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Influence of Recovery Method and Centrifugation on Epididymal Sperm fromCollared Peccaries (Pecari tajacu Linnaeus, 1758)Author(s): José Artur Brilhante Bezerra , Andréia Maria da Silva, Gislayne Christianne XavierPeixoto, Mariana de Araújo da Silva, Moacir Franco de Oliveira, and Alexandre Rodrigues SilvaSource: Zoological Science, 31(5):338-342. 2014.Published By: Zoological Society of JapanDOI: http://dx.doi.org/10.2108/zs130149URL: http://www.bioone.org/doi/full/10.2108/zs130149

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2014 Zoological Society of JapanZOOLOGICAL SCIENCE 31: 338–342 (2014)

Influence of Recovery Method and Centrifugation on Epididymal

Sperm from Collared Peccaries (Pecari tajacu Linnaeus, 1758)

José Artur Brilhante Bezerra, Andréia Maria da Silva,

Gislayne Christianne Xavier Peixoto, Mariana de Araújo da Silva,

Moacir Franco de Oliveira, and Alexandre Rodrigues Silva*

Laboratory of Animal Germplasm Conservation – LCGA, Universidade Federal

Rural do Semi-Árido – UFERSA, Mossoró, RN, Brazil

In order to establish protocols for gamete recovery from accidentally killed wild animals, or to take

advantage of those slaughtered by captive breeders, we assess the influence of two methods on

the recovery of epididymal sperm from collared peccaries, and verify the effect of centrifugation on

such gametes. Genitalia from nine animals were used. For each animal, one epididymis was pro-

cessed by flotation and the other was processed by retrograde flushing, both using a buffered

media based on Tris. Following recovery, sperm were evaluated for motility, vigor, viability, func-

tional membrane integrity, and morphology. A 1-mL aliquot of each sample was centrifuged, the

supernatant removed, and the pellet suspended and evaluated as fresh samples. The sperm char-

acteristics did not differ between the samples collected by flotation or retrograde flushing (P < 0.05).

Centrifugation promoted an increase in head and tail defects, thus reducing the percentage of via-

ble sperm (P < 0.05). No other parameter assessed for both methods was affected by centrifugation.

In conclusion, epididymal sperm from collared peccaries can be efficiently collected through flota-

tion or retrograde flushing, but not when either is followed by centrifugation.

Key words: epididymis, sperm, centrifugation, collared peccary, Tayassu tajacu

INTRODUCTION

In Latin America, captive breeding of collared peccaries

(Pecari tajacu) has been stimulated in order to supply the

hides to international leather markets, particularly those in

Europe and United States (Nogueira and Nogueira-Filho,

2011). The International Union for Conservation of Nature

classifies the peccaries as species of least concern, but

emphasizes that the population is disappearing in some nat-

ural habitats (IUCN, 2013). In spite of the commercial and

conservational interests regarding this species, there is a

lack of information regarding the application of reproductive

biotechnologies in collared peccaries, and studies on male

are limited to the establishment of protocols for cryopreser-

vation of ejaculated sperm (Castelo et al., 2010a; Castelo et

al., 2010b; Silva et al., 2012).

Obtaining sperm from the cauda epididymis of valuable

wild animals and captive breeders with high genetic value,

in particular from those accidentally killed, appears to be an

additional option to the preservation of genetic material. It

is important to highlight that such sperm is morphologically

viable, has an adequate degree of maturity, and preserves

the ability to fertilize (Foote, 2000; Monteiro et al., 2011).

Different factors, such as size of the epididymis and vas def-

erens diameter, influence the success of the technique for

epididymal sperm recovery (Chatdarong et al., 2010). In

addition, the possibility of contamination of samples by red

blood and epithelial cells, which can be removed by centrif-

ugation, are limiting factors (Rijsselaere et al., 2004).

Several methods have been described for epididymal

sperm recovery, but none of these have been applied in the

collared peccary to date. The flotation method, in which the

epididymis is sliced into a medium, is preferable for small

animals (Chatdarong et al., 2010), due to the size of the

epididymis. However, there have been several reports of its

use for large animals, such as the giant panda (Pérez-

Garnelo et al., 2004), the European bison (Kozdrowski et al.,

2011) and the Cantabrian brown bear (Anel et al., 2011).

Retrograde flushing of epididymis by injecting a buffered

medium into the vas deferens, is widely used for domestic

animals, such boar (Malo et al., 2011), and has also been

applied in wild species, such as red deer (Comizzoli et al.,

2001), the African buffalo (Herold et al., 2004) and the ago-

uti (Silva et al., 2011).

In order to establish protocols for gamete recovery from

accidentally killed wild animals, or even to take advantage

of those slaughtered by captive breeders, the aim of the

present study was to compare flotation and retrograde flush-

ing methods, followed or not by centrifugation, for the recov-

ery of epididymal sperm from collared peccaries.

MATERIALS AND METHODS

The ethics committee of the UFERSA approved the experimen-

tal protocols as well as the animal care procedures used (Process

n° 23091.000254/11-88). All reagents were obtained from Sigma

* Corresponding author. Tel. : +55-84-88571964;

Fax : +55-84-33151778;

E-mail: legio2000@yahoo.com

doi:10.2108/zs130149

Epididymal sperm recovery from peccaries 339

Aldrich (St. Louis, MO, USA).

Animals

The animals used belonged to the Center of

Multiplication of Wild Animals from UFERSA

(Mossoró, RN, Brazil; 5°10′S, 37°10′W). The cli-

mate is typical semiarid, with an average annual

temperature of 27°C. This center has approxi-

mately 200 collared peccaries, with an annual

planned slaughter for population control, which is

conducting in compliance with ethical and animal

welfare standards. Carcasses are designated for

experimental use. For the present study, nine sex-

ually mature male collared peccaries with an aver-

age age of 4.3 ± 0.6 years and weighing 21.2 ±1.16 kg were used. Before slaughter, they were

subjected to similar management and fed a bal-

anced swine ration plus vegetables. Freshwater

was available ad libitum.

Obtaining epididymal sperm

After slaughtering, the testes and annexes

were collected and immediately examined in order

to exclude any pathology. The complexes were

covered with gauze humidified with physiologic salt

solution (NaCl 0.9%), stored in beakers, and imme-

diately transported to the laboratory at room tem-

perature.

For each complex, the cauda epididymis,

including 4 to 5 cm of the proximal vas deferens,

was dissected and washed in physiologic salt solution. For each

animal, cauda epididymis plus vas deferens were randomly submit-

ted to sperm recovery by flotation or retrograde flushing methods,

as follows.

Flotation was conducted as described by Cary et al. (2004).

The cauda epididymis and the vas deferens were sliced with a scal-

pel in a Petri dish containing 5 mL of a Tris-based extender [3.028-g

Tris-hydroxymethyl-aminomethane, 1.78-g monohydrated citric acid,

and 1.25-g D-fructose dissolved in 100 mL of ultrapure water (Tris;

295 mOsm/L)] warmed at 37°C. After 5 min in static position, the

tissues were removed and the sperm suspension was evaluated.

For retrograde flushing, the cauda epididymis and vas deferens

were isolated. A blunt 26-gauge needle connected to a 5-mL sterile

plastic syringe was inserted into the vas deferens. Retrograde flush-

ing was conducted by injection of 5-mL Tris-based extender

warmed at 37°C, and the expelled fluid was collected in a 10-mL

sterile plastic tube. As the epididymal ducts became distended with

the flushing medium, stab incisions were made to improve recovery

of the fluid. The recovered suspension was immediately evaluated

(Silva et al., 2011).

Sperm evaluation

The volume recovered from each epididymis was measured

with a micropipette. The sperm concentration (× 106 sperm/mL) for

each sample was determined using a Neubauer counting chamber,

and the number of recovered sperm was calculated. The color of

the samples was also noted. Sperm motility and vigor (strength of

sperm flagellum beating on a 0 to 5 scale) were assessed immedi-

ately using light microscopy at × 100 and × 400 magnification.

Brome-phenol blue-stained smears were prepared with 5 μL of

semen to evaluate sperm viability and morphology using light

microscopy (× 1000), based on analysis of 200 cells per slide. Func-

tional integrity of the sperm membrane was evaluated with a hypo-

osmotic swelling (HOS) test, using distilled water (0 mOsm/L) as the

hypo-osmotic solution as reported by Silva et al. (2012). Two hun-

dred sperm were examined, and those with a swollen, coiled tail

were considered to have a functional membrane.

Centrifugation procedure

After initial assessment, 1 mL of each sample containing

epididymal sperm was centrifuged at 3 G for 10 min. The superna-

tant was discarded and the pellet was extended in 1 mL of Tris at

37°C. Motility, vigor, viability, morphology, and functional membrane

integrity were assessed as previously described.

Statistical analysis

Statistical analysis was performed using the StatView software

for Windows (SAS Institute Inc., copyright 1992–1998, Version 5.0).

The results were expressed as mean ± SEM. Data for sperm motil-

ity, viability, functional integrity of the sperm membrane and mor-

phology were arcsine transformed and submitted to analysis of vari-

ance followed by Fisher PSLD test (P < 0.05). The number of sperm

and vigor were evaluated by the Mann-Whitney nonparametric test

(P < 0.05).

RESULTS

In general, both methods were similarly efficient for

sperm recovery from the cauda epididymis of collared pec-

caries. However, the presence of red blood cells, which con-

fer a reddish color to samples obtained from flotation, was

noted; this was not observed in samples collected by retro-

grade flushing.

An average of 635.9 ± 188.6 × 106 and 1096.1 ± 462.5 ×106 sperm were recovered by retrograde flushing and flota-

tion, respectively (P = 0.4529). Sperm characteristics are

shown in Table 1. For all parameters assessed, no differ-

ences were found between flotation and retrograde flushing

(P > 0.05).

The manipulative processes did not influence the general

percentage of sperm presenting normal morphology (P >

0.05). However, the centrifugation promoted an increase in

the percentage of the tail defects in flotation (P = 0.0003)

and retrograde flushing samples (P = 0.0012). This increase

Table 1. Values for epididymal sperm characteristics in collared peccaries (Pecari

tajacu; n = 9), collected by flotation and retrograde flushing, after recovery (AR) and

after centrifugation (AC).

Sperm characteristicsFlotation Retrograde Flushing

AR AC AR AC

Motility (%) 64.4 ± 10.6a 61.1 ± 10.5a 57.8 ± 13.2a 40.0 ± 10.8a

Vigor (0 – 5) 3.1 ± 0.3a 2.9 ± 0.4a 2.9 ± 0.3a 2.2 ± 0.4a

Viability (%) 63.1 ± 5.2a 50.5 ± 3.4b 68.2 ± 5.7a 53.8 ± 3.8b

Functional membrane integrity (%) 64.0 ± 5.2a 50.5 ± 3.9a 60.3 ± 7.3a 56.2 ± 4.9a

a. b Within a row, means without a common superscript differed (P < 0.05).

Table 2. Morphologic analysis of collared peccary (Pecari tajacu; n = 9) epididymal

sperm, recovered by flotation and retrograde flushing, after recovery (AR) and after

centrifugation (AC).

ParametersFlotation Retrograde Flushing

AR AC AR AC

Normal sperm (%) 57.3 ± 4.4ab 66.6 ± 2.5a 52.3 ± 3.1b 63.7 ± 3.9a

Total defects (%) 42.7 ± 4.4ab 33.4 ± 2.5b 47.7 ± 4.4a 36.3 ± 3.9b

Distal droplets (%) 23.1 ± 3.7a 9.5 ± 3.1b 29.6 ± 3.9a 7.8 ± 2.7b

Proximal droplets (%) 8.1 ± 3.3a 3.2 ± 1.1a 7.6 ± 2.9a 3.2 ± 1.3a

Tail defects (%) 7.2 ± 1.0b 15.1 ± 1.9a 9.4 ± 1.2b 19.2 ± 3.9a

Mid-piece defects (%) 0.0 ± 0.0a 0.1 ± 0.1a 0.2 ± 0.1a 0.1 ± 0.1a

Head defects (%) 4.2 ± 2.1ab 5.6 ± 2.1ab 0.9 ± 0.2b 6.0 ± 2.3a

a. b Within a row, means without a common superscript differed (P < 0.05).

J. A. B. Bezerra et al.340

of tail defects was however countered in part by a decrease

in the percentage of sperm presenting distal droplets both in

flotation (P = 0.0076) and in retrograde flushing (P = 0.0003)

samples. Detailed morphology of normal or altered sperm is

presented in Fig. 1.

By comparing both recovery methods, observable

amounts of red blood cells were noted in the bottom of the

pellet derived from flotation samples after centrifugation. In

addition, the separation of sperm and red blood cells was

not possible during the aspiration of the pellets for all the

samples.

DISCUSSION

To the best of our knowledge, this is the first study

describing the characteristics of epididymal sperm recovered

by different methods from collared peccaries. Similar to stal-

lion (Cary et al., 2004) and Spanish ibex (Capra pyrenaica)

(Santiago-Moreno et al., 2009), no differences were observed

in epididymal sperm recovered by flotation or retrograde

flushing from collared peccaries.

We note that values for motility and vigor of epididymal

sperm in the present study were lower than those previously

reported for ejaculated samples in collared peccaries

(Castelo et al., 2010a; Peixoto et al., 2012). This may be

due to the absence of seminal plasma, which contains com-

ponents responsible for stimulating sperm metabolism, and

which function in the maintenance of motility and plasma

membrane protection against oxidative stress (Schoneck et

al., 1996). Alternatively, this may be attributable to the pres-

ence of immobilin, a protein in the cauda epididymis, which

increases the viscosity of epididymal fluid and reduces the

motility of spermatic cells in mammals (Usserlman and

Cone, 1983). We emphasize however that our results for

viability and functional mem-

brane integrity of epididymal

sperm were higher than

those reported for ejaculated

sperm from the same spe-

cies (Castelo et al., 2010a).

In Iberian red deer, no

differences were observed

in the characteristics of

epididymal sperm immedi-

ately after collection using

both methods; however, the

values found for sperm motil-

ity after freezing-thawing

were higher for sperm

recovered by retrograde

flushing than those recov-

ered by flotation. The

authors of that study attrib-

uted the observed differ-

ences to the presence of

higher amounts of red blood

cells in samples obtained

by flotation (Martinez-Pastor

et al., 2006). In fact, in the

flotation procedure, the

epididymal tissue is first

subjected to slicing, which

usually releases debris and blood cells that collect in the

bottom of the pellet after centrifugation. Since aspiration and

resuspension of the samples does not provide a good sep-

aration of sperm and blood cells, the use of more accurate

separation techniques, such as density gradient separation,

is recommended for future manipulations of peccary epididy-

mal sperm, independent of the recovery technique used

(Phillips et al., 2012).

Additionally, damage caused by blood on sperm quality

mainly occurs after cooling and freezing. It was previously

reported that the presence of blood, even in low concentra-

tions, can be deleterious to sperm after freezing and thaw-

ing. The detrimental effect of blood on sperm is likely due to

the action of hemoglobin, released after hemolysis, which is

mainly responsible for damage to sperm (Rijsselaere et al.,

2004). In collared peccaries, we verified the presence of red

blood cells in all samples collected by flotation, but further

studies should evaluate the effect of these cells after cryo-

preservation of epididymal sperm.

In both methods, the morphological defect most cur-

rently observed in samples immediately after recovery was

the presence of distal droplets, which are typical of sperm

found in cauda epididymis (Gloria et al., 2011). In ejacu-

lates, these droplets are physically removed at the moment

of ejaculation (Sostaric et al., 2008) or become detached

when exposed to seminal plasma (Matousek and Kysilka,

1989). Droplets can also be removed by centrifugation

(Tebet et al., 2006), as this promotes the rupture of the drop-

let membrane and release of its contents. In fact, we

observed that centrifugation promoted a reduction in the

number of sperm presenting distal droplets, and as a result,

the percentage of total morphological defects decreased.

However, centrifugation also promoted an increase in

Fig. 1. Morphology of collared peccary (Pecari tajacu) epididymal sperm stained with Brome-phenol

Blue. (A) normal sperm, (B) abnormal acrosome (arrow), (C) bent midpiece (arrow), (D) proximal cyto-

plasmic droplet (arrow), (E) distal cytoplasmic droplet (arrow), (F) bent coiled tail, (G) broken neck (left

arrow) and coiled tail (arrow pointing down), (H) detached head, (I) micro cephalic sperm (arrow). (light

microscopy – 100 ×).

Epididymal sperm recovery from peccaries 341

head and tail abnormalities. In collared peccary ejaculate, it

was previously verified that centrifugation increases the per-

centage of such morphological defects after thawing. The

authors of that report speculated that collared peccary

sperm could be as sensitive to centrifugal forces (Castelo et

al., 2010a) as described for humans (Ng et al., 1990) and

rats (Schreuders et al., 1996).

Centrifugation is commonly used for sperm processing

in order to concentrate the sperm population, or in sperm

washing to remove the seminal plasma or extenders before

and after cryopreservation (Carvajal et al., 2004). However,

centrifugal forces can exert mechanical and physical stress

on sperm (Varisli et al., 2009). During salvage, sperm

obtained from cauda epididymis can be contaminated by

blood cells and cellular debris; this may be reduced by cen-

trifugation, which would consequently improve sperm quality

(Melo et al., 2010). However, no improvement derived from

centrifugation was observed for epididymal sperm of col-

lared peccaries in the present study; on the contrary, it neg-

atively influenced sperm morphology by increasing head

and tail defects. Such influence was reflected on a decrease

of sperm viability.

In general, our results are in agreement with previous

findings that retrograde flushing is associated with a low

level of contamination and high sperm recovery rate, as

reported for stallions (Cary et al., 2004), red deer (Martinez-

Pastor et al., 2006) and Spanish ibex (Santiago-Moreno et

al., 2009). On the other hand, we also agree with Martinez-

Pastor et al. (2006), who associated flotation with a higher

level of sperm contamination by blood in red deer.

In conclusion, both flotation and retrograde flushing can

be used for recovery of collared peccary epididymal sperm,

but we recommend the use of retrograde flushing for future

studies on epididymal sperm cryopreservation, as this

method was associated with a lower level of sperm contam-

ination by red blood cells. In addition, we do not recommend

the centrifugation of samples, due to the risk of increasing

the percentage of sperm tail defects, leading to a decrease

in the viability of epididymal sperm.

ACKNOWLEDGMENTS

CNPq (Process. 507533/2010-0) provided financial support for

the research and grants for José Artur B. Bezerra, Andréia M. Silva,

Moacir F. Oliveira and Alexandre R. Silva. None of the authors have

any conflict of interest to declare.

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(Received July 9, 2013 / Accepted January 15, 2014)