Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 April 2020 | 12(5): 15557–15564
ISSN 0974-7907 (Online) | ISSN 0974-7893
(Print)
doi: https://doi.org/10.11609/jott.5744.12.5.15557-15564
#5744 | Received 29 January 2020 | Finally
accepted 13 April 2020
Field friendly method for wild feline semen cryopreservation
Gediendson Ribeiro de Araujo 1, Thyara de Deco-Souza 2, Letícia
Coelho Ferreira Bergo 3, Leanes Cruz da Silva 4, Ronaldo Gonçalves Morato 5, Pedro Nacib Jorge-Neto 6, Maitê Cardoso
Coelho da Silva 7, Gustavo Guerino Macedo
8 & Tarcízio
Antônio Rego De Paula 9
1,3,4,9 Federal University of Viçosa (UFV) / Avenida Peter Henry Rolfs, s/n - Campus Universitário, Viçosa / MG,
36570-900, Brazil.
2,7,8 Faculty
of Veterinary Medicine and Animal Science, Federal University of Mato Grosso do
Sul (UFMS) / Rua Senador Filinto Muller, 2443 - Vila Ipiranga,
Campo Grande / MS, 79070-900, Brazil.
5 Centro
Nacional De Pesquisa E Conservação
De Mamíferos Carnívoros, ICMBio/MMA / Estrada Municipal Hisaichi
Takebayashi, 8600 - Bairro da Usina,
Atibaia / SP, 12952-011, Brazil.
6 Department of
Animal Reproduction, Faculty of Veterinary Medicine and Animal Science,
University of São Paulo (USP) / Av. Prof. Dr. Orlando Marques de Paiva, 87 - Cidade
Universitária, São Paulo / SP, 05508-270, Brazil.
1 gediendson@gmail.com
(corresponding author), 2 thyara.araujo@ufms.br, 3 letbergo@gmail.com,
4 leanes.c.s@gmail.com,
5 ronaldo.morato@icmbio.gov.br,
6 pepovet@usp.br, 7 maitecoelhocardoso@gmail.com, 8 gustavo.macedo@ufms.br,
9 tarcizio@ufv.br
Editor:
Anonymity requested. Date of
publication: 26 April 2020 (online & print)
Citation: Araujo, G.R.D., T.D. Deco-Souza, L.C.F. Bergo, L.C.D. Silva, R.G. Morato,
P.N. Jorge-Neto, M.C.C.D. Silva, G.G. Macedo &
T.A.R.D. Paula (2020). Field friendly method for wild
feline semen cryopreservation. Journal of Threatened
Taxa 12(5): 15557–15564. https://doi.org/10.11609/jott.5744.12.5.15557-15564
Copyright:
© Araujo et al. 2020.
Creative Commons Attribution 4.0 International License. JoTT allows
unrestricted use, reproduction, and distribution of this article in any medium
by providing adequate credit to the author(s) and the source of publication.
Funding: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES) – Finance Code 001; Fundação de Amparo à Pesquisa do Estado de Minas Gerais; Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul.
Competing interests: The authors declare no competing interests.
Author
details: Gediendson Ribeiro de Araujo, DVM, MSc, PhD, Postdoc. Veterinarian at
Bioscience Institute (UFMS). Expert in scientific capture and reproduction of
free-living felids. Member of REPROCON research group. Thyara de
Deco-Souza, DVM, MSc, PhD. Professor at FAMEZ/UFMS. Expert in morphophysiology of reproduction of wild and domestic
animals. Member of REPROCON research group.
Letícia Coelho Ferreira Bergo,
DVM, MSc, PhD. Expert in carnivore conservation. Professor at Centro Universitário de Viçosa. Leanes Cruz da Silva, DVM, MSc, PhD. Expert in
carnivore conservation. Ronaldo Gonçalves Morato,
DMV, MSc, PhD. Coordinator of the National Center for Research and Conservation
of Terrestrial Mammals (ICMBio/MMA). Associate
researcher at Instituto Pró-Carnívoros and
Smithsonian Conservation Biology Institute.
Maitê Cardoso Coelho da Silva, DMV. Actually
master’s degree student (PPGCV-FAMEZ / UFMS). Member of REPROCON research
group. Gustavo Guerino
Macedo, DVM, MSc, PhD, Postdoc. Professor at FAMEZ/UFMS. Expert in morphophysiology of reproduction of wild and domestic
animals. Expert in Reproductive Physiology and Biotechnologies. Pedro Nacib Jorge Neto, DVM, MBA, MSc. Actually, PhD student
(PPGRA-FMVZ / USP) and Technical-Commercial Director of IMV Technologies
Brazil. Member of REPROCON research group.
Author contribution: GR Araujo, T Deco-Souza and TAR Paula conceived,
designed and directed the study. GR Araujo, T Deco-Souza, LCF Bergo, LC Silva and TAR Paula performed the experiments. GR
Araujo, T Deco-Souza, RG Morato, MCC Silva, GG Macedo
and TAR Paula analyzed and interpreted the data. GR Araujo, T Deco-Souza and GG
Macedo wrote the manuscript. PN Jorge-Neto critically
revised the manuscript. All authors approved the manuscript for publication.
Abstract: The aim of this study was to
develop a field-friendly method for free-living jaguar and cougar semen
cryopreservation. Six captive Jaguars Panthera onca and
three captive Cougars Puma concolor were
chemically restrained with a combination of medetomidine (0.08–0.1 mg/kg) and
ketamine (5 mg/kg). Semen was collected
through a tomcat urinary catheter with an open end, diluted for a final
concentration of 50 x 106 sperm/mL in a TRIS-egg yolk extender and
packaged into 0.25 mL straws. We
compared two cooling methods: CoolA - in which straws
were placed in a glass tube that was placed in a glass bottle containing water
(600mL at 38°C) and transferred to a polystyrene container (12L) containing an
11cm column of ice and water at room temperature; CoolB
– where the glass bottle – straws kit was transferred to a 4.26L cooler
containing nine blocks (81cm3) of Ice Foam recyclable ice,
previously frozen in liquid nitrogen.
The sperm volume varied from 2 to 720 µl for the jaguars and from 80 to
140 µl for the cougars. Sperm
concentration varied from 224 to 5,115 x106 sperm/mL for the jaguars
and from 485.7 to 562.5 x 106 sperm/mL for the cougars. Concerning the cooling treatments, there was
no difference in frozen-thawed sperm quality between the methods, in both
species. Thereby, the cooling method
using recyclable ice frozen in liquid nitrogen can be used for semen cryopreservation
in wild felines, eliminating the need for electric energy.
Keywords: Cryopreservation, free-living, Panthera onca, Puma
concolor, spermatozoa.
Resumo: O objetivo
deste estudo foi desenvolver um método de campo amigável para a criopreservação de sêmen de onça-pintada e onça-parda de vida livre. Seis onças-pintadas em cativeiro (Panthera
onca) e três onças-pardas em cativeiro (Puma concolor) foram anestesiados com uma combinação de medetomidina (0,08 a 0,1mg/kg) e cetamina
(5mg/kg). O sêmen
foi coletado através de um cateter urinário tomcat com extremidade aberta, diluído para uma concentração final de 50 x 106
spz / mL em diluente de gema de ovo TRIS e embalado em palhetas de 0,25mL. Comparamos dois métodos de resfriamento: CoolA - em que as palhetas foram colocados em um tubo de vidro
que foi colocado em uma garrafa
de vidro contendo água (600mL a 38°C) e transferido
para um recipiente de poliestireno
(12L) contendo uma coluna de 11cm de gelo e água à temperatura ambiente; CoolB - onde as palhetas em garrafa de vidro
foram transferidas para uma caixa térmica
de 4,26 L contendo nove blocos (81cm3) de gelo reciclável, previamente congelado em nitrogênio
líquido. O
volume espermático variou
de 2 a 720µl para as onças-pintadas e de 80 a 140µl
para os pumas. A concentração
espermática variou de 224 a
5.115 x106 spz/mL para as onças-pintadas e de 485,7 a 562,5 x 106 spz/mL para as onças-pardas. Em relação aos tratamentos
de resfriamento, não houve diferença na qualidade dos espermatozoides descongelados
entre os métodos em ambas as espécies. Desse modo, o método de resfriamento usando gelo reciclável congelado em nitrogênio
líquido pode ser usado para a criopreservação de sêmen em felinos selvagens,
eliminando a necessidade de
energia elétrica.
INTRODUCTION
The Jaguar Panthera
onca and the Cougar Puma concolor
are apex predators and play a crucial role in the prey population control, thus
both are considered keystone species for the ecosystems conservation (Crawshaw
Jr. 1991). Threats such as deforestation
and human activity are resulting in a reduced Jaguar and Cougar population in
Brazil, and both species are classified as Vulnerable by the Brazilian Red Book
of Threatened with Extinction Fauna (ICMBio
2018a). The conservation of such species
depends on several actions that can reduce their vulnerability. These actions are defined in the National
Action Plan for Big Cats Conservation (NPBigcat) (ICMBio 2018b) produced by the Brazilian Ministry of
Environment.
One of the recommended actions by the NPBigcat is to develop assisted reproduction programs,
which aim to help increase the genetic variability of the species. Sperm cryopreservation is an assisted
reproduction technique that enables keeping viable sperms for an indeterminate
period (Silva et al. 2004). In addition,
semen cryopreservation allows translocation of genetic material among
populations, dispensing the transport of individuals, which reduces the stress
caused by the translocation and the risks of transmission of infectious diseases
(Wildt 1990).
For cryopreservation, sperm must be cooled from body
temperature to 5°C and only then frozen in nitrogen vapor (-70°C) and finally
stored in liquid nitrogen at -196°C (Budhan Pukazhenthi et al. 1999; D. Zambelli
et al. 2010). Several automatic cooling
and freezing equipment are available in the market, however, they are large and
require electricity. There are also
portable containers for sperm cooling and transportation, which use recyclable
ice. Nevertheless, they also need
electricity to freeze the ice for 12h before being used. Thus, these devices are not feasible for use
in free-living felines, since capture sites are often difficult to access and
without electricity. This difficulty is
clearly demonstrated when we evaluate the articles published in scientific
journals, in which only two papers describe the characteristics of fresh sperm
in free-living Jaguars, but they did not cryopreserve the samples (Morato et al. 2001; Araujo et al. 2018). Therefore, one of the challenges in developing assisted reproduction techniques in
free-living cats is the lack of portable and electricity-free devices. Thus, this study was aimed to develop a field
friendly method for Jaguar and Cougar semen cryopreservation.
MATERIALS AND METHODS
Animals
Captive Jaguars (n=6) and cougars (n=3) were used from
three different institutions: two Jaguars and two Cougars at Mata Ciliar
Association (Jundiaí – SP; -23.1780S,
-46.9410W), one Jaguar and one Cougar at Paulínia Zoo (Paulínea – SP; -22.7640S, -47.1530W)
and three Jaguars at a non-governmental organization NEX - No Extinction (Corumbá de Goiás – GO; -15.8590,
-48.4760W). The animals were
housed in enclosures with natural lighting, with water ad libitum and fed a
meat-based diet. Animal ages were
estimated based on medical records of the respective maintainers.
The present study had authorization for scientific
activities issued by SISBIO / ICMBio / MMA under no.
46031-4, approved by the Ethic Committee on Animal Use of the School of the
Federal University of Viçosa (CEUA-UFV) under
protocol no. 79/2015 and was registered in the SISGEN National System for the
Management of Genetic Heritage and Associated Traditional Knowledge (Register
A327AAC).
Semen collection
Males were fasted for 12 hours without food and water
before chemical restraint, that was performed using anesthetic darts fired with
a blowpipe and containing medetomidine (0.08–0.1 mg/kg, Precision Pharmacy, CA,
USA) and ketamine (5mg/kg, Dopalen, Vetbrands, SP, Brazil).
After semen collection, anesthesia was reversed using Atipamezole
(0.25mg/kg, Precision Pharmacy).
The semen was collected by urethral catheterization as
described by Araujo et al. (2018).
Briefly, 20–40 min after medetomidine administration a semi-rigid tomcat
urinary catheter (w/ open end, 3FR, 130mm long) was introduced into the urethra
and negative pressure was applied (by a 1mL syringe) to increase suction effect
and semen collection. The semen was then
placed in a pre-warmed (38°C) 2mL plastic tube and kept in a water bath at
38°C.
Semen evaluation and processing
Immediately after collection, the semen was diluted
(2:1) in maintenance medium (MM; TRIS 24g/L; citric acid 14g/L; glucose 8 g/L;
amikacin 2g/L; egg yolk 200g/L; Nutricell, SP,
Brazil). Then, subjectively evaluated
for forward progressive motility (FPM) on a scale from 0 to 5, where 0
represented no forward movement and 5 represented steady, rapid forward
progression; and progressive motility (PM) from 0% to 100%, in increments of 5%
under a 200x magnifying microscope (CBRA 2013).
The sperm concentration was measured using a Neubauer chamber.
An aliquot of each diluted semen sample was fixed in Karnovsky fixative (Karnovsky
1965) and later evaluated for sperm morphology (200 cells/ejaculate) under
phase-contrast microscopy (1000× magnification). Individual cells were
classified as normal, major defects or minor defects in terms of their
perceived adverse effects on male fertility (Blom
1973).
The sperm plasma membrane function was accessed by the
hypo-osmotic swelling test (HOST), as described by Araujo et al. (2015). Semen was incubated in 100 mOsmol/kg sucrose solution (1:4) at 38 °C for 30 min, and
fixed in Karnovisk fixative (Karnovsky
1965). One hundred sperms were evaluated
under phase-contrast microscopy (1000× magnification) and those with bent or
coiled tail were considered functional – this number was corrected by excluding
the sperms with bent and coiled tail in the morphology test.
Sperm cryopreservation
After evaluation, the semen concentration was
standardized for 100 x 106 sperm/mL in MM and then diluted (1:1) in
cryopreservation media (12% glycerol, 1% de Equex STM
Paste in MM). Thus, semen was
cryopreserved in TRIS-egg yolk extender with a final concentration of 50 x 106
sperm/mL, 6% glycerol, 0.5% de Equex STM Paste. Samples were package into 0.25mL straws (IMV
Technologies, NOR, France).
For each ejaculate, two cooling methods were
evaluated: Cooling A (CoolA); we used the previously
described method (Deco-Souza et al. 2013; Araujo et al. 2015) in which straws
were placed in a glass tube that was placed in a glass bottle containing water
(600 mL at 38 °C) and transferred to a polystyrene container (12L) containing
an 11cm column of ice and water at room temperature, for 1.5h. The cooling rate was -0.53°C/min (from 38 to
5°C). Cooling B (CoolB); where straws were cooled for
1.5h in a 4.26L cooler container containing nine blocks – 81cm3 each
– of Ice Foam recyclable ice, previously frozen in liquid nitrogen (Image
1). For this the straws were placed in a
glass tube that was placed in a glass bottle containing water (600mL at 38°C)
and transferred to the cooler. This
glass bottle was surrounded by the ice foam blocks. The amount of Ice Foam was previously defined
to reach a cooling rate similar to the CoolA group.
Cryopreservation was performed by placing the straws
horizontally over a freezing rack inside a Styrofoam container filled with
liquid nitrogen and exposed to nitrogen vapor at 10cm above liquid for 15
minutes (Deco-Souza et al. 2013).
Afterwards, the straws were immersed in liquid nitrogen (-196o).
The straws were thawed in a water bath at 38°C for 30s
and transferred to a plastic tube where they were maintained during the
evaluation. Each frozen-thawed sample
was assessed as the fresh semen and for sperm motility, using a computer
assisted sperm analysis (CASA) system and staining with fluorescent probes.
Frozen-thawed semen evaluation
The plasmatic and acrosomal membranes were assessed
using a combination of three fluorescent probes: propidium iodide (PI;
Sigma–Aldrich Co. LLC.– P4170), Hoechst 33342 (H342; Molecular Probes–H1399)
and Peanut agglutinin conjugated with fluorescein isothiocyanate (FITC-PNA;
Sigma–Aldrich Co. LLC. –L7381). The frozen-thawed semen (10µl) was incubated
with 10µl of H342 (25µg/mL in DPBS) and 60µl of FITC-PNA (10.3 µg/mL in sodium
citrate 3% in DPBS) at 38°C. After 8min,
2µl of PI (0.5mg/mL in DPBS) were added and incubated for another 2min. The sperm were evaluated by epifluorescence
microscopy (Nikon H550S, excitation: 365nm; emission: 410nm) and were
classified based on the fluorescence emitted from each probe as: DI – damaged
plasma membrane and intact acrosome (only the nucleus emitting red
fluorescence); II – intact plasma membrane and intact acrosome (only the
nucleus emitting blue fluorescence); DD – damaged plasma membrane and damaged
acrosome (the nucleus emitting red fluorescence and the acrosomal region
emitting green fluorescence); and ID – intact plasma membrane and damaged acrosome
(the nucleus emitting blue fluorescence in acrosome region and emitting green
fluorescence).
The sperm motility was accessed using the sperm class
analyzer CASA system (Microptic S.L., Spain) with the
following settings described by Lueders et al. (2012)
in African lions: negative phase (Ph-) with green filter; particle size 5–85;
connectivity 14 at of capture of 50fps and 40/50 images; drifting 10; static
VCL 25μm/s; slow/medium VCL 65μm/s; rapid 100μm/s; STR 75%; and VAP setting
7μm/s. Semen sample (4μL) at 25 x 106 sperm/ mL was loaded onto a
pre-warmed disposable Leja 4 Chamber Slides (Leja Products BV, The Netherlands) and accessed by total motily (%), progressive motility (%), velocity average
pathway – VAP (μm/s), velocity straight line – VSL (μm/s), velocity curved line – VCL (μm/s),
amplitude lateral head – ALH (mm), beat cross–frequency – BCF (Hz),
straightness – STR (%), and linearity –
LIN (%).
Statistical analysis
Data on sperm quality from CoolA
versus CoolB groups were analyzed using Bayesian
t-test with unequal variances (Kery 2010). Data from fresh semen versus CoolA and CoolB groups were
analyzed using simple variance (one-way ANOVA) with fixed effect with
hierarchical Bayesian modeling. This
method of analysis allows inferences about the population and is indicative of
the probability that the parameters estimated for each group are derived from
the same distribution. According to
McCarthy (2007) and Kery (2010) the specification
model was:
yijk = αj(i) + εi
εi∼Normal(0,σ2)
In this model, yijk
corresponds to the data K observed from animal i in
the population j, aj(i)
corresponds to the expected value for the data in the population j, and the
residual εi corresponds
to the random deviation of the sperm parameter of the animal i of the mean of its population αj (i).
Observations that did not meet the assumptions of
normality were assessed using a Shapiro–Wilk test (Royston 1982) with a
significance of p<0.05 and were log-transformed. Marginal posterior distributions of
parameters were estimated using Markov Chain Monte Carlo (MCMC) methods. Analyses were implemented in program R (R
Development Core Team 2011) using the rjags package,
JAGS version 3.2.0. Each of the MCMC
chains was run for 100,000 iterations; the first 20,000 iterations were
discarded to allow for burn-in.
Convergence was assessed by visually inspecting trace plots to ensure a
reasonable exploration of the parameter space, and a potential scale reduction
factor of <1.02 for each variable (Gelman & Rubin 1992). Results were back-transformed, if
necessary. At each MCMC step, we
calculated the Bayesian equivalent to a p-value by assessing whether the mean
of one group was greater than the other.
RESULTS
Semen collection by urethral catheterization was
effective in all animals, with good volume and concentration (Table 1),
however, one Jaguar and one Cougar only ejaculated seminal fluid and thus were
not considered for statistical analysis in frozen-thawed semen.
In Jaguars there were differences (p<0.05) in sperm
FPM, sperm PM and HOST between fresh and frozen-thawed sperm, there was no
difference (p<0.05) between CoolA and CoolB parameters (Table 2).
*Data accessed by sperm class analyzer. Means ± S.D. Means within columns with different
letters differ significantly (p<0.05). FPM – Sperm forward progressive
motility; PM – sperm progressive motility; HOST – hypo-osmotic swelling test;
velocity average pathway – VAP (μm/s); velocity
straight line – VSL (μm/s); velocity curved line – VCL
(μm/s); amplitude lateral head – ALH (mm); beat
cross-frequency – BCF (Hz); straightness – STR
(%) and linearity – LIN (%). DI: damaged plasma membrane and intact
acrosome; II: intact plasma-membrane and intact acrosome; DD: damaged plasma
membrane and damaged acrosome; ID: intact plasma membrane and damaged acrosome.
As we saw in Jaguars, there were no differences
(p>0.05) in sperm quality between the CoolA and CoolB for the cougars (Table 3), however, HOST and minor
defects increased in frozen-thawed semen.
*Data accessed by sperm class analyzer. Means ± S.D.
Means within columns with different letters differ significantly (p<0.05).
FPM – sperm forward progressive motility; PM – sperm progressive motility; HOST
– hypo-osmotic swelling test; velocity average pathway – VAP (μm/s); velocity straight line – VSL (μm/s);
velocity curved line – VCL (μm/s); amplitude lateral
head – ALH (mm), beat cross-frequency – BCF (Hz); straightness – STR (%) and linearity – LIN (%). DI: damaged
plasma membrane and intact acrosome; II: intact plasma-membrane and intact
acrosome; DD: damaged plasma membrane and damaged acrosome; ID: intact plasma
membrane and damaged acrosome.
DISCUSSION
The results for fresh semen quality shows that
urethral catheterization after medetomidine administration (CT) was effective
for semen collection in Jaguars and Cougars.
Thus, this may be an alternative method for electroejaculation.
In Jaguars and in Cougars, the semen volume was lower
than previously described (5.3 to 11 mL and 0.45 to 3.4 mL, respectively) (Wildt et al. 1988; Morato et al.
1998, 1999, 2001, 2004; Paz et al. 2000,
2003, 2006, 2007; Swanson et al. 2003; Deco et al. 2010). All those studies, however, used the
electroejaculation (EE) for semen collection.
It is well know that EE stimulates contractions of the smooth muscles
and subsequently the accessory sex glands, which increases the seminal volume
(Ball 1986), resulting in more diluted semen samples. On the other hand, with the CT we collected
more concentrated semen samples than described in literature in both
species. The total number of
spermatozoa, however, was smaller than described for Cougars (Wildt et al. 1988; Deco et al. 2010). Because of the small number of Cougars used
in this study, we cannot state if this result was related to the collection
method or to the animals. The semen
volume and concentration were good enough for cryopreservation and the CT was
much more practical to be used than EE.
The SPM and PM (3.6 and 76%, respectively) in Jaguars
were superior than previously described (2.2 to 3.3 and 50.6 to 64%,
respectively) (Morato et al. 1998, 1999; Swanson et al. 2003; Silva et al. 2004;
Paz et al. 2006).
On the other hand, in Cougars the
SPM and PM were superior to the 2.5–3 and 40–50 % described by Miller et al.
(1990) and similar to the 3.5 and 75% described by Deco et al. (2010). Both parameters were considered good quality
for cryopreservation.
In the present study, Jaguars had more normal sperm
(60.7%) than Cougars (40.5%); as well as more normal sperm than described in
literature (46.7% (Morato et al. 1998); 49% (Morato et al. 1999); 31.7% (Paz et al. 2000); 50% (Morato et al. 2001); 57.3% (Swanson et al. 2003); 48.7%
(Paz et al. 2003)). Cougars had higher
or even similar normal sperm than described for the specie (26% (Wildt et al. 1988); 1–18 % (Miller et al. 1990); 8.6% (B. Pukazhenthi et al. 2001); 46.13% (Deco et al. 2010)). Felines usually have high proportion of
pathologic sperm in the ejaculate, however, the etiology and impact of those in
fertility is controversy (Howard et al. 1986). Several factors may affect sperm
morphology; although, nutrition and stress are the main factors in captive
animals.
After cryopreservation sperm quality reduced in both
species. This is expected for any
species as cryopreservation damages sperm, impairing their ability to fertilize
oocyte. Despite the reduction in the
quality of frozen-thawed sperm, SPM and PM values were similar to those
described for Jaguars (SPM: 2.7 and PM 30% (Paz et al. 2000); SPM 3.1 and PM 26.7% (Paz et al. 2007)) and for Cougars (SPM 2.5 and PM 42%
(Deco-Souza et al. 2013)). To obtain
semen samples from wild animals is always a challenge, because of the reduced
number of captive animals (several of them are vasectomized) and the difficulty
of accessing free-living animals.
Therefore, frozen-thawed semen must be used, even if they are of
poor-quality. For this, we can use
artificial insemination via laparoscopy – depositing sperm closer to the site
of fertilization – or even the intracytoplasmic sperm injection – ICSI. In addition, studies should be done to
increase sperm quality after thawing, thus increasing the efficacy of its use
for assisted reproduction programs.
For sperm cryopreservation sample must be cooled (from
body temperature to 5°C), frozen (in liquid nitrogen vapor at -70°C) and stored
(in liquid nitrogen at -196°C). Sperm
cell also may be stored at the cooling temperature, however, it remains viable
only for a few days. Several protocols and equipment are evaluable for
carnivore semen cryopreservation (and cooling) (Zambelli
et al. 2002; Luvoni et al. 2003; Tsuitsui
et al. 2003; Macente et al. 2012). Some of those are also used for wild felids
(Paz et al. 2007; Deco-Souza et al. 2013; Araujo et al. 2015; Jorge Neto et al. 2019). In
these cases, cooling was performed using refrigerators, automatic cooling and /
or freezing equipment, or even in portable containers using previously frozen
recyclable ice. All these methods depend
on electricity and cannot be used in the field, as in several places there is
no electricity available.
The CoolA method was
successfully used for cougar and ocelot semen cryopreservation (Deco-Souza et
al. 2013; Araujo et al. 2015), however, it still needs electricity to store ice. Thus, we used nontoxic recyclable ice to
reach the same cooling rate (CoolB), with the
advantage of being frozen and kept in liquid nitrogen – which is necessary for
the later stages of semen freezing. This
enables this method to be used in fields where there is no energy available.
There was no difference in sperm quality in both cooling methods,
demonstrating that the CoolB may be used for semen
cryopreservation from the felines. This
makes it feasible for sperm banks to use
semen from free-living animals, increasing the genetic resources of
these species.
CONCLUSION
The cooling method using recyclable ice frozen in
liquid nitrogen offers good semen quality and may be used for feline semen
cryopreservation, eliminating the need of electricity. Thus, this is a more practical method to be
used in the field.
Table 1. Quality of fresh semen collected by urethral
catheterization after medetomidine administration in captive Jaguars (Panthera onca, N=6)
and Cougars (Puma concolor, N=3).
|
|
Jaguar |
Cougar |
|
Volume (µl) |
292.0 ± 326.6 |
106.7 ± 30.6 |
|
Concentration (x 106 sperm/ mL) |
2091.4 ± 1816.2 |
524.1 ± 54.3 |
|
Total number of spermatozoa (x106) |
316.6 ± 399.0 |
56.5 ± 16.3 |
Table 2. Fresh and frozen-thawed Jaguar (Panthera onca, N=5)
sperm evaluation.
|
|
Fresh |
Frozen-thawed |
|
|
|
|
CoolA |
CoolB |
|
FPM |
3.6 ± 0.4a |
2.3 ± 0.3 b |
2.4 ± 0.3 b |
|
PM (%) |
73.0 ± 14 a |
31.0 ± 19 b |
38.6 ± 17.7 b |
|
HOST (%) |
55.0 ± 9.5 a |
26.4 ± 5.8 b |
24.3 ± 6.5 b |
|
Normal sperm (%) |
60.7 ± 6.8 a |
46 ± 11.4 b |
47.8 ± 5.3 b |
|
Major defects (%) |
21 ± 6.6 a |
24.6 ± 12.6 a |
24.8 ± 6.4 a |
|
Minor defects (%) |
18.3 ± 12.2 a |
29.4 ± 7.6 a |
27.4 ± 3.9 a |
|
DI |
|
55 ± 18.7 a |
39 ± 10.0 a |
|
II |
|
21.2 ± 15.7 a |
23.4 ± 13.8 a |
|
DD |
|
23.6 ± 15.3 a |
37.6 ± 10.5 a |
|
ID |
|
0.4 ± 0.9 a |
0.0 ± 0.0 a |
|
Total Motility* |
|
28.4 ± 14 a |
28.8 ± 5.9 a |
|
Progressive motility* |
|
2.0 ± 1.9 a |
1.8 ± 1.2 a |
|
VAP* |
|
10.5 ± 4.5 a |
10.5 ± 3.4 a |
|
VSL* |
|
6.6 ± 4.3 a |
6.8 ± 3.1 a |
|
VCL* |
|
23.5 ± 6.4 a |
23.5 ± 3.8 a |
|
ALH* |
|
2.2 ± 1.8 a |
2.1 ± 1.4 a |
|
BCF* |
|
7.2 ± 6.2 a |
8.5 ± 6.2 a |
|
STR* |
|
58.6 ± 13.4 a |
62.0 ± 12.5 a |
|
LIN* |
|
26.3 ± 11.4 a |
27.8 ± 9.7 a |
Table 3. Fresh and frozen-thawed Cougar (Puma concolor, N=2) sperm evaluation.
|
|
Fresh |
Frozen-thawed |
|
|
|
|
CoolA |
CoolB |
|
FPM |
3.0 ± 0 a |
2.8 ± 0.3 a |
2.8 ± 0.3 a |
|
PM (%) |
70.0 ± 0 a |
50.0 ± 14.1 a |
50.0 ± 14.1 a |
|
HOST (%) |
39.5 ± 6.4a |
13.5 ± 3.5 b |
25.0 ± 1.4 b |
|
Normal sperm (%) |
40.5 ± 7.8 d |
23.5 ± 2.1 d |
31.5 ± 0.7 d |
|
Major defects (%) |
41.0 ± 12.7 d |
26.5 ± 2.1 d |
36 ± 5.6 d |
|
Minor defects (%) |
18.5 ± 4.9d |
44.5 ± 6.4e |
32.5 ± 6.4d.e |
|
DI |
|
38.0 ± 1.4 a |
50.5 ± 6.4 a |
|
II |
|
44.0 ± 9.9 a |
39.0 ± 12.7 a |
|
DD |
|
17.0 ± 7.1 a |
9.5 ± 4.9 a |
|
ID |
|
1.0 ± 1.4 a |
1.0 ± 1.4 a |
|
Total Motility* |
|
40.0 ± 4.7 a |
36.3 ± 3.2 a |
|
Progressive motility* |
|
6.3 ± 1.7 a |
5.8 ± 4.3 a |
|
VAP* |
|
20.8 ± 4.6 a |
21.3 ± 4.1 a |
|
VSL* |
|
13.7 ± 4.3 a |
14.7 ± 3.9 a |
|
VCL* |
|
40.7 ± 6.7 a |
39.6 ± 9.2 a |
|
ALH* |
|
3.6 ± 0.1 a |
3.1 ± 0.3 a |
|
BCF* |
|
12.9 ± 2.8 a |
14.3 ± 1.7 a |
|
STR* |
|
65.3 ± 6.3 a |
68.4 ± 5.3 a |
|
LIN* |
|
33.3 ± 5.1 a |
36.9 ± 1.3 a |
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