO (%) 
AV (n = 7) 6597 ± 1125 47.86 ± 
EPI (n = 10) 8520 ± 1056 15.50 ± 
EPI R (n = 10) 8036 ± 1289 15.50 ± 
EPI RR (n = 8) a 25.63 ± 
eans in the same column with different superscripts, differ (p 0.05). The mean
testicular and epididymal tail weights obtained in groups
EPI and EPI R are presented in Table 2. No differences
(p > 0.05) were detected for any of these parameters
between EPI and EPI R groups. No correlations (p > 0.05)
were found between testicular and epididymal tail weight,
as well as between the total number of sperm cells recov-
ered (Table 3) and the epididymal tail weight. The mean
weight of paired testes was 340.30 ± 24.89 g while the
mean number of spermatozoa harvested per stallion was
16,550 ± 2352 × 106 (5445 × 106 to 26,005 × 106).
In Table 3, the mean values obtained for PMO, viability
and DEF are presented for all experimental groups. After
two attempts to obtain an ejaculate by artificial vagina,
three out of ten stallions did not ejaculate. Semen of the
7 ejaculates (VA group) had more than 30% PMO and a
total number of sperm cells ranging from 1700 × 106 to
9696 × 106. The PMO of epididymal spermatozoa samples
(EPI, EPI R and EPI RR) varied between 0 and 40%, while
the total number of spermatozoa harvested per epididymis
entages of progressive motility (PMO), viability and of total sperm cells
roups.
Viability (%) DEF (%)
4.61a 88.71 ± 2.86 27.43 ± 2.96
4.31b 86.60 ± 1.56 24.50 ± 2.70
4.80b 86.40 ± 2.02 28.5 ± 3.15
4.67b 83.50 ± 3.11 33.75 ± 2.21
lated sperm obtained with artificial vagina; EPI, spermatozoa collected
d from the epididymal tail stored for 24 h at 4 ◦C; and EPI RR, spermatozoa
 hat 4 ◦C.
iately frozen, but instead was refrigerated for 24 h before freezing.
88 T. Guimarães et al. / Animal Reproduction Science 136 (2012) 85– 89
Table 4
Mean (±SEM) percentages of total motile sperm cells (TM), rapid cells (RAP), viable cells (viability), cells with abnormal morphology (DEF) and mean
(±SEM) straight line velocities (VSL).
TM (%)a RAP (%)a Viability (%) DEF (%) VSL (�m/s)a
AV (n = 7) 34.63 ± 5.57 2.31 ± 0.61 63.71 ± 2.55a 42.71 ± 2.61 18.24 ± 0.70
EPI (n = 10) 54.10 ± 4.15 7.04 ± 1.70 75.60 ± 2.32b 41.00 ± 1.72 18.10 ± 1.36
EPI R (n = 10) 40.91 ± 4.41 3.91 ± 0.79 73.50 ± 2.19a,b 40.80 ± 2.58 17.97 ± 1.94
EPI RR (n = 8) 48.90 ± 6.62 6.21 ± 1.56 71.88 ± 3.69a,b 42.50 ± 2.49 18.24 ± 1.12
Means in the same column with different superscripts differ (p 0.05) was found between the AV
group and the EPI, EPI R and EPI RR groups for the per-
centage of TM of frozen–thawed spermatozoa.
4. Discussion
Novice and even experienced stallions can exhibit dif-
ferences in their response to semen collection with artificial
vagina, reflecting preferences or aversions to specific char-
acteristics of this collection technique (McDonnel, 2011). In
the present study 3 out of 10 healthy stallions did not ejacu-
late suggesting that more attempts using different settings
of the artificial vagina (temperature, inner pressure, gel and
others) or a different mare in heat might have been needed
to obtain an ejaculate.
To our knowledge, data on weights of left and right epi-
didymal tail have not been reported before, as opposed to
testicular weights where differences have been detected
(Johnson and Thompson, 1986). The lack of weight differ-
ences (p > 0.05) between right and left epididymis found in
this study is in agreement with the similar number of sperm
cells obtained from right and left epididymis, indicating
that both epididymal tails have the same storing ability.
No differences (p > 0.05) were found between EPI and
EPI R groups in regard to testis and epididymal tail weight.
The mean weight of paired testes was similar to that
reported by Pickett et al. (1989), 329 ± 104 g, but the mean
weight per testis was lower than that obtained by James
et al. (2002), 231 ± 0.01 g. These variations can probably be
due to differences in age, breed of the stallions and sea-
son (breeding/non breeding). In fact, it has been reported
ted from the epididymal tails stored for 24 h at 4 ◦C; EPI RR, spermatozoa
 h at 4 ◦C.
that stallions, on average, have seasonal variation of up to
17% in size for the same testis, and that older stallions have
in average larger testis than young stallions (Pickett et al.,
1989).
The epididymal tail weight obtained in this study was
almost twice the weight 7.80 ± 0.60 g reported by James
et al. (2002). However, it is not clear if in that study the
epididymal tail was weighted before or after the dissection
of the connective tissue and fascias.
The total number of spermatozoa recovered per
individual epididymal tail (EPI-8520 ± 1056 × 106;
EPI R-8036 ± 1289 × 106) were slightly higher than
5565 ± 1008 × 106 the reported by James et al. (2002),
but lower than 12.90 ± 9.20 × 106 obtained by Monteiro
et al. (2011b). Nonetheless, the total epididymal sperm
obtained for both epididymal tails was similar to those,
15–25 billion sperm cells, reported by Bruemmer (2006),
and similar to what has been reported by Monteiro et al.
(2011b). As expected, the total spermatozoa recovered
from both epididymides was higher (p 0.05) with the number of sperm harvested per epi-
didymal tail or with the testicle weight, indicating that
in these animals the epididymal tail weight was not
directly related to the total number of spermatozoa recov-
ered or to the testis weight. In agreement with Monteiro
et al. (2011b), no differences were found between the
total number of spermatozoa collected from the epi-
didymal tail (EPI) immediately after castration or after
24 hrs of refrigeration of the organ, for the same stallion
(EPI R).
Before cryopreservation, differences between experi-
mental groups were only observed for PMO. As expected,
a lower percentage of PMO was observed in the epi-
didymal groups (EPI, EPI R and EPI RR) than in the AV
group. In addition, and similarly to findings by others (Neil
et al., 2006), immediately after collection some epididy-
mal sperm suspensions did not have forward progressive
motility, but after processing and freeze–thawing the per-
centage of sperm cells with forward motility increased.
This is due to the existence a motility inhibiting factors
in the epididymis that keep sperm cells in an immotile
and metabolic quiescent state (Usselman and Cone, 1983).
Conversely, after ejaculation, spermatozoa are diluted
product
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Turner, T.T., Reich, G.W., 1985. Cauda epididymal sperm motility: a com-
T. Guimarães et al. / Animal Re
n seminal plasma, and epididymal sperm suspensions
rocessed for cryopreservation are centrifuged and re-
iluted in a cryoextender, allowing the elimination or
ilution of these inhibiting factors (Turner and Reich,
985).
In the present study no differences were observed in the
perm cells defects (DEF) and viability between ejaculated
nd epididymal sperm cells in agreement with Monteiro
t al. (2011b). But Heise et al. (2011) reported a higher per-
entage of abnormalities and higher percentage of viable
perm cells in the epididymal tail (Heise et al., 2011). The
ifference in viability could be attributed to alterations
ccurring in the plasma membrane of ejaculated sperm
hen in contact with seminal plasma, which could affect
perm viability (Johnson et al., 1980), but a solid explana-
ion for the difference in sperm abnormalities remains to
e given. Additional research on viability and DEF between
jaculated and epididymal sperm cells are warranted,
iven the contradictory results obtained by different
uthors.
A strong association between the total motility of
rozen–thawed ejaculated and frozen–thawed epididymal
perm was reported by Magistrini et al. (1988). In the
resent study, no such correlation (p > 0.05) was detected.
After freezing/thawing, no differences between exper-
mental groups were observed for all the motility
arameters assessed, as well as for the percentage of mor-
hological defects recorded. Still, a higher percentage of
iable spermatozoa were observed in the EPI group than
n the VA group, which could be due to a higher resistance
o cold shock of epididymal rather than ejaculated sperm
Johnson et al., 1980; Monteiro et al.,2011a,b).
For all parameters (TM, VSL, RAP, DEF and viability)
valuated in frozen/thawed samples the EPI R group did
ot differ from the EPI and AV groups, which is in agree-
ent with others results (Bruemmer et al., 2002; Monteiro
t al., 2011b), indicating that 24 h refrigeration of epididy-
al sperm within the epididymal tail (EPI R) does not affect
ryopreservation.
In this study we also demonstrated, for the first time in
he stallion, that cold preservation of extended epididymal
perm for 24 h before freezing was neither deleterious nor
eneficial for the quality of the frozen–thawed semen as
ompared to the other experimental groups.
The same fertility was obtained with epididymal sperm
ells cryopreserved immediately after castration or after
4 h of refrigeration of the epididymis, and for both cases
he fertility was equivalent to the one obtained with cryo-
reserved ejaculated sperm (Monteiro et al., 2011b). In
he present study, the comparative fertilizing ability of
rozen–thawed epididymal sperm diluted and stored at 4◦
or 24 h before freezing remains to be tested.
. Conclusion
The immediate collection and refrigeration of epididy-
al spermatozoa was as efficient as the refrigeration of
he testis–epididymis complex. Thus, the choice of either
ethod to ship epididymal sperm cells for cryopreserva-
ion will depend on the operator preferences.
ion Science 136 (2012) 85– 89 89
The present study also showed that both right and left,
epididymal tails had a similar weight and number of stored
sperm cells.
Acknowledgements
This work was partially financed by Projects
PTDC/CVT/108456/2008 (FCT) and COMPETE: FCOMP-
01–0124-FEDER-009565 (FEDER).
References
Bruemmer, J.E., Reger, H., Zibinski, E.L., Squires, E.L., 2002. Effect of storage
at 5 ◦C on the motility and cryopreservation of stallion epididymal
spermatozoa. Theriogenology 58, 405–407.
Bruemmer, J.E., 2006. Collection and freezing of epididymal stallion sperm.
Vet. Clin. North Am. 3, 677–682.
Caballero, J., Frenette, Sullivan, R., 2011. Post testicular sperm matura-
tional changes in the bull: important role of the epididymosomes and
prostasomes. Vet. Med. Int., ID 757194.
Heise, A., Thompson, P.N., Gerber, D., 2011. Influence of seminal plasma on
fresh and post-thaw parameters of stallion epididymal spermatozoa.
Anim. Reprod. Sci. 123, 192–201.
James, A.N., Green, H., Hoffman, S., Landry, A.M., Paccamonti, D., Godke,
R.A., 2002. Preservation of equine sperm stored in the epididymis at
4 ◦C for 24, 48, 72 and 96 h. Theriogenology 58, 401–404.
Johnson, L., Amann, R.P., Pickett, B.W., 1980. Maturation of equine
spermatozoa. Am. J. Vet. Res. 41, 1190–1196.
Johnson, L., Thompson, D.L., 1986. Seasonal variation in the total volume of
Leydig cells in stallions is explained by variation in cell number rather
than cell size. Biol. Reprod. 35, 971–979.
Magistrini, M., Tinel, C., Noue, P., Palmer, E., 1988. Correlations between
characteristics of frozen spermatozoa from ejaculates or perfusates
from cauda epididymes and proximal deferent duct in a group of stal-
lions. In: University College Dublin (Ed.), 11th International Congress
on Animal Reproduction and Artificial Insemination. University Col-
lege, Dublin, UK, p. 273.
McDonnel, S., 2011. Abnormal sexual behaviour. In: Mckinnon, A.O.,
Squires, E.L., Vaala, W.E., Varner, D.D. (Eds.), Equine Reproduction. ,
2nd ed. Wiley-Blackwell, Chichester, UK, p. 1407.
Monteiro, G.A., Freitas-Dell’Aqua, C.P., Guasti, P.N., Landim-Alvarenga,
F.C., Dell’Aqua Junior, J.A., Alvarenga, M.A., Papa, F.O., 2011a. Com-
parison of apoptotic index between cryopreserved ejaculated and
epididymal sperm in stallions. In: AAEP (Ed.), 57th Annual Convention
of American Association of Equine Practitioners. San Antonio, USA,
p. 226.
Monteiro, G.A., Papa, F.O., Zahn, F.S., Dellaqua, J.A., Melo, C.M., Maziero,
R.R.D., Avanzi, B.R., Alvarenga, M.A., Guasti, P.N., 2011b. Cryopreser-
vation and fertility of ejaculated and epididymal stallion sperm. Anim.
Reprod. Sci. 127, 197–201.
Muradás, P.R., Weiss, R.R., Kozicki, L.E., Granemann, L.C., Santos, I.W., Pim-
pão, C.T., 2006. Some viability parameters from equine spermatozoa
harvested by artificial vagina and epididymal tail washing. Arch. Vet.
Sci. 11, 69–74.
Neil, D., Miragaya, M., Chaves, G., Pinto, M., Alonso, A., Gambarotta, M.,
Losinno, L., Aguero, A., 2006. Cryopreservation of cauda epididymis
spermatozoa from slaughterhouse testicles 24 h after ground trans-
portation. Anim. Reprod. Sci. 94, 92–95.
Pickett, B.W., Amann, R.P., McKinnon, A.O., Squires, E.L., Voss, J.L., 1989.
Management of the stallion for maximum reproductive efficiency II.
In: Colorado State University (Ed.), Colorado State University, USA. ,
pp. 59–65 (Animal Reproduction Laboratory Bulletin no. 5).
Sostaric, E., Aalberts, M., Gadella, B.M., Stout, T.A.E., 2008. The roles of the
epididymis and prostasomes in attainment of fertilizing capacity by
stallion sperm. Anim. Reprod. Sci. 107, 237–248.
Tiplady, C., Morris, L.H.A., Allen, W.R., 2001. Stallion epididymal spermato-
zoa: pre-freezing and post thaw motility and viability after 3
treatments. Havemeyer Foundation Monography Series, vol. 5, pp.
63–65.
parison among five species. Biol. Reprod. 32, 120–128.
Usselman, M.C., Cone, R.A., 1983. Rat sperm are mechanically immobilised
in the cauda epididymis by immobilin, a high molecular weight gly-
coprotein. Biol. Reprod. 29, 1241–1253.
	Characteristics of stallion epididymal spermatozoa at collection and effect of two refrigeration protocols on the quality ...
	1 Introduction
	2 Materials and methods
	2.1 Animals and experimental design
	2.2 Collection of ejaculated spermatozoa
	2.3 Harvesting of epididymal tail spermatozoa
	2.4 Cryopreservation and thawing
	2.5 The evaluation of ejaculated and epididymal tail spermatozoa
	2.6 Statistical analysis
	3 Results
	3.1 The epididymal sperm characteristics before freezing
	3.2 Epididymal sperm quality after freezing
	4 Discussion
	5 Conclusion
	Acknowledgements
	References