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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, Contents
Jaboticabal, So Paulo, Brazil This study aimed to evaluate tissue damage of feline testicles sectioned in two differ-
2
Programa de Mestrado em Cincia
ent sizes (0.3 or 0.5cm3) and submitted to different cryoprotectants (propanediol or
Animal,Universidade de Franca, UNIFRAN,
Franca, So Paulo, Brazil glycerol). Testicles obtained from 12 domestic cats were sectioned in 0.3 and 0.5cm3
3
CDVE Centro de Diagnstico Veterinrio sized pieces and immediately evaluated by TBARS and semi-quantitatively by histo-
Especializado, So Joo da Boa Vista, So
morphology. The remaining fragments were placed in cryotubes with 1ml Egg yolk
Paulo, Brazil
Tris Equex STM extender containing 3% glycerol or 3% propanediol and cryopreserved
Correspondence
by fast-freezing technique. Frozen-thawed fragments were also evaluated by TBARS
Beatrice I. Macente, Departamento
de Medicina Veterinria Preventiva e and histomorphology. Statistical analysis was performed using one-way ANOVA with
Reproduo Animal, UNESP, Jaboticabal,
StudentNewmannKeuls post hoc test, with p<.05. Fresh and cryopreserved tis-
So Paulo, Brazil.
Email: beatrice.vetuel@yahoo.com.br 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,


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

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

at the time of freezing and passage out during the thawing process fertilizing ability of feline testicular spermatozoa. Animal Reproduction
(Honaramooz, 2012). The tolerance to cryoprotectants by the tissues Science, 131, 219227.
Buarpung, S., Tharasanit, T., Comizzoli, P., & Techakumphu, M. (2013). Feline
is limited, and its excessive exposure may cause serious damage (Pegg,
spermatozoa from fresh and cryopreserved testicular tissues have
2002); however, this toxicity is very difficult to be evaluated (Fuller, comparable ability to fertilize matured oocytes and sustain the embryo
2004). A possible explanation is the molecular difference between development after intracytoplasmic sperm injection. Theriogenology,
these two structures; even though these two cryoprotectants have 79, 149158.
Buege, J. A., & Aust, S. D. (1978). Microsomal lipid peroxidation. Methods in
the same biochemical origin, glycerol is a larger and denser molecule
Enzymology, 52, 302310.
than propanediol, and therefore, its infusion through testicular frag- Comizzoli, P., & Wildt, D. E. (2012). On the horizon for fertility preserva-
ment might be lower and/or slower (Abrishami etal., 2010). tion in domestic and wild carnivores. Reproduction in Domestic Animals,
Our results expose two points to consider: (i) 0.3-cm3 fragments 47(Suppl. 6), 261265.
Comizzoli, P., Wildt, D. E., & Pukazhenthi, B. S. (2006). In vitro development
showed the most serious cell damage (pyknosis) in histomorphologi-
of domestic cat embryos following intra-cytoplasmic sperm injection
cal assessment, which might have been caused by the cryoprotectants with testicular spermatozoa. Theriogenology, 66, 16591663.
toxicity; (ii) propanediol might have reached intracellular toxic levels Crabb, E., Verheyen, G., Tournaye, H., & Van Steirteghem, A. (1999).
resulting from a greater and faster diffusion between cells under the Freezing of testicular tissue as a minced suspension preserves sperm
quality better than whole-biopsy freezing when glycerol is used as
conditions of the present study. Therefore, we conclude that glycerol
cryoprotectant. International Journal of Andrology, 22, 4348.
can be considered more efficient for cryopreservation of 0.5-cm3
Demirci, B., Salle, B., Frappart, L., Franck, M., Guerin, J. F., & Lornage, J.
fragments. However, additional research is needed to verify the pen- (2002). Morphological alterations and DNA fragmentation in oocytes
etration rate of these cryoprotectants through the testicular tissue, as from primordial and primary follicles after freezing-thawing of ovarian
well as new investigations testing other types of intracellular cryopro- cortex in sheep. Fertility and Sterility, 77, 595600.
Fleck, R. A., Benson, E. E., Bremner, D. H., & Day, J. G. (2000). Studies of free
tectants, because the morphology, architecture and amount of lipids in
radical-mediated cryoinjury in the unicellular green alga Euglena graci-
testicular tissue can interfere with the cryoprotectants performance, lis using a non-destructive hydroxyl radical assay: A novel approach for
depending on the species. developing protistan cryopreservation strategies. Free Radical Research,
32, 157170.
Fuller, B. J. (2004). Cryoprotectants: The essential antifreezes to protect life
ACKNOWLE DGE ME N TS in the frozen state. Cryoletters, 25, 375388.
Honaramooz, A. (2012). Cryopreservation of testicular tissue, current frontiers in
Authors would like to thank Roberta Vantini and Ivo Luiz de Almeida cryobiology. InTech. Available from: http://www.intechopen.com/books/
Jnior for technical and laboratory support and also Professor Jeffrey currentfrontiers-in-cryobiology/cryopreservation-of-testicular-tissue.
Hori, T., Ichikawa, M., Kawakami, E., & Tsutsui, T. (2004). Artificial insemi-
Frederico Lui (CECG) and his team for the biological material.
nation of frozen epididymal sperm in beagle dogs. Journal of Veterinary
Medicine Science, 66, 3741.
Hori, T., Matsuda, Y., Kobayashi, M., Kawakami, E., & Tsutsui, T. (2011).
CO NFLI CT OF I NTERE S T
Comparison of fertility on intrauterine insemination between cryo-
preserved ejaculated and cauda epididymal sperm in dogs. Journal of
None of the authors have any conflict of interest to declare.
Veterinary Medicine Science, 73, 16851688.
Jahnukainen, K., Ehmcke, J., Hergenrother, S. D., & Schlatt, S. (2007). Effect
of cold storage and cryopreservation of immature non-human primate
AUT HOR CONTRI BUTI O N S
testicular tissue on spermatogonial stem cell potential in xenografts.
Human Reproduction, 22, 10601067.
BIM and MA have designed the study, and participated in the acqui-
Keros, V., Hultenby, K., Borgstrm, B., Fridstrm, M., Jahnukainen, K., &
sition, interpretation of data and drafting of the manuscript. GHT,
Hovatta, O. (2007). Methods of cryopreservation of testicular tissue
CFMM, HET, CLC, MEGT, RRG have participated in the acquisition with viable spermatogonia in pre-pubertal boys undergoing gonado-
and interpretation of data. All authors have read and approved the toxic cancer treatment. Human Reproduction, 22, 13841395.
final version of the manuscript. Keros, V., Rosenlund, B., Hultenby, K., Aghajanova, L., Levkov, L., & Hovatta,
O. (2005). Optimizing cryopreservation of human testicular tissue:
Comparison of protocols with glycerol, propanediol and dimethylsul-
phoxide as cryoprotectants. Human Reproduction, 20(6), 16761687.
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