DOI: 10.1111/rda.

12828

ORIGINAL ARTICLE

Evaluation of different fragment sizes and cryoprotectants for

cryopreservation of feline testicular tissues

BI Macente1|GH Toniollo1|M Apparicio2|CFM Mansano1|HE Thom3|

CL Canella3|MEG Tozato1|RR Gutierrez1

1
Departamento de Medicina Veterinria
Preventiva e Reproduo Animal,UNESP,
Jaboticabal, So Paulo, Brazil
2
Programa de Mestrado em Cincia
Animal,Universidade de Franca, UNIFRAN,
Franca, So Paulo, Brazil
3
CDVE Centro de Diagnstico Veterinrio
Especializado, So Joo da Boa Vista, So
Paulo, Brazil
Correspondence
Beatrice I. Macente, Departamento
de Medicina Veterinria Preventiva e
Reproduo Animal, UNESP, Jaboticabal,
So Paulo, Brazil.
Email: beatrice.vetuel@yahoo.com.br
Contents
This study aimed to evaluate tissue damage of feline testicles sectioned in two differ-
ent sizes (0.3 or 0.5cm3) and submitted to different cryoprotectants (propanediol or
glycerol). Testicles obtained from 12 domestic cats were sectioned in 0.3 and 0.5cm3
sized pieces and immediately evaluated by TBARS and semi-quantitatively by histo-
morphology. The remaining fragments were placed in cryotubes with 1ml Egg yolk
Tris Equex STM extender containing 3% glycerol or 3% propanediol and cryopreserved
by fast-freezing technique. Frozen-thawed fragments were also evaluated by TBARS
and histomorphology. Statistical analysis was performed using one-way ANOVA with
StudentNewmannKeuls post hoc test, with p<.05. Fresh and cryopreserved tis-
sues generally exhibited a similar morphology concerning detachment of cells from the
basement membrane and observation of nucleoli, with a great proportion scored as 0
(no alteration). When present, alterations were slight and the morphology was consid-
ered to be good (most classified in scores 1). Pyknosis was the main anomaly observed
as score 2 in 54.6% and 58.4% of 0.3-cm3 fragments cryopreserved in propanediol and
glycerol, respectively (16.7% scored 2 in fresh tissue). In TBARS evaluation, 0.5-cm3
fragments cryopreserved in glycerol produced less free radical compared to the
0.3cm3 cryopreserved in glycerol or propanediol. Our results showed that glycerol
was more efficient than propanediol to cryopreserve 0.5-cm3 fragments; this might be
attributed to the fact that glycerol molecular weight is larger than propanediol and so
its perfusion in the testicular tissue is slower.

1| INTRODUCTION to therapeutic orchiectomy or that died unexpectedly (Buarpung,

Assisted reproductive techniques (ARTs) are very important for genetic
preservation and maintenance of endangered species, especially cryo-
preservation that allows the genetic material to be storage indefinitely
(Pukazhenti, Neubauer, Jewgenow, Howard, & Wildt, 2006).
In human medicine, cryopreservation of testicular tissue has
been studied with the purpose to be used in xenotransplanta-
tion (Shinohara etal., 2002). In veterinary medicine, this technique
has been shown to be a reliable and innovative alternative to con-
serve male genetic material of any animal that has to be subjected
Tharasanit, Comizzoli, & Techakumphu, 2012). Moreover, reports
have shown that the sperm cells obtained after the freezingthawing
process and submitted to testicular sperm extraction (TESE) and in-
tracytoplasmic sperm injection (ICSI) resulted in the development of
embryos and the birth of viable offsprings (Abrishami, Anzar, Yang, &
Honaramooz, 2010; Bono-Mestre, Cardona-Costa, & Garca-Ximnez,
2009; Buarpung, Tharasanit, Comizzoli, & Techakumphu, 2013; Hori,
Ichikawa, Kawakami, & Tsutsui, 2004; Hori, Matsuda, Kobayashi,
Kawakami, & Tsutsui, 2011; Jahnukainen, Ehmcke, Hergenrother,
& Schlatt, 2007; Marks, Dupuis, Mickelsen, Memon, & Platz, 1994;

Reprod Dom Anim 2016; 51 (Suppl. 3): 16 wileyonlinelibrary.com/journal/rda 2016 Blackwell Verlag GmbH | 1
 |
2 Macente etal.
Milazzo etal., 2008, 2010; Schlatt, Foppiani, Rolf, Weinbauer, &
Nieschlag, 2002; Shinohara etal., 2002; Thuwanut etal., 2013; Wu
etal., 2011).
Investigations on testicular tissue cryopreservation of the
domestic cat are new, and it is justified by the fact that this species
is considered an experimental model for human disorders and for
wild felids threatened of extinction (Luvoni, 2006; Pukazhenti etal.,
2006). Additionally, recent studies have reported satisfactory results
using the obtained spermatozoa on in vitro fertilization of autolo-
gous oocytes (Buarpung etal., 2012; Comizzoli, Wildt, & Pukazhenthi,
2006; Tharasanit etal., 2012). However, the methodology and proto-
cols, as well as the successful production of embryos, have not been
established yet (Comizzoli & Wildt, 2012). Among the factors that
need further investigation are the cryoprotectants, once they ensure
the nutrition of cells and prevent morphological damage from thermal
and osmotic shock (Watson, 2000). Nevertheless, the type, concen-
tration and/or association of cryoprotectants depend on the type of
tissue to be cryopreserved, the freezing rate and the size of the frag-
ment and animals species (Buarpung etal., 2013; Crabb, Verheyen,
Tournaye, & Van Steirteghem, 1999; Keros etal., 2007).
This study aimed to evaluate the cryopreservation of testicular tis-
sue obtained from domestic cats comparing two fragment sizes (0.3
and 0.5cm3) and two cryoprotectants (propanediol 3% and glycerol
3%) through histomorphology and lipid peroxidation production using
thiobarbituric acid reactive substances test (TBARS) for the first time.
2| MATERIALS AND METHODS
2.1|Testicle collection, cryopreservation and
thawing
Testicles were obtained from 12 domestic cats (cross-breed, 15years)
submitted to elective orchiectomy in the Centro de Esterilizao de
Ces e Gatos of the Faculdade de Cincias Agrrias e Veterinrias,
Jaboticabal campus, Brazil. The testicles were transported into a 50-
ml falcon tube containing NaCl 0.9% inside an isothermal box (18C)
to the laboratory within 2h. Testicular tissue was separated from the
vessels, tunica albuginea and epididymides, washed three times in sa-
line solution supplemented with streptomycin (100g/ml)+penicillin
(100UI/ml) and sectioned in 0.3 and 0.5cm3 sized pieces (measured
with a digital pachymeter).
One fragment of each size (0.3 and 0.5cm3) was immediately eval-
uated by TBARS and semi-quantitatively by histomorphology. Details
of these procedures are described separately below. The remaining
0.3- and 0.5-cm3 fragments were placed in cryotubes with 1ml Egg
yolk Tris Equex STM extender containing 3% of glycerol or 3% pro-
panediol for 10min at room temperature. Then, they were maintained
at 4C for 10min and finally were laid horizontally on a rack 4cm
above the liquid nitrogen vapour (90C) for 30min, before being
plunged into liquid nitrogen (196C) for cooling and storage.
For thawing, the samples were exposed to air for 10s and sub-
merged into 37C water bath for 30s. The testicular tissues were
placed in TRIS extender medium at 37C for 5min. Frozen-thawed
fragments were also evaluated by TBARS and histomorphology.
2.2|Histomorphological evaluation
The fresh and frozen-thawed testicular fragments were fixed in
Bouin solution for 610h at 48C, dehydrated in 70% ethanol and
then embedded in paraffin wax. The samples were sectioned, stained
with haematoxylin and eosin (HE) dye and semiquantitatively evalu-
ated for integrity and structural alterations under a light microscope.
Five randomized areas of the sectioned tissue were classified accord-
ing to Milazzo etal. (2008) with modifications: (i) detachment of cells
from the basement membrane was scored as 0 if absent, 1<75% and
2>75% of the circumference; (ii) gap formation and shrinkage were
scored as 0 if absent, as 1 if slight and as 2 if more obvious; (iii) distinc-
tion between Sertoli cells and spermatogonia nuclei was scored as 0 if
easy, 1 if difficult and 2 if impossible; (iv) observation of nucleoli was
scored as 0 if easy (visible in 40% of cells) and scored as 1 if indistin-
guishable; (v) pyknotic nuclei was scored as 0 if absent, as 1 if <40%
and as 2 if >40% were pyknotic.
2.3|Lipid peroxidation
Lipid peroxidation reaction was determined using thiobarbituric
acid reactive substances (TBARS) test, following the protocol de-
scribed by Buege and Aust (1978): a homogenized testicular tissue
was prepared by slicing it for 30s with KCl and 1.15% phenylmeth-
anesulfonyl fluoride (PMSF) at a concentration of 100mmol/L in
isopropanol and 10l/ml in KCl. Then, the homogenate was centri-
fuged at 1610 g for 10min in a cooled centrifuge at 04C. After
centrifugation, 0.25ml of the supernatant was added to the trichlo-
roacetic acid (TCA) at 10% (w/v), to denature the proteins and acid-
ify the reaction. This mixture was then stirred and centrifuged at
1,000g for 3min. An amount of 0.5ml of this supernatant was
added to 0.5ml of thiobarbituric acid (TBA) 0.67% (w/v) to react
with the lipoperoxidation products forming a pink compound. The
mixture was incubated at 100C for 15min and then was cooled
in ice. TBARS were quantified on a spectrophotometer (Thermo
Fisher ScientificGenesy 20, China), using wavelength of 532nm.
The value was assessed directly as production of lipid peroxidation.
2.4|Statistical analysis
Statistical analysis was performed using the Statistical Analysis
Systems software package (Version 9.2; SAS Institute Inc., 2008, NC,
USA). Chi-square was used for histomorphological evaluation and
Fischers exact test when the expected number was inferior to five.
For lipid peroxidation, the statistical difference for repeated measures
was evaluated by one-way ANOVA, and the StudentNewmann
Keuls post hoc test was used to evaluate the interaction of fresh and
frozen-thawed testicular tissue. p-Value was considered statistically
significant when <.05.
Macente etal. |
3

was the primary abnormality observed for cryopreserved tissue; 54.6%

3|RESULTS
and 58.4% of the 0.3-cm3 fragments cryopreserved in propanediol and
glycerol, respectively, were classified as score 2 and differed signifi-
In histomorphological assessment of testicular fragments (Tables1
cantly from the control (16.7% classified as score 2; Tables1 and 2).
and 2), fresh samples showed lower rates in the detachment and re-
Concerning lipid peroxidation production evaluated by TBARS
traction of the basal cell membrane scores, as well as for the distinction
test, statistical analysis of means showed no significant difference be-
between the Sertoli cells and spermatogonia; a great proportion was
tween the 0.3-cm3 fragments cryopreserved in glycerol or propanediol
classified as score 0 (Figure1a). All fresh samples showed pyknosis;
and the fresh samples. On the other hand, there was a significant dif-
however, most were classified as score 1 (less than 40% alterations).
ference between the 0.5-cm3 cryopreserved fragments and the fresh
The cryopreserved tissue showed similar morphology to the fresh
ones, with glycerol presenting lower TBARS values (Table3). There
tissue when we compared the detachment of cells from the basement
was no significant difference between the 0.3-cm3 and 0.5-cm3 frag-
membrane and nucleus visibility. Significant alterations were observed
ments cryopreserved with glycerol or propanediol.
for the 0.3-cm3 fragments cryopreserved with propanediol with re-
gard to distinction of Sertoli cells and spermatogonia; the majority of
the samples was classified as score 1, and it was difficult to differenti-
ate these two types of cells (Figure1b). The 0.5-cm3 fragments cryo- 4|DISCUSSION
preserved with glycerol also showed a higher percentage of samples
classified as score 1, with the presence of retraction of the basement It is well known that the quality of testicular cells may be affected by
membrane; the majority presented with a slight retraction. Pyknosis factors such as the freezing technique (Keros etal., 2007), the type of

T A B L E 1 Frequency of scores (percentage) in histomorphological evaluation of domestic cat testicular tissue sectioned in two sizes and
cryopreserved in 3% glycerol or 3% propanediol

Detachment of basement membrane of tubular

cells Retraction of basement membrane

Fragment size Groups 0 (None) 1 (<75%) 2 (>75%) 0 (None) 1 (slight) 2 (obvious)

3
0.3cm Fresh (control) 66.67 33.33 0.00 100.00 0.00 0.00
Glycerol 3% 63.64 36.36 0.00 72.73 27.27 0.00
PrOH* 3% 50.00 50.00 0.00 66.67 33.33 0.00
p-Value 0.0967** 0.0121**
0.5cm3 Fresh (control) 66.67 25.00 8.33 91.67 0.00 8.33
Glycerol 3% 75.00 25.00 0.00 16.67 83.33 0.00
PrOHa 3% 75.00 25.00 0.00 66.67 33.33 0.00
p-Value .0377** .0008*

Differences by: *Chi-square test, **Fishers exact test.

a
Propanediol.

T A B L E 2 Frequency of scores (percentage) of cellular characteristics and differentiation of domestic cat testicular tissue sectioned in two
sizes and cryopreserved in 3% glycerol or 3% propanediol evaluated by histomorphology

Distinction Sertolispermatogonia Visibility of nucleus Pyknosis

Fragment 1
size Groups 0 (easily) 1 (difficult) 2 (impossible) 0 (easily) (indistinguishable) 0 (absent) 1<40% 2>40%
3
0.3cm Fresh (control) 83.33 16.67 0.00 91.67 8.33 0.00 83.33 16.67
Glycerol 3% 54.55 27.27 18.18 90.91 9.09 0.00 45.45 54.55
PrOH* 3% 25.00 58.33 16.67 75.00 25.00 0.00 41.67 58.33
p-Value .0580* .4243* .0074**
0.5cm3 Fresh (control) 75.00 8.33 16.67 75.00 25.00 0.00 75.00 25.00
Glycerol 3% 66.67 25.00 8.33 83.33 16.67 0.00 75.00 25.00
PrOHa 3% 41.67 58.33 0.00 75.00 25.00 0.00 66.67 33.33
p-Value .0821* .8515* .0943**

Differences by: *Chi-square test, **Fishers exact test.

a
Propanediol.
 |
4 Macente etal.

F I G U R E 1 Photomicrography of
histological assessment of testicular tissue
sectioned in 0.3-cm3 fragments before
(control) and after cryopreservation. (a,
b) Fresh integrate tissue. The presence
of germinal epithelial cells connected
to Sertoli cells, overlapping the tubular
basement membrane and interstitial
cells in harmony with other tubules.
Pyknotic nucleus (arrows) is sparse. (c, d)
Cryopreserved tissue. The presence of
well-defined seminiferous tubules, with
several no apparent or pyknotic nucleus
(arrows). Haematoxylin and eosin dye. Scale
bars represent 100nm (a, c) and 1,000nm
(b,d)

T A B L E 3 Evaluation of lipid peroxidation (through thiobarbituric quality sperm after thawing, but they have not assessed the quality of
acid reactive substances (TBARS) test) produced by testicular tissue the thawed tissue, only the recovered spermatozoa.
of domestic cat evaluated before and after cryopreservation In contrast, Thuwanut etal. (2013) evaluated the structure of fe-
Fragment sizes line testicular tissue sectioned in 0.3-cm3 fragments after thawing,
comparing the slow- and fast-freezing techniques using DMSO 11%.
Groups 0.3cm3 0.5cm3
The authors reported no difference between the two techniques with
Fresh (control) 0.290.08a,A 0.320.06c,A regard to tissue damage, but highlighted the necessity of the material
Glycerol 3% 0.220.1a 0.210.1a to be rapidly transported to the laboratory, ensuring a better integrity
PrOH 3% 0.220.09a,A 0.250.08b,A of the tissue morphology. In our study, this assumption was confirmed,

PrOH, propanediol.
because the period of two hours between collection and evaluation
Mean in the same column followed by different superscript letters differs caused little cell damage in the fresh fragments analysed histologi-
significantly by the StudentNewmanKeuls test (p<.05). SEM=standard cally. Similarly, at post-thawing histomorphological evaluation, both
deviation, n=12. Mean in the same line followed by different superscript fragment sizes showed moderate scores of detachment and retraction
letters differs significantly by the StudentNewmanKeuls test (p<.05).
of the tubular cell basement membrane. However, when cell charac-
SEM=standard deviation. n=12.
teristics and differentiation were evaluated, the 0.3-cm3 fragments
showed a higher percentage of pyknosis, an irreversible damage that
cryoprotectant (Keros etal., 2005) and the size of the fragment to be could compromise their future use (Demirci etal., 2002), such as in
frozen (Crabb etal., 1999). For some species, such as mice, the small xenotransplantation.
size and weight (12g) of the testicle require the use of the whole The cryoprotectants act on the production of free radicals and ox-
dissected testicle (Milazzo etal., 2008). For others, there is a need to idative stress in cells by blocking the unstable intermediates products,
section the testicular tissue into fragments selected by the weight (in such as free radicals and lipid peroxidation, by linking them to their
swine, 5mg, Abrishami etal., 2010) or size (in cattle, 0.30.5cm3, Wu hydrogen atoms (Benson, 2004; Fleck, Benson, Bremner, & Day, 2000;
3
etal., 2011; wild cats, 0.3cm , Thuwanut etal., 2013). In the present Fuller, 2004). Our study also provides the first description of the eval-
study, two cryoprotectant agents were tested to preserve the testicu- uation of lipid peroxidation reaction in testicular tissue through TBARS
lar tissue obtained from domestic cats, sectioned in fragments of two test. The production of lipid peroxides marked a significant difference
different sizes (0.3 and 0.5cm3). To our knowledge, this is the first between the two sizes of fragments: the highest rate was obtained for
study to investigate the interference of testicular size fragments of the smaller fragment when propanediol was used. In contrast, glyc-
domestic cats over the freezing protocol. There are only three papers erol proved to be effective for both fragment sizes, with no significant
about feline testicular tissue cryopreservation, in which 0.3-cm3 frag- difference, but with a numerically superior response of the 0.5-cm3
ments were cryopreserved with different cryoprotectants (Buarpung fragment. This difference between the cryoprotectants on the differ-
etal., 2013; Tharasanit etal., 2012; Thuwanut etal., 2013). Tharasanit ent sizes of the fragments might be a result from their toxicity into the
etal. (2012) and Buarpung etal. (2013) employed a 235mm tissues with respect to the equilibration time employed in the current
fragments, cryopreserved with glycerol at 7% by slow freezing and at study (30min at 4C). For a successful cryopreservation, an adequate
5% by two-step freezing technique. Both authors have obtained good tissue distribution of cryoprotectants is needed for both penetration
Macente etal.
at the time of freezing and passage out during the thawing process
(Honaramooz, 2012). The tolerance to cryoprotectants by the tissues
is limited, and its excessive exposure may cause serious damage (Pegg,
2002); however, this toxicity is very difficult to be evaluated (Fuller,
2004). A possible explanation is the molecular difference between
these two structures; even though these two cryoprotectants have
the same biochemical origin, glycerol is a larger and denser molecule
than propanediol, and therefore, its infusion through testicular frag-
ment might be lower and/or slower (Abrishami etal., 2010).
Our results expose two points to consider: (i) 0.3-cm3 fragments
showed the most serious cell damage (pyknosis) in histomorphologi-
cal assessment, which might have been caused by the cryoprotectants
toxicity; (ii) propanediol might have reached intracellular toxic levels
resulting from a greater and faster diffusion between cells under the
conditions of the present study. Therefore, we conclude that glycerol
can be considered more efficient for cryopreservation of 0.5-cm3
fragments. However, additional research is needed to verify the pen-
etration rate of these cryoprotectants through the testicular tissue, as
well as new investigations testing other types of intracellular cryopro-
tectants, because the morphology, architecture and amount of lipids in
testicular tissue can interfere with the cryoprotectants performance,
depending on the species.
ACKNOWLE DGE ME N TS
Authors would like to thank Roberta Vantini and Ivo Luiz de Almeida
Jnior for technical and laboratory support and also Professor Jeffrey
Frederico Lui (CECG) and his team for the biological material.
CO NFLI CT OF I NTERE S T
None of the authors have any conflict of interest to declare.
AUT HOR CONTRI BUTI O N S
BIM and MA have designed the study, and participated in the acqui-
sition, interpretation of data and drafting of the manuscript. GHT,
CFMM, HET, CLC, MEGT, RRG have participated in the acquisition
and interpretation of data. All authors have read and approved the
final version of the manuscript.
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