Cryobiology xxx (xxxx) xxx

Contents lists available at ScienceDirect

Cryobiology
journal homepage: http://www.elsevier.com/locate/cryo

Cryopreservation of Spix’s yellow-toothed cavy epididymal sperm using

Tris- and coconut water-based extenders supplemented with egg yolk or
Aloe vera
Samara Sandy Jeronimo Moreira a, Andreia Maria da Silva a, Ana Liza Paz Souza a,
Erica Camila Gurgel Praxedes a, João Batista Freire de Souza Junior a,
Alexsandra Fernandes Pereira b, Alexandre Rodrigues Silva a, *
a
Laboratory of Animal Germplasm Conservation, Federal Rural University of Semi-Arid, Mossoro-RN, Brazil
b
Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid, Mossoro-RN, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Addressing the establishment of biobanks for the conservation of wild hystricomorph rodents’ germplasm, we
Wildlife verified the effects of different extenders and distinct concentrations of non-permeant cryoprotectants on the
Biobank sperm parameters of Spix’s yellow-toothed cavies. Nine testis-epididymis complexes were used for sperm
Rodentia
collection by retrograde washing using Tris or a powdered coconut water extender (ACP®-116c). Spermatozoa
Epididymis
were diluted and frozen with the same extenders supplemented with egg yolk or Aloe vera at a 10% or 20%
concentration. After recovery and cryopreservation, all samples were evaluated for sperm kinetic parameters,
morphology, membrane integrity, osmotic response, and sperm-binding capability using an egg yolk perivitelline
membrane assay. After recovery, no differences were observed between Tris and ACP®-116c that provided
515.4 × 106 sperm/mL and 561.6 × 106 sperm/mL, presenting >65% motile sperm, respectively. After cryo­
preservation, most effective preservation of sperm kinetic parameters (68.1 ± 5.9% motile sperm) and membrane
integrity (48.2 ± 7.4%) was provided by Tris extender supplemented with 10% egg yolk. However, both ex­
tenders supplemented with any concentration of egg yolk or Aloe vera presented similar preservation of osmotic
response and sperm-binding ability after cryopreservation. In summary, we suggest the use of a Tris extender
supplemented of 10% egg yolk for cryopreservation of Spix’s yellow-toothed cavy epidydimal sperm.

1. Introduction
The Spix’s yellow-toothed cavies (Galea spixii Wagler, 1831) are wild
rodents belonging to the hystricomorph suborder. They are distributed
throughout the Brazilian territory [28], contributing for the mainte­
nance and functionality of the ecosystems they inhabit [16]. Although
cavy population is considered stable [12], South American rodents are
seriously threatened, mainly by environmental disturbance and inten­
sive hunting, and few attempts have been made to utilize sustainably the
local native mammals [18,34]. Moreover, cavies are interesting models
for the development of strategies that will help in the conservation of
critically threatened hystricomorph rodents as the G. monasteriensis
[14].
Among wildlife conservation strategies, the formation of biobanks
has been widely highlighted [23]. Since the electroejaculation protocols
are not yet available for the species, our team recently reported the re­
covery of epididymal sperm from Spix’s yellow tooth cavy using a
Tris-based extender for retrograde flushing [33]. Moreover, the same
extender was used in the initial attempt to cryopreserve the cavy sperm
[32].
Even if Tris-based extenders are extensively used for sperm recovery
and conservation in various species [9,35], variations related to
species-specific sperm responses to different extenders have been also
reported. In red-rumped agouti (Dasyprocta leporina), another wild
hystricomorph rodent, the use of a powdered coconut water-based
extender (ACP®-116c) was demonstrated for providing significantly
better post-thawing sperm motility results than Tris [37]. In fact, the
rich biochemical composition of coconut water [2,41], as well as the
presence of an auxin that contribute for cells conservation [37], makes
the ACP®-116c an interesting alternative to be tested as a plant origin

* Corresponding author. Federal Rural University of Semi-Arid, Av. Francisco Mota, 572, Mossoró-RN, 59625, Brazil.
E-mail address: alexrs@ufersa.edu.br (A.R. Silva).

https://doi.org/10.1016/j.cryobiol.2021.01.016
Received 29 September 2020; Received in revised form 20 January 2021; Accepted 22 January 2021
Available online 27 January 2021
0011-2240/© 2021 Elsevier Inc. All rights reserved.

Please cite this article as: Samara Sandy Jeronimo Moreira, Cryobiology, https://doi.org/10.1016/j.cryobiol.2021.01.016
S.S. Jeronimo Moreira et al.
extender for sperm from other wild rodents.
Another interesting point related to the improvement of sperm
cryopreservation protocols is the search for adequate non-permeant
cryoprotectants. Despite the extensive use of egg yolk in the composi­
tion of semen extenders [32,37], it clearly interferes with microscopic
observations or biochemical assays, as it contains granular material of
the same size and shape as the sperm [27]. Moreover, as a component of
animal origin, egg yolk may represent a potential risk of bacterial
contamination during semen transportation and storage [27]. A reduc­
tion in the amount of egg yolk in the extender composition would be
welcome, or even its complete replacement with an alternative cryo­
protectant. In this way, Aloe vera appears as an alternative agent of plant
origin to be used in the cryopreservation of sperm, especially due to its
rich composition and the presence of polysaccharides that can present
cryoprotective action [13,21]. Its positive effect has already been proven
for domestic [19] and wild [39] ungulate species. However, a toxic ef­
fect of Aloe vera on rat spermatogenesis was reported to cause an
excessive expression of nitric oxide, whose role is to act in cell apoptosis
[4]. Since the rat is also a rodent like the cavy, the investigation of the
efficiency of Aloe vera as a non-permeant cryoprotectant is warranted.
Therefore, the aim of the present study was to evaluate the effects of
different extenders (Tris and ACP®-116c) supplemented with different
concentrations (10% and 20%) of egg yolk or Aloe vera on the param­
eters of Spix’s yellow-toothed cavy epididymal sperm.
2. Materials and methods
2.1. Bioethics and chemicals
The experiments were conducted in accordance with the Animal
Ethics Committee (CEUA/UFERSA n. 23.091.002006/2013–78) and
Chico Mendes Institute for Biodiversity Conservation (Authorization n.
66,618/3). Unless otherwise stated, all chemicals were obtained from
Sigma Chemical Co. (St. Louis, MO, USA).
2.2. Animals
The animals used in this study were from the population maintained
by the Center of Multiplication of Wild Animals, UFERSA (Mossoró,
Brazil; 5◦ 10′ S, 37◦ 10′ W), registered at the Brazilian Institute of Envi­
ronment and Renewable Natural Resources (IBAMA, n. 1,478,912). A
programmed slaughter is conducted every year for population control,
and the animals are then allocated for use in different experiments.
For the present study, nine sexually mature male Spix’s yellow-
toothed cavies, aging ~2.0 years and weighing ~300 g, were used.
The animals were isolated from females for 15 days before the beginning
of the study and maintained in a 12 h natural photoperiod. They were
grouped and maintained in a covered paddock (3 × 3 m) and fed a
commercial rabbit diet.
2.3. Epididymal sperm recovery
The animals were pre-medicated with an association of xylazine (1
mg/kg, Rompun, Bayer, São Paulo, SP, Brazil) and ketamine (15 mg/kg,
Ketalar, Pfizer, São Paulo, SP, Brazil) administered IM. After 15 min,
anesthesia was induced with 50 mg/kg IM sodium thiopental (Thio­
pentax; Cristalia, São Paulo, SP, Brazil) given IV. After induction of
anesthesia, animals were euthanized with 1 mL/kg potassium chloride,
IV [42].
The testis-epididymis complexes were recovered immediately after
euthanasia and transported to the laboratory in physiological salt solu­
tion (NaCl 0.9%) at 37 ◦ C within 15 min. Collection of sperm samples
was performed by retrograde washing according to Silva et al. [31]. For
this purpose, Tris and ACP®-116c were prepared as previously reported
for cavies [32] and agoutis [37], respectively. Briefly, each
testis-epididymis complex from the same individual was dissected for
2
Cryobiology xxx (xxxx) xxx
isolation of the tail of the epididymis and the vas deferens, followed by
insertion of a 26 G needle connected to a 5 mL sterile syringe filled with
1.5 mL washing solution. For sperm obtaining, an epididymis was
washed with Tris extender, and the other with ACP®-116c. The expelled
fluid was collected and immediately evaluated.
2.4. Sperm evaluations
The volume recovered from each epididymis was verified using
monochannel micropipettes (Eppendorf, Hambug, Germany). Sperm
concentration was determined using a Neubauer counting chamber. The
number of sperm recovered was obtained by multiplying the volume by
concentration.
Sperm motility was assessed by computer-assisted sperm analysis
(CASA; IVOS 12.0, Hamilton-Thorne, Beverly, MA, USA) using 3 μL of
sample in 4-channel Leja slides – 20 μm (IMV technologies, L’Aigle,
France), according to the mouse sperm Setup 1, as reported at the HTM-
IVOs Installation and Getting Started Guide Version 14.0 (2011). The
settings of the instrument were temperature 37 ◦ C; 60 frames/s; mini­
mum contrast, 50; progressive cells path velocity (VAP) 10.0 μ/s, and
straightness, 0.0%; low-velocity VAP cutoff 5.0 μm/s and VSL cutoff 0.0
μm/s. Five independent and nonconsecutive microscopic fields were
randomly selected. The following parameters were analyzed: number of
counted cells, total motility (%), VAP (μm/s), velocity straight line (VSL;
μm/s), curvilinear velocity (VCL; μm/s), amplitude of lateral head (ALH;
μm), beat cross frequency (BCF; Hz), straightness (STR), and linearity
(LIN). According to the low (LVV; 5 μm/s) and medium (MVV; 10 μm/s)
VAP cut-off values, the overall sperm population was classified ac­
cording to four categories: rapid, medium, slow, and static. For reliable
evaluation of sperm tracks, samples were diluted (1:1) in a 0.9% NaCl
solution and the Edit Tracks Option of the IVOS 7.4G system was used to
exclude debris [32].
For morphological analysis, Bengal rose stained smears were pre­
pared and evaluated using light microscopy (1000 × ). Two hundred
cells per slide were counted using light microscopy [31].
To evaluate plasma membrane integrity of the spermatozoa, an
aliquot (10 μL) of sperm samples was incubated at 37 ◦ C for 10 min with
40 μL of 6-carboxyfluorescein diacetate (0.46 mg/mL C-FDA) and pro­
pidium iodide (0.5 mg/mL PI). After incubation, the samples were
evaluated using an epifluorescence microscope (400 × ; BX51TF,
Olympus Co., Tokyo, Japan). Two hundred cells were counted. Sper­
matozoa marked in green (C-FDA) were classified as having an intact
sperm membrane, and those marked in red (PI) were classified as non-
intact [32].
Functional integrity of the sperm membrane was verified by evalu­
ating the sperm osmotic response under a hypo-osmotic swelling test
using distilled water (0 mOsm/L) as the hypo-osmotic solution [32]. A
volume of 5 μL of semen was added to 95 μL of a hypo-osmotic solution
and incubated at 37 ◦ C for 60 min. After incubation, slides were
mounted and visualized under a phase-contrast microscope (JG40, All­
tion, Guangxi, China). Spermatozoa with swollen or coiled tails were
recorded. Two hundred cells were counted.
2.5. Egg perivitelline membrane binding assay
Sperm binding was assessed as described by Silva et al. [32], using
perivitelline membranes from fresh, unfertilized hen’s eggs. Egg yolks
were separated from albumen and placed on parafilm. Briefly, peri­
vitelline membranes were isolated, washed with saline placed in a Petri
dish and cut into squares (1 × 1 cm). Each square was placed in four-well
plates containing incubation medium comprised of: 114 mM NaCl; 3.1
mM KCl; 0.4 mM NaH2PO4; 10 mM calcium lactate; 25 mM NaHCO3; 10
μg/mL phenol red; 1.4 mM caffeine; 2.0 mM CaCl2⋅2H2O; 0.5 mM
MgCl2; 10 mM Hepes; 6 mg/mL BSA; 0.45 mM sodium pyruvate; 5.5 mM
glucose; 50 μg/mL gentamicin sulfate; pH 7.4–7.8.
After evaluation, sperm samples were washed in incubation medium
S.S. Jeronimo Moreira et al. Cryobiology xxx (xxxx) xxx

(1:1), and centrifuged (700×g for 10 min) at room temperature. The
supernatant was discarded, and the pellet diluted in incubation medium
(200 μL) and then the concentration adjusted to 1.0 × 106 motile sperm/
mL. The sperm cell suspension was added to the perivitelline membrane
at a final volume of 500 μL, for 20 min (in a water bath at 38.5 ◦ C). At
least two perivitelline membranes (two droplets) were used per repli­
cate. After co-incubation, perivitelline membranes were washed for
removal of loosely attached sperm, stretched over a slide, and stained
with Hoechst 33,358 (10 μg/mL). Sperm bound to the membrane were
counted at 400 × , using an epifluorescence microscopy (BX51TF,
Olympus Co., Tokyo, Japan). For each sample, all sperm attached to the
membrane in six fields were counted and the mean of adhered sperm
recorded.
2.6. Freezing-thawing procedures
After recovery, epididymal sperm were diluted in Tris [32] or
ACP®-116c [37]. The Tris extender was composed of 3.028 g
Tris-hydroxymethyl-aminomethane (Sigma-Aldrich Co., St. Louis, MO,
USA), 1.78 g monohydrated citric acid (Sigma-Aldrich Co.) and 1.25 g
Dfructose (Sigma-Aldrich Co.) dissolved in 100 mL ultrapure water
(Tris; 295 mOsm/L) [32]. The ACP®-116c extender (290 mOsm/L) was
obtained by the atomization process in a spray dryer and dissolved in
ultrapure water, as recommended by its manufacturer (ACP Bio­
tecnologia, Fortaleza, Brazil) [37].
Diluted samples were then cryopreserved with egg yolk or Aloe vera,
both at concentrations of 10% and 20%. The Aloe vera was obtained
from a plantation at the UFERSA campus (Mossoró, RN, Brazil). It was
cultured in a sandy clay soil type, under a typical semiarid climate. The
crude extract of Aloe vera was extracted from the parenchyma of its
leaves as a colorless gel, that was filtered through a sieve (25 mesh/1
mm). Finally, the gel was stored in a glass container till its use [39].
Cryopreservation procedure consisted in cooling diluted samples at
15 ◦ C for 40 min in isothermal boxes and subsequently in an incubator
(Q315M26, Quimis, Diadema, Brazil) for an additional 30 min until
reaching 5 ◦ C. Subsequently, they were subjected to glycerol addition
with 12% glycerol aiming at a final concentration of 6% of the internal
cryoprotectant, and then filled in 0.25 mL plastic straws, reaching a final
concentration of 100 × 106 sperm/mL. The straws were subjected to
nitrogen vapor (3 cm) for approximately 5 min and finally placed in a
cryogenic cylinder. After at least one week, the samples were exposed to
thawing in a 37 ◦ C water bath for 1 min and sperm was immediately
evaluated as reported for fresh samples.
2.7. Statistical analysis
All data were expressed as mean ± standard error and analyzed using
a general linear model using the PROC GLM procedure of the Statistical
Analysis System, version 8.0 (SAS Institute Inc., Cary, NC, USA). All
results were verified for normality by the Shapiro-Wilk test and for
homoscedasticity by Levene’s test. Values expressed as percentages were
transformed into arcsine. The comparisons were performed using a one-
way ANOVA followed by the Duncan’s multiple range test. The rela­
tionship between sperm osmotic response and the sperm-binding rate
was verified by Pearson’s correlation and simple linear regression test
using the OriginPro® 8 software (OriginLab Corporation, Northampton,
MA).
3. Results
After sperm recovery, the volume obtained was 1.29 ± 0.05 mL and
1.35 ± 0.05 mL for Tris and ACP®-116c, respectively. Both extenders
were effective in providing an adequate number of sperm, as 515.4 ×
106 sperm/mL and 561.6 × 106 sperm/mL for Tris and ACP®-116c,
respectively, presenting >65% motile sperm (Table 1). Moreover, there
were no differences between extenders related to sperm parameters
evaluated (Tables 1–3).
After sperm cryopreservation, the use of Tris with 10% egg yolk
provided higher values of total motility (48.2 ± 7.4%) and straight
linear velocity (23.4 ± 2.8 mm/s) when compared to other treatments
(Table 1). Even with the use of Tris extender, reducing the egg yolk
concentration to 10% provided better preservation of most kinetic pa­
rameters of the sperm than when using the 20% egg yolk (P < 0.05).
Regardless of non-permeant cryoprotectants, the lowest values of sperm

Table 1
Evaluation of kinetic motility patterns after recovery and cryopreservation of Spix’s yellow-toothed cavy (n = 9) epididymal sperm using Tris or ACP®-116c sup­
plemented with different concentrations (10 or 20%) of egg yolk or Aloe vera.
Parameters Recovery Cryopreservation

Tris ACP®-116c

Tris ACP®-116c 10% egg 20% egg 10% 20% 10% egg 20% egg yolk 10% 20%
yolk yolk Aloe vera Aloe vera yolk Aloe vera Aloe vera

Total motility 67.2 ± 4.4a 68.4 ± 5.2a 48.2 ± 7.4ab 38 ± 5.2bc 25.9 ± 6.5cd 23.5 ± 5.3cd 13.5 ± 3.8de 13.1 ± 4.0de 13 ± 5.4ef 3.2 ± 1.5f
(%)
VAP (mm/s) 60.8 ± 3.9a 59.17 ± 40.41 ± 38.7 ± 1.9b 33.7 ± 3.8bc 29.1 ± 3.6bcd 23.0 ± 2.8cd 29.5 ± 5.2bcd 19.3 ± 5.6d 19.2 ± 2.3d
5.4a 3.8b
VSL (mm/s) 26.5 ± 3.5a 27.1 ± 3.4a 23.4 ± 2.8a 19.6 ± 1.9ab 14.7 ± 2.2bc 13.7 ± 2.3bc 13.7 ± 1.4bc 15.4 ± 2.9bc 10.6 ± 3.4c 12.0 ±
2.2bc
b b bc bc cd
VCL (mm/s) 123.0 ± 122.6 ± 80.3 ± 6.8 80.9 ± 4.8 74.6 ± 6.2 71.1 ± 4.6 51.3 ± 6.7 63.3 ± 43.7 ± 45.1 ± 5.6d
6.0a 9.6a 11.2bcd 11.6d
ALH (mm) 7.2 ± 0.2a 7.8 ± 0.3a 8.2 ± 0.4a 8.2 ± 0.2a 7.2 ± 0.4a 2.1 ± 0.6b 7.4 ± 0.6a 7.8 ± 0.6a 1.7 ± 0.6b 0.2 ± 0.2c
BCF (Hz) 40.7 ± 1.5a 39.6 ± 2.0a 34.4 ± 1.9ab 34.2 ± 0.7ab 40.3 ± 2.8a 35.2 ± 2.9ab 30.1 ± 3.5b 34.8 ± 2.5ab 33.6 ± 41.3 ± 3.9a
3.1ab
STR 36.7 ± 2.0e 39.4 ± 56.8 ± 4.5ab 50.6 ± 43.5 ± 47.1 ± 63.5 ± 4.2a 54.3 ± 4.2abc 37 ± 61.9 ±
2.4ed 2.2bcd 4.2cde 3.8bcde 10.2cde 6.5ab
c
LIN 20.7 ± 1.2 20.9 ± 1.5c 31.4 ± 25.5 ± 1.0bc 22.4 ± 2.4bc 22.4 ± 2.5bc 33.4 ± 3.8 a
28.2 ± 2.7 abc
21 ± 5.1bc 30.1 ±
4.6abc 5.3ab
Sperm subpopulations
Rapid (%) 38.1 ± 5.0a 39.7 ± 7.5a 15.7 ± 3.9b 9.7 ± 1.7bc 6.2 ± 2.1cd 4.5 ± 1.8dc 1.3 ± 0.6de 2.2 ± 0.9de 1.2 ± 0.8de 0.0 ± 0.0e
Medium (%) 29 ± 5.9ab 28.8 ± 32.3 ± 4.4a 28 ± 3.5ab 19.6 ± 4.6 19 ± 3.8bc 12.2 ± 3.5c 10.9 ± 3.5cd 11.7 ± 3.2 ± 1.5d
4.7ab bc
4.9cd
Slow (%) 3.9 ± 2.6a 1.7 ± 1.1a 0.5 ± 0.2a 0.5 ± 0.2a 0.4 ± 0.2a 0.5 ± 0.2a 0.6 ± 0.2a 11.3 ± 10.9a 0.4 ± 0.2a 0.4 ± 0.2a
Static (%) 29 ± 4.2e 30 ± 5.6e 51.5 ± 7.5de 61.5 ± 5.2cd 73.9 ± 6.6bc 76.2 ± 5.2bc 85.9 ± 4.3b 85 ± 4.3b 86.6 ± 96.1 ± 1.8a
5.5ab
a,b,c,d
Within a row, values with different superscripts differ (P < 0.05).

3
S.S. Jeronimo Moreira et al. Cryobiology xxx (xxxx) xxx

Table 2
Evaluation of sperm morphology, membrane integrity, and osmotic response after recovery and cryopreservation of Spix’s yellow-toothed cavy (n = 9) epididymal
sperm using Tris or ACP®-116c supplemented with different concentrations (10 or 20%) of egg yolk or Aloe vera.
Parameters Recovery Cryopreservation

Tris ACP®-116c

Tris ACP®-116c 10% egg 20% egg 10% 20% 10% egg 20% egg 10% 20%
yolk yolk Aloe vera Aloe vera yolk yolk Aloe vera Aloe vera

Normal morphology 92.3 ± 91.3 ± 2.3a 87.0 ± 2.5ab 86.7 ± 2.8ab 80.2 ± 73.3 ± 86.2 ± 2.1ab 87.5 ± 1.8ab 80.9 ± 84.0 ±
(%) 1.5a 2.3bc 4.6c 2.1bc 2.6b
a ab bc
Membrane integrity 85.6 ± 87.9 ± 2.7 68.1 ± 5.9 52.1 ± 6.7 31.2 ± 8.1c 44.5 ± 38.1 ± 8.8c
42.6 ± 5.9bc
37.5 ± 31.0 ±
(%) 4.7a 6.4c 10.3c 6.8c
Osmotic response (%) 68.1 ± 67.88 ± 28.3 ± 5.4b 28.0 ± 5.9b 27.71 ± 16.5 ± 24.7 ± 6.3b 18.4 ± 3.7b 18.83 ± 24.2 ±
3.3a 5.3a 3.8b 2.4b 4.7b 4.9b
a,b,c
Within a row, values with different superscripts differ (P < 0.05).

Table 3
Evaluation of number of sperm bound to the egg perivitelline membrane after recovery and cryopreservation of Spix’s yellow-toothed cavy (n = 9) epididymal sperm
using Tris or ACP®-116c supplemented with different concentrations (10 or 20%) of egg yolk or Aloe vera.
Parameters Recovery Cryopreservation

Tris ACP®-116c

Tris ACP®-116c 10% egg 20% egg yolk 10% 20% 10% egg 20% egg 10% 20%
yolk Aloe Aloe vera yolk yolk Aloe vera Aloe vera
vera

Number of bound sperm 250.2 ± 229.5 ± 9.8a 98.9 ± 111.6 ± 71.9 ± 80.6 ± 94.4 ± 112.9 ± 104.1 ± 71.0 ±
(Mean ± SEM) 18.4a 18.9b 15.5b 6.6b 15.3b 15.8b 16.5b 18.5b 9.5b
Range 162.2–319.7 187.5–274.5 11.5–168.6 44.16–198.3 55 – 9.1 – 32.5–173.9 59.2–184.3 26.6–198.7 37.3 ±
85.3 131.6 126.3
ab
Within a row, values with different superscripts differ (P < 0.05).

motility were provided by the ACP®-116c.

Regardless of the extender used for cryopreservation, egg yolk in any
concentration provided a more effective conservation of sperm
morphology, with values similar to non-cryopreserved samples, than
Aloe vera (Table 2). For membrane integrity, Tris extender supplemented
with 10% egg yolk provided significantly higher values (68.1 ± 5.9%)
than other treatments (Table 2). Regarding the osmotic response, Tris
and ACP®-116c supplemented with any concentration of egg yolk or
Aloe vera provided similar preservation of membrane functionality
(Table 2).
Additionally, the cryopreservation procedure caused a decrease in
sperm-binding rates regardless of the extender or cryoprotectant used,
when compared to non-cryopreserved samples, in which the rates
ranged from 229.5 ± 9.8 to 250.2 ± 18.4 bound sperm (Table 3). There
were no differences related to extenders or cryoprotectants in relation to
post-thaw sperm binding rates, which ranged from 71.0 ± 9.5 to 111.6
± 15.5 bound sperm.
There were significant linear relations between the osmotic response
and the sperm binding rates using both Tris (r = 0.80; P < 0.0001) or
ACP (r = 0.65; P < 0.0001) extenders as demonstrated at the dispersion
diagram (Fig. 1). Fig. 1. Dispersion diagram demonstrating the relationship between the relative
number of osmotic responsive sperm and the number of bound sperm per egg’s
4. Discussion perivitelline membrane for Galea spixii (n = 9) epididymal samples cry­
opreserved in Tris (black) and ACP® (red) extenders. (For interpretation of the
In order to establish strategies for the conservation of wild hys­ references to color in this figure legend, the reader is referred to the Web
tricomorphic rodents, this study evaluated, for the first time, the effects version of this article.)
of extenders (Tris and ACP®-116c) and non-permeant cryoprotectants
(10% and 20% of egg yolk or Aloe vera) on the cryopreservation of the the recovery of cavy epididymal sperm, since they were able to provide
epididymal sperm from Spix’s yellow tooth cavy. Although we have an excellent amount of sperm with good qualitative parameters. Similar
shown the possibility of using components of plant origin such as ACP®- results have been reported for agoutis [37], thus confirming the possi­
116c and Aloe vera to preserve the sperm membrane functionality and bility of using these solutions for retrograde washing of the epididymis
binding ability after cryopreservation, the effect of Tris supplemented in wild hystricomorphic rodents. In fact, both extenders have
with 10% of yolk egg is noted for providing the most effective conser­ biochemical properties that can provide a suitable environment for
vation of most sperm parameters. sperm. While the Tris extender consists of a buffer used to maintain the
We also found that Tris and ACP®-116c were effective solutions for ionic balance and pH of the solution [15], the ACP®-116c extender is

4
S.S. Jeronimo Moreira et al.
composed by salts, sugars, vitamins, proteins, neutral fats and important
nutrients for sperm viability and fertility [10,25]. It also contains an
auxin called 3-indole acetic acid, which is known to contribute to the
conservation of sperm in several species [37,41]. In plant cells, auxins
such as 3-indole acetic acid bind to soluble proteins, thus forming a
complex that can bind to membrane receptors causing changes in cell
permeability, respiration pattern and nucleic acid metabolism [6,17]. In
animal cells, the mechanism is not elucidated, but a similar path is hy­
pothesized [3].
During cryopreservation of the cavy sperm, the use of the Tris
extender resulted in a more adequate conservation of most parameters
when compared to the ACP®-116c. In previous research, we had already
shown that Tris was more effective in promoting longevity of cavy sperm
than the TES-based extender [33]. The effectiveness of Tris is supported
by its ability to reduce fructose metabolism [29] and, due to the pres­
ence of citric acid, which acts as an antioxidant protecting the sperm
from reactive oxygen species (ROS) and lipid peroxidation [38]. Similar
results were also observed in epididymal sperm from collared peccaries
(Pecari tajacu), with higher values for sperm motility, vigour, viability,
osmotic response and various kinetic parameters having been found in
those samples cryopreserved in Tris when compared to those diluted in
ACP®-116c [8]. Interestingly, this effect in peccaries was reversed when
the sperm obtained by electroejaculation was cryopreserved in the
ACP®-116c extender [36], showing that the response to the extender
may vary as to the origin of the sperm.
On the other hand, Silva et al. [37] confirmed the efficacy of a similar
coconut water-based extender when compared to the Tris-based
extender in conserving several qualitative parameters of epididymal
sperm obtained from agouti (Dasyprocta leporina). These results show
the existence of differences related to the species-specific sperm
response to different extenders, even between different rodent species of
the hystricomorphic suborder. In general, it was known that the sper­
matozoa of the cavies [30] showed marked morphological differences
compared to rats and mice, which belong to the suborder Myomorph
[43] and now, it has been shown that there are also differences
regarding the spermatozoa of agouti, a member of the suborder hys­
tricomorph [37].
Regarding non-permeant cryoprotectants, most of the spermatic
parameters of the cavy were better cryopreserved with the use of egg
yolk than Aloe vera. In addition, a negative effect of Aloe vera on the cavy
sperm morphology was evidenced, similar to that previously described
for goat sperm [26]. The mechanism by which Aloe vera can damage the
sperm morphology, especially after freezing and thawing procedures,
remains to be elucidated. Despite this, Aloe vera has been reported to
provide effective conservation of various qualitative parameters of
sperm in various mammals, such as peccaries [39] and canines [24]
during sperm cooling and freezing. In fact, Aloe vera consists of a rich
combination of vitamins, minerals, enzymes, anthraquinones, lignin,
saponins, sterols, amino acids and polysaccharides, particularly those
derived from mannose and glucose [13,21], which can contribute to the
preservation of sperm.
About the use of egg yolk, it was evident that the reduction in con­
centration from 20 to 10% was adequate for the preservation of the
sperm parameters of the cavy. Interestingly, only the combination of
Tris with 10% egg yolk was able to maintain the integrity of the sperm
membrane. In fact, the low-density lipoprotein present in the egg yolk
can restore the phospholipids and cholesterol present in cell membranes,
which are lost due to the thermal shock of the temperature change
during cryopreservation [7,22]. In high concentrations, however, some
of the components of the egg yolk have been shown to impair sperm
respiration and motility [1].
The Tris extender with 10% egg yolk was also efficient in preserving
the parameters of kinetic movement of the cavy sperm after cryopres­
ervation. According to Awad et al. [5], kinetic parameters such as VAP,
VSL, VCL and LIN can be influenced by the type and concentration of the
cryoprotectant. The reduction of the egg yolk concentration plays an
5
Cryobiology xxx (xxxx) xxx
important role in the optimization of cryopreservation protocols in
cavies, mainly because it is well known that the egg yolk debris can
interfere in the subsequent analyses [27].
Interestingly, both extenders using any cryoprotectants in any con­
centration provided a similar effect in the preservation of the osmotic
response and in the binding rates of the cavy spermatozoon, two
strongly correlated functional parameters. For years, the functional
integrity of the membrane, represented by its osmotic response, has been
suggested as a good indicator of male fertility [20]. This aspect supports
our current findings, since the orthologous similarity between glyco­
proteins of the mammalian oocyte zona pellucida and chicken egg per­
ivitelline membrane proteins [40] allows the estimation of male fertility
through this sperm-binding assay [11]. At this point, we must consider
the possibility of developing an extender composed mainly of contents
of plant origin, such as ACP®-116 and Aloe vera. However, we highlight
the need for further research to improve its use as an alternative for
sperm bioprocessing.
5. Conclusions
In conclusion, we recommend the use of a Tris extender supple­
mented with 10% egg yolk for cryopreservation of epididymal sperm
from Spix’s yellow tooth cavy. The current results were encouraging and
provided important information for cavy germplasm preservation.
Declaration of competing interest
None of the authors have conflict of interest.
Acknowledgments
The authors thank the CEMAS/UFERSA for providing biological
material. This study was supported by Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior – Brasil (CAPES, Financial Code 001). AR
Silva and AF Pereira are CNPq investigators.
Data Availability Statement
The data that support the findings of this study are available from the
corresponding authors upon reasonable request.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.cryobiol.2021.01.016.
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