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Theriogenology xxx (2015) 1–6
Contents lists ava
Theriogenology
journal homepage: www.ther io journal .com
Characteristics of frozen epididymal spermatozoa from
stallions that died 12 to 36 hours after colic surgery
A. Gloria*, A. Carluccio, L. Petrizzi, F. Noto, A. Contri
Faculty of Veterinary Medicine, University of Teramo, Teramo, Italy
a r t i c l e i n f o
Article history:
Received 23 April 2015
Received in revised form 3 September 2015
Accepted 3 September 2015
Keywords:
Horse
Epididymal semen
Cryopreservation
Colic
* Corresponding author. Tel.: þ39 861 266995; fa
E-mail address: gloriaalessia@libero.it (A. Gloria)
0093-691X/$ – see front matter � 2015 Elsevier Inc
http://dx.doi.org/10.1016/j.theriogenology.2015.09.0
a b s t r a c t
Equine spermatozoa from the cauda epididymis were previously collected and frozen, and
the fertility was assessed. Most studies were performed on healthy stallions that had
undergone routine castration or on the epididymis collected at the abattoir, but there are
no studies on the quality of epididymal semen in subjects which have died from colic or
which underwent intensive care. The present study was designed to verify whether a
severe illness could affect epididymal semen quality and freezability in the stallion.
Therefore, epididymal semen characteristics during the freezing process in stallions which
had died from colic and in healthy stallions submitted to elective castration were
compared. Five stallions that had died from colic (ill stallions [ISs]) and seven stallions that
had undergone elective castration (healthy stallions) were castrated, and cauda epididymis
spermatozoa were collected and processed. Sperm quality was tested after collection, after
washing procedures, at the end of the equilibration (5 �C for 75 minutes), and after
freezing/thawing. Sperm quality was measured by objective motility characteristics,
membrane and acrosome integrity, and mitochondrial activity. After collection, sperm in
ISs showed low kinetic parameters (total motility: 17.3 � 3%, progressive motility: 6 � 1%,
average path velocity: 57.4 � 35.4 mm/s, straightness: 74.2%) compared with healthy
stallions (total motility: 90.8 � 3.7%, progressive motility: 70 � 4%, average path velocity:
118.1 � 12.6 mm/s, straightness: 82.4%) but demonstrated similar membrane and acrosome
integrity (85 � 2.8% vs. 87.6 � 3.1%). Sperm kinetic parameters increased after washing
procedures and cooling in ISs, reaching comparable values after equilibration (5 �C for
75 minutes) and freezing/thawing. The data reported in this study suggest that the quality
of the equine epididymal spermatozoa cryopreserved in stallions that had died from colic
was similar to that reported in epididymal sperm after elective castration and was also
similar to the data reported in literature for cryopreserved equine semen.
� 2015 Elsevier Inc. All rights reserved.
1. Introduction
Traumatic injuries or colic conditions can prematurely
end the breeding career of a male. In such circumstances,
owners may request a final semen collection to allow the
propagation of valuable genetics. In specific cases, the
epididymal sperm is the only available source of male
gametes for use in assisted reproduction programs.
x: þ39 861 266962.
.
. All rights reserved.
06
Collection of epididymal sperm further offers the oppor-
tunity to acquire and use genetic material from elite males,
even postmortem. Epididymal semen can be used fresh or
cryopreserved and stored in a genetic resource bank [1].
Successful epididymal sperm collection with resulting
pregnancies has been reported in a number of species,
including goats, red deer, dogs, and humans [2–6].
Furthermore, frozen equine epididymal spermatozoa have
been proven to be fertile [7,8]. The pregnancy rate after the
use of frozen epididymal sperm ranges from 17% to 30%
using hysteroscopic insemination [8].
mailto:gloriaalessia@libero.it
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http://dx.doi.org/10.1016/j.theriogenology.2015.09.006
http://dx.doi.org/10.1016/j.theriogenology.2015.09.006
A. Gloria et al. / Theriogenology xxx (2015) 1–62
Recently, articles reported the collection of spermatozoa
from the cauda epididymis in both horses and donkeys
[9–13]. These studies were performed in experimental
conditions, with normal and healthy males undergoing
elective castration. However, no studies are available on the
collection and handling of epididymal sperm in critical
patients or in stallions which died after intense care, or the
impact of these conditions on the quality of cryopreserved
semen. If sperm recovery from the cauda epididymismeans
the last chance to obtain viable spermatozoa from a stal-
lion, the success of this biotechnology in stallions which
died after intensive care is an important area of study [10].
In this article, we reported the procedures for epidid-
ymal sperm collection and cryopreservation from stallions
which died from colic complications, and we compared
these characteristics with those recorded in stallions that
underwent elective castration. Furthermore, unlike other
studies [12,13], differences between sperm characteristics
in epididymal samples from ill or healthy stallions (ISs and
HSs, respectively) were evaluated using objective systems,
such as the computer-assisted sperm analyzer (CASA)
system and flow cytometry, which allow the detection of
fine differences between samples [14].
2. Materials and methods
2.1. Animals and surgery
Twelve sexually mature stallions, Quarter Horse and
Standardbred, aged between 5 and 8 years and weighing
between 450 and 550 kg, were enrolled in this study. For all
stallions, explicit informed consent for the owners was
acquired.
Five stallions were referred to the Veterinary Teaching
Hospital of Teramo (Italy) for severe colic (ISs), evaluated by
clinical examination, abdominal distention and topography
by transrectal palpation, heart rate, aspect of mucosae and
capillary refilling time, decreased or absent gut sounds,
liquid characteristics after abdominocentesis, and lack of
response to analgesic–sedative treatment. The symptoms
developed in an acute manner, and the diagnosis was
performed within 12 hours. A surgical approach to confirm
the suspected diagnosis and to treat the colic was
attempted. Systemic preoperative antibiotics were admin-
istered (cefazolin: 10 mg/kg intravenous [IV]; gentamicin
sulfate, 8 mg/kg IV). The horses were sedated with mede-
tomidine hydrochloride (Domitor: 1 mg/mL; Pfizer Animal
Health, Kirkland, Quebec, Canada), 0.007 mg/kg IV. Induc-
tion was performed using diazepam (5 mg/mL; Sandoz,
Boucherville, Quebec, Canada), 0.02mg/kg IV and ketamine
hydrochloride (Vetalar; 100 mg/mL; Bioniche, Belleville,
Ontario, Canada), 2.2 mg/kg IV. Anesthesia was maintained
by 3% isoflurane inhalant in oxygen (10 L/min) and a
constant-rate infusion of medetomidine (0.0035 mg/kg/h).
Ringer lactate and colloids were infused, and dobutamine
(maximum dose: 0.00125 mg/kg/min) was administered to
maintain a mean arterial blood pressure of between 70 and
90 mmHg. After surgery, flunixin meglumine (0.25 mg/kg
q6 hours) and DMSO infusion (10% solution in polyonic
fluids q12 hours) were administered to prevent endotox-
emia. Boluses of xylazine (0.4 mg/kg) and butorphanol
tartrate (0.01 mg/kg) were administered when the horse
showed acute abdominal pain. In all cases, the horses died
12 to 36 hours after recovery. All the stallions were
orchidectomized soon after death.
Seven healthy stallions were submitted to the Veteri-
nary Teaching Hospital of Teramo, Italy, for routine orchi-
ectomy (HSs). In these cases, the surgery was performed
following the same anesthetic protocol as described for the
ISs, except that the postsurgical treatment consisted of only
flunixin meglumine (1.1 mg/kg q12 hours). In both ISs and
HSs, local anesthesia was not performed.
2.2. Collection and cryopreservation of epididymal sperm
The gonads were individuallytransferred to sterile
beakers and transported to the laboratory in a Styrofoam
box and processed within 30 minutes. The cauda epidid-
ymis of each testis was dissected [11], and spermatozoa
from the cauda epididymis were collected by retrograde
flushing via the deferent ductus. Briefly, each deferent
ductus was cannulated and flushed using 20 mL of
INRA96. Samples were collected in a 200-mL sterile glass
beaker and transferred to a 50-mL centrifugation tube.
Total sperm per sample was calculated as the product of
the concentration, estimated by a Burker chamber, and the
volume of the flushing. The samples were washed twice,
with INRA96 and MAXI Freeze (IMV Technologies, L’Aigle,
France) by centrifugation at 1000 � g for 20 minutes, as
reported by the manufacturer. After washing, the samples
were diluted with INRA-Freeze (IMV Technologies) at the
final concentration of 200 � 106 sperm/mL and equili-
brated at 5 �C for 75 minutes. The samples were then
packaged in 0.5-mL french straws, frozen using a nitrogen
vapor programmable freezer as previously described
(freeze rate of �60 �C/min from 5 �C to �140 �C) [15], and
plunged into liquid nitrogen and stored (�196 �C). After
5 days of storage, ten straws for each stallion were thawed
(37 �C for 1 minute in a water bath). Sperm evaluations
were performed after collection (C), after the first (W1)
and the second washing (W2), after equilibration (EQ),
and after cryopreservation (FT).
2.3. Motility evaluation
Objective sperm motility evaluation was performed by
the CASA system, IVOS 12.3 (Hamilton Thorne Biosciences,
Beverly, MA, USA) as previously described [16]. Semen was
diluted at 30 � 106 sperm/mL concentration using INRA96.
Each sample was rewarmed at 38 �C for at least 10 minutes,
a 6-mL dropwas loaded into aMakler chamber (SefiMedical
Instruments, Haifa, Israel), and 12 nonconsecutive fields
were analyzed [17]. The CASA settings were 60 frames/s
(Hz) and 45 frames per field. In this study, the following
parameters were considered: total motility (TM, %), pro-
gressive motility (PM, %), average path velocity (VAP, mm/s),
straight line velocity (VSL, mm/s), curvilinear velocity (VCL,
mm/s), amplitude of lateral head displacement (mm), beat
cross frequency (Hz), straightness (STR, as VSL/VAP, %), and
linearity (as VSL/VCL, %). On the basis of their VAP, sper-
matozoa were classified as rapid (VAP, >75 mm/s), medium
(75 mm/s > VAP > 25 mm/s), slow (VAP,mitochondrial
activity were recorded after the W2, EQ, and FT phases
(Table 1).
4. Discussion
The collection and cryopreservation of epididymal
spermatozoa from stallions has been proposed over
several years [7,9,10,20–23], to allow the conservation of
semen from stallions with high genetic value which had
died suddenly or become permanently unable to
Table 1
Sperm parameters (mean � standard deviation) after collection (C), first washing (W1), second washing (W2), equilibration at 5 �C for 75 minutes (EQ), and freezing/thawing (FT), in samples collected from the
epididymis of stallions after colic (IS group, n ¼ 5) or routine castration (HS group, n ¼ 7).
Parameter C W1 W2 EQ FT
IS group HS group IS group HS group IS group HS group IS group HS group IS group HS group
TM (%) 17 � 3a 90.8 � 3.7b 67 � 4c 87 � 6b 78 � 3c 89 � 8b 90 � 2b 91 � 7b 57 � 7c 59 � 11c
PM (%) 6 � 1a 70 � 4b 26 � 3c 71 � 5b 54 � 4d 72 � 5b 71 � 4b 72 � 6b 37 � 6cd 42 � 8cd
VAP (mm/s) 57.4 � 35.4a 118.1 � 12.6b 104.6 � 43.8c 116.8 � 11.4b 109 � 32.8c 121.4 � 14.7b 110.6 � 24.2c 119.3 � 13.1b 89.2 � 31.2c 94.5 � 19.4c
VSL (mm/s) 42.6 � 30.7a 113.6 � 16.1b 72.7 � 41.3c 108.7 � 19.2b 83.5 � 32.7c 104.8 � 28.2b 87 � 29.3c 96.6 � 15.4bc 71.5 � 32.4c 82.5 � 21.5c
VCL (mm/s) 115 � 72a 250.6 � 20.8b 208.4 � 82.6c 238.6 � 23.4b 212.3 � 68.7bc 246.3 � 37.2b 219.6 � 46.4bc 239.5 � 26.6b 163.9 � 62.1c 172.6 � 58.8c
ALH (mm) 7.4 � 3.2a 8.5 � 0.9a 8.2 � 3.3a 8.6 � 1.2a 8.1 � 2.3a 8.4 � 1.3a 8.3 � 2.4a 8.2 � 1.2a 6.6 � 2.1b 6.9 � 1.8b
BCF (Hz) 34.5 � 12.3a 40.3 � 1.5b 35.6 � 14.7a 42.4 � 8.7b 39.1 � 10.4ab 40.8 � 9.6b 40.6 � 6.8b 41.1 � 8.7b 40.9 � 14.8b 40.4 � 11.8b
STR (%) 74.2 � 17a 82.4 � 1.1b 67.8 � 21.6c 82.8 � 2.1b 75 � 19.2ab 82.9 � 3.6b 81 � 14b 81.7 � 4.9 80.4 � 19.6ab 79.7 � 13.7ab
LIN (%) 41 � 15ab 46.6 � 1.1a 35 � 16b 45.2 � 5.3a 42 � 11ab 47.2 � 6.7a 43 � 10ab 42.9 � 6.4ab 44 � 15.9ab 45.2 � 10.8ab
Rapid sperm (%) 6 � 1.1a 89.8 � 4.3b 55 � 4.8c 84.6 � 5.1b 74 � 5.1bc 88.6 � 5.2b 83 � 3.8b 85.7 � 4.1b 46 � 2.9c 45 � 4.3c
Medium sperm (%) 10 � 0.8a 1.0 � 0.9b 14 � 2.1a 2.4�1b 6 � 0.7ab 1.3 � 0.7b 3 � 0.5b 2.7 � 0.9b 11 � 2.4a 11.8 � 3.1a
Slow sperm (%) 9 � 1a 3.2 � 2.3b 17 � 1.6c 5.1 � 1.9b 6 � 1.3b 3.2 � 1b 3 � 0.8b 3.8 � 1.1b 11 � 1.9a 8.6 � 2.4a
Static sperm (%) 75 � 5a 6.0 � 2.2b 14 � 2.2bc 7.9 � 1.4b 14 � 2.6bc 6.9 � 1.6b 5 � 1.2b 7.8 � 1.4b 32 � 5.9c 34.6 � 7.1c
PI�, PSA� (%) 85 � 2.8a 87.6 � 3.1a 86 � 2a 85.9 � 3.3a 86 � 2.1a 87.1 � 2.6a 86 � 3a 85.3 � 4.2a 51 � 3.5b 54.9 � 5.1b
PI�, PSAþ (%) 2 � 0.3 1.7 � 0.2 1 � 0.3 1.4 � 0.5 1 � 0.4 1.2 � 0.3 1 � 0.3 1.4 � 0.4 1 � 0.4 1.5 � 0.5
PIþ, PSA� (%) 12 � 1.1a 9.8 � 1.3a 11 � 0.9a 11.6 � 1.2a 11 � 1.2a 10.7 � 1.3a 12 � 3.3a 11.6 � 2.1a 38.6 � 4.7b 35.7 � 5.4b
PIþ, PSAþ (%) 1 � 0.2 0.9 � 0.1 2 � 0.4 1.1 � 0.3 2 � 0.2 1 � 0.3 1 � 0.3 1.7 � 0.5 9 � 0.4 7.9 � 0.5
MitoTR (%) 18 � 9.8a 78 � 8.2b 46 � 16.2c 84 � 9.7b 78.9 � 13.7b 82.8 � 9.3b 82.6 � 9.4b 83.6 � 12.5b 47.9 � 10.6c 49.3 � 7.9c
In the same row, values with different superscript letters (a, b, c, d) differ significantly (P � 0.05).
MitoTR represents spermatozoa with membrane activity; PI�, PSA�: membrane integrity and acrosome integrity; PIþ, PSA�, membrane damage and acrosome integrity; PIþ, PSA�, membrane damage and
acrosome integrity.
Abbreviations: ALH, amplitude of lateral head displacement; BCF, beat cross frequency; HS, healthy stallions; IS, ill stallions; LIN, linearity; PI, propidium iodide; PM, progressive motility; PSA, Pisum sativum
agglutinin; STR, straightness; TM, total motility; VAP, average path velocity ; VCL, curvilinear velocity; VSL, straight line velocity.
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A. Gloria et al. / Theriogenology xxx (2015) 1–6 5
reproduce. However, all these studies were performed on
HSs that had undergone routine castration, whereas no
experiments were conducted in field conditions of
animals which had died after severe illness and intensive
care. Our data, reported here, showed that the character-
istics of spermatozoa collected from stallions that had
died from colic (ISs) could be successfully collected and
cryopreserved. Furthermore, neither the total sperm nor
the sperm quality was different in the left and right
epididymis, which correlates with most studies
[11,21,24,25] but differs from others using similar devices
for sperm kinetic evaluation [26].
Epididymides were considered the most important
extragonadal reservoir of spermatozoa, ensuring the sur-
vival of these cells for several weeks [27]. During the stor-
age period, the cauda epididymis accumulates sperm to
ensure that a “sufficient”’ number is available at the time of
ejaculation [28]. The total sperm collectible from each
epididymis was similar to that reported in some studies
[23,29], confirming that the epididymis should be consid-
ered a reliable source of a large amount of spermatozoa.
Epididymal sperm in the IS group showed a very low
kinetic level soon after collection compared with the HS
group and with the data reported in the literature on
stallions [10,22,30] and jackasses [16] in comparable con-
ditions. In our study, the different characteristics recorded
in stallions which had died after severe illness and inten-
sive care could be related to endotoxic conditions during
colic and surgery [31] and to the drugs used during inten-
sive therapy [32]. Indeed, DMSO was found to negatively
affect sperm motility [33]. The presence of a toxic and/or
drug-related effect was corroborated by the improvement
of sperm characteristics after sperm washing. It was
possible that the progressive dilution of the molecules
loaded in spermatozoa during the washing procedure
resulted in the improvement of the sperm kinetic level
during cooling. On the other hand, the characteristics of
spermatozoa after equilibration and after cryopreservation
in stallions that had undergone intensive care were similar
to those of healthy animals after elective castration in
controlled conditions. The data reported in the present
study showed similar postthawing values compared with
those of equine ejaculated semen with the same extender
[34]. On the other hand, the data on sperm characteristics
after cryopreservation in both ISs and HSs were higher than
those reported in recent studies on equine epididymal
spermatozoa [12,13]. In these studies, a similar base
extender was used (INRA96), but a higher concentration of
glycerol was used (5% vs. 2.5%) making a direct comparison
of the results difficult. It is well known that an increased
concentration of glycerol in the stallions could negatively
affect sperm characteristics after cryopreservation. Con-
centrations greater than 3.5% showed a significant effect on
sperm characteristics, and the maximal toxic effect was
found at 5% [35]. Thus, the most effective glycerol con-
centration in INRA-based extenders was 2.5% [36], as
specified for the INRA-Freeze by the manufacturer.
In conclusion, the data reported in this study showed
that the collection of spermatozoa from the cauda epidid-
ymis could be a feasible procedure that allows the recovery
of a large quantity of good-quality spermatozoa. This
procedure could also be performed on stallions that have
undergone intensive therapy in field conditions, with
similar results after cryopreservation compared with stal-
lions that had undergone elective castration. Sperm
collected from ISs or stallions that had received pharma-
cological treatments should be treated with repeated
washing procedures to be acceptable for cryopreservation.
The data reported in this study suggested that acute illness
and the correlated treatment could affect epididymal
semen characteristics. However, no conclusions could be
drawn regarding long-term or chronic disease.
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	Characteristics of frozen epididymal spermatozoa from stallions that died 12 to 36 hours after colic surgery
	1. Introduction
	2. Materials and methods
	2.1. Animals and surgery
	2.2. Collection and cryopreservation of epididymal sperm
	2.3. Motility evaluation
	2.4. Flow cytometry analysis
	2.5. Statistical analysis
	3. Results
	4. Discussion
	References