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Sperm Cryopreservation in American Flamingo (Phoenicopterus Ruber): Influence of Cryoprotectants and Seminal Plasma Removal

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The American flamingo is a useful model for the development of successful semen cryopreservation procedures to be applied to threatened related species from the family Phoenicopteridae, and to permit genetic material banking. Current study sought to develop effective sperm cryopreservation protocols through examining the influences of two permeating cryoprotectants and the seminal plasma removal. During two consecutive years (April), semen samples were collected and frozen from American flamingos. In the first year, the effect of two permeating cryoprotectants, DMA (dimethylacetamide) (6%) or Me2SO (dimethylsulphoxide) (8%), on frozen–thawed sperm variables were compared in 21 males. No differences were seen between DMA and Me2SO for sperm motility, sperm viability, and DNA fragmentation after thawing. In the second year, the role of seminal plasma on sperm cryoresistance was investigated in 31 flamingos. Sperm samples were cryopreserved with and without seminal plasma, using Me2SO (8%) as a cryoprotectant. The results showed that samples with seminal plasma had higher values than samples without seminal plasma for the following sperm variables: Straight line velocity (22.40 µm/s vs. 16.64 µm/s), wobble (75.83% vs. 69.40%), (p < 0.05), linearity (62.73% vs. 52.01%) and straightness (82.38% vs. 73.79%) (p < 0.01); but acrosome integrity was lower (55.56% vs. 66.88%) (p < 0.05). The cryoresistance ratio (CR) was greater in samples frozen with seminal plasma than without seminal plasma for CR-progressive motility (138.72 vs. 54.59), CR-curvilinear velocity (105.98 vs. 89.32), CR-straight line velocity (152.77 vs. 112.58), CR-average path velocity (122.48 vs. 98.12), CR-wobble (111.75 vs. 102.04) (p < 0.05), CR-linearity (139.41 vs. 113.18), and CR-straightness (124.02 vs. 109.97) (p < 0.01). This research demonstrated that there were not differences between Me2SO and DMA to successful freezing sperm of flamingos; seminal plasma removal did not provide a benefit for sperm cryopreservation.
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Article
Sperm Cryopreservation in American Flamingo
(Phoenicopterus Ruber): Influence of Cryoprotectants and
Seminal Plasma Removal
María Gemma Millán de la Blanca 1, Eva Martínez-Nevado 2, Cristina Castaño 1, Juncal García2, Berenice Bernal 1,
Adolfo Toledano-Díaz 1, Milagros Cristina Esteso 1, Paula Bóveda 1, Lucía Martínez-Fresneda 1,
Antonio López-Sebastián1and Julián Santiago-Moreno 1, *


Citation: Millán de la Blanca, M.G.;
Martínez-Nevado, E.; Castaño, C.;
García, J.; Bernal, B.; Toledano-Díaz,
A.; Esteso, M.C.; Bóveda, P.;
Martínez-Fresneda, L.;
López-Sebastián, A.; et al. Sperm
Cryopreservation in American
Flamingo (Phoenicopterus Ruber):
Influence of Cryoprotectants and
Seminal Plasma Removal. Animals
2021,11, 203.
https://doi.org/10.3390/ani11010203
Received: 24 November 2020
Accepted: 14 January 2021
Published: 15 January 2021
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Copyright: © 2021 by the authors.
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This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1Department of Animal Reproduction, Instituto Nacional de Investigación y Tecnología Agraria y
Alimentaria (INIA), 28040 Madrid, Spain; gemma.millan@inia.es (M.G.M.d.l.B.);
cristina.castano@inia.es (C.C.); berenice.bernal@inia.es (B.B.); toledano@inia.es (A.T.-D.);
esteso.milagros@inia.es (M.C.E.); boveda.paula@inia.es (P.B.); lumf4@msn.com (L.M.-F.);
alopezs@inia.es (A.L.-S.)
2Zoo-Aquarium Madrid, 28011 Madrid, Spain; emartinez@grpr.com (E.M.-N.); jgarcia@grpr.com (J.G.)
*Correspondence: moreno@inia.es
Simple Summary:
The Phoenicopteridae family is made up of six species of which 50% are in
near-threatened status and 17% are in vulnerable status because their population is decreasing. The
American flamingo is a useful model for development of successful semen cryopreservation proce-
dures to be applied to threatened related species from the family Phoenicopteridae, and to permit
genetic material banking. The current study sought to develop effective sperm cryopreservation
protocols through examining the influences of two permeating cryoprotectants and the seminal
plasma removal. In addition, morphometric and functional sperm characteristics were studied
for the first time in this species. Head morphometric data provide relevant information in future
studies about sperm cryobiology in these species because the head size is related with the ability
of sperm to survive the freeze/thawed process. There was no apparent effect of cryoprotectants
(DMA (dimethylacetamide) 6% and Me
2
SO (dimethylsulphoxide) 8%) on frozen–thawed flamingo
sperm variables. The removal of the seminal plasma provided lower sperm quality after thawing
than samples containing seminal plasma. This research demonstrated that there were no differences
between Me
2
SO and DMA to successful freezing sperm of flamingos, but we recommend the use of
Me2SO because the cell toxic effect of DMA.
Abstract:
The American flamingo is a useful model for the development of successful semen cryop-
reservation procedures to be applied to threatened related species from the family Phoenicopteridae,
and to permit genetic material banking. Current study sought to develop effective sperm cryop-
reservation protocols through examining the influences of two permeating cryoprotectants and the
seminal plasma removal. During two consecutive years (April), semen samples were collected and
frozen from American flamingos. In the first year, the effect of two permeating cryoprotectants, DMA
(dimethylacetamide) (6%) or Me
2
SO (dimethylsulphoxide) (8%), on frozen–thawed sperm variables
were compared in 21 males. No differences were seen between DMA and Me
2
SO for sperm motility,
sperm viability, and DNA fragmentation after thawing. In the second year, the role of seminal plasma
on sperm cryoresistance was investigated in 31 flamingos. Sperm samples were cryopreserved with
and without seminal plasma, using Me
2
SO (8%) as a cryoprotectant. The results showed that samples
with seminal plasma had higher values than samples without seminal plasma for the following sperm
variables: Straight line velocity (22.40
µ
m/s vs. 16.64
µ
m/s), wobble (75.83% vs. 69.40%), (p< 0.05),
linearity (62.73% vs. 52.01%) and straightness (82.38% vs. 73.79%) (p< 0.01); but acrosome integrity
was lower (55.56% vs. 66.88%) (p< 0.05). The cryoresistance ratio (CR) was greater in samples frozen
with seminal plasma than without seminal plasma for CR-progressive motility (138.72 vs. 54.59),
CR-curvilinear velocity (105.98 vs. 89.32), CR-straight line velocity (152.77 vs. 112.58), CR-average
path velocity (122.48 vs. 98.12), CR-wobble (111.75 vs. 102.04) (p< 0.05), CR-linearity (139.41 vs.
Animals 2021,11, 203. https://doi.org/10.3390/ani11010203 https://www.mdpi.com/journal/animals
Animals 2021,11, 203 2 of 13
113.18), and CR-straightness (124.02 vs. 109.97) (p< 0.01). This research demonstrated that there were
not differences between Me
2
SO and DMA to successful freezing sperm of flamingos; seminal plasma
removal did not provide a benefit for sperm cryopreservation.
Keywords:
cryopreservation; American flamingo; spermatozoa; freezing/thawing; cryoprotectant;
seminal plasma; morphometry
1. Introduction
The Phoenicopteridae family is made up of six species which the 50% is in near
threatened status and the 17% is in vulnerable status because their population is decreasing.
The principal threats are the collection of eggs as food, mining activities, unfavorable
water-levels, hyper-salinity of water, erosion of nest-sites, human disturbance, pollution
and hunting food [
1
]. Currently, the Chilean flamingo (Phoenicopterus chilensis) is near
threatened and Andean flamingo (Phoenicoparrus andino) is included in the IUCN red list
of threatened species as vulnerable. [
1
]. In this context, reproductive biotechnologies,
particularly cryopreservation of gametes, and the development of Genetic Resource Banks
provide us with an indefinite gene resource of these species. The American flamingo
(Phoenicopterus ruber) is a good animal model for the study of the sperm cryopreservation
of flamingo species because its phylogenetic relationship with threatened species and its
least concern status [1].
The storage of sperm in genetic resource banks allows maximizing the genetic diversity,
and the sustainability of captive populations [
2
]. Some aspects to take into account for
semen cryopreservation are the cooling rate [
3
], the semen packaging type (e.g., straws or
pellets) [
4
], the presence or absence of seminal plasma [
5
], and the cryoprotectant agent
used [
6
]. To the best of our knowledge, there are no studies on the semen cryopreservation
of flamingo species.
Glycerol is generally thought to be the most effective cryoprotective agent in avian and
mammalian sperm cells against cold shock and it is the least toxic toward them [
7
]. Glycerol
is the most effective cryoprotectant in low fertility lines of poultry [
7
]. Although the use
of glycerol is associated in poultry with high sperm motility and viability after thawing,
its use has been shown to have contraceptive effects in birds, and therefore glycerol must
be eliminated from frozen–thawed semen before insemination [
6
,
8
,
9
]. Some alternatives
to consider are the Me
2
SO (dimethyl sulphoxide) [
10
] or DMA (dimethylacetamide) [
3
,
4
].
The Me
2
SO is a permeable cryoprotectant that increases the fluidity lipid membrane’s
hydrophobic core (but a high concentration can disintegrate the lipid bilayer); enhances
the permeability of hydrophilic molecules [
11
]; and decreases the ice crystallization [
12
].
The use of 8% Me
2
SO improved the frozen–thawed sperm quality and the fertility (73%
fertility) in Indian red jungle fowl compared with other concentrations [10], and has been
successfully used in crane sperm, obtaining a higher post-thaw survival and capacity to
bind to inner perivitelline membrane than with DMA [
13
]. On the other hand, DMA
has provided high levels of fertility after artificial insemination with frozen–thawed fowl
(85–93% fertility) and sandhill crane (42–48% fertility) sperm [
4
,
14
,
15
]. Its use at 6% in a
2-step freezing method allows a good preservation of acrosome and membrane integrity,
and 41% fertility in chicken sperm [3].
Seminal plasma is a key biological fluid that modulates sperm function in all ani-
mal species. In mammals, the seminal plasma may have some components that protect
of cryoinjury and improve the sperm resistance to cold-shock damage [
16
18
], but in
some species (e.g., goat) the seminal plasma removal is routine in sperm cryopreservation
protocols, because contains components that reduces the chances of efficient sperm preser-
vation [
19
]. Some of these components include proteins, ions, free amino acids, lipids,
carbohydrates, polyamines, steroid hormones, and prostaglandins [
5
,
20
24
]. The role of
seminal plasma on bird semen
in vitro
storage remains largely a matter of speculation as
Animals 2021,11, 203 3 of 13
both inhibitory and stimulating effects have been found [
20
]. The intense sperm metabolism
and the high proteolytic activities of seminal enzymes imply very rapid dilution of bird
semen in buffered diluents to avoid the rapid degradation of sperm
in vitro
[
20
]. Previous
reports showed contrasted effects of seminal plasma fractions on sperm cryopreserved or
incubated, showing a global deleterious effect on chickens and turkey sperm [
25
27
]. In
chickens the fertilization rate was lowest in older animals when the semen was stored with
seminal plasma, and in turkey the sperm membrane integrity, sperm motility, energy status,
and fertility were lower in semen samples stored in presence of seminal plasma [
25
,
27
].
Whereas the presence of seminal plasma did not seem to affect the rooster sperm survival
after freezing and thawing, the DNA was less damaged without plasma [5].
Considering all this information, the aim of this study was to develop a suitable
method for cryopreservation of flamingo’s semen by comparing the effect of two cryopro-
tectant agents, Me
2
SO and DMA, and evaluating the effect of seminal plasma removal on
sperm cryoresistance. Morphometric and functional sperm characteristics were studied for
the first time in this species.
2. Materials and Methods
2.1. Animals
American flamingo (Phoenicopterus ruber) males were housed in an outdoor exhibition
enclosure at Madrid Zoo-Aquarium (Madrid, Spain) in natural condition of photoperiod
and temperature. For the first experiment, 21 flamingos were sampled and for the second
experiment 31 males were sampled. All animals of the first experiment also participated
in the second experiment. The management of the bird and the semen collection was
done during the annual routine health check and was applied the Spanish Policy for
Animal Protection (RD53/2013), which conforms to European Union Directive 2010/63/UE
regarding the protection of animals used in scientific experiments. Animals were handled
according to procedures approved by the INIA Ethics Committee (Reference number
ORCEEA 2016/001). In addition, the internal animal welfare committee of Madrid Zoo-
Aquarium, based on the EAZA (European Association of Zoos and Aquariums) Code of
Ethics, evaluated and approved all procedures of this research.
2.2. Semen Collection
The semen samples were collected for two consecutive reproductive seasons, once a
year in April. All samples for each experiment were collected in only one day. The first
year the samples were used to investigate the effect of two cryoprotectants, Me
2
SO and
DMA. The second year the samples were used to evaluate the effect of seminal plasma
removal on sperm cryoresistance. All birds were subjected to the massage technique of
Burrows and Quinn, adapted to this species [
28
]. The collection of semen required some
research staff. One individual immobilized the body, the head and the neck, other held the
legs in the floor, and other stimulated the bird and collected the sample. The stimulation
was done massaging the abdomen with the left hand, the back with the right hand and
finally the copulatory organ protruded by mild stimulation and then gripping its base with
the thumb and index fingers of the right hand. Semen was recovered by capillarity using
a microhematocrit tube (Brand
®
GMBH + Co KG, Wertheim, Germany), and the volume
was evaluated measuring the length of the semen column in the microcapillary tube with a
plastic ruler (accuracy
±
1 mm) and calculating the equivalent in volume units (
µ
L). The
microcapillary was emptied into a 1.5 mL Eppendorf microcentrifuge tube (Eppendorf
Ibérica SLU, Madrid, Spain), and the semen sample was diluted 1:1 (v:v) at ambient
temperature with Lake-Ravie medium composed of sodium-L-glutamate (1.92 g), glucose
(0.8 g), magnesium acetate 4H2O (0.08 g), potassium acetate (0.5 g), polyvinylpyrrolidone
(PVP, relative molecular mass = 10,000; 0.3 g), and H2O (100 mL) (final pH 7.08, final
osmolarity 343 mOsm/kg). The diluted semen samples were immediately refrigerated at 5
C (cooling rate: 0.2 C/min) and transported to the laboratory.
Animals 2021,11, 203 4 of 13
2.3. Semen Freezing
Experiment 1: All the semen samples were divided in two aliquots and diluted with
Lake-Ravie medium to a concentration of 800 million spermatozoa/mL. The aliquots were
incubated 5
C for 1 h. Afterwards, the cryoprotectant was added to the respective aliquots
to a final concentration of 6% DMA and 8% Me
2
SO. The aliquots kept for equilibration at 5
C for 10 min. After equilibration, these samples were loaded into 0.25 mL French straws
(Minitub, Landshut, Germany). These straws were then frozen by a two-step cooling
method (from 5
C to
35 at 7
C/min, and from
35
C to
140
C at 60
C/min) [
3
].
First, the straws were frozen by placing them in nitrogen vapor 17 cm above the surface
of a liquid nitrogen bath for 4 min, and after were placed in nitrogen vapor 1 cm above
the surface of a nitrogen bath for 2 min (box’s volume 4.24 L). These samples were then
plunged into the liquid nitrogen.
Experiment 2: All the semen samples were divided into two aliquots and diluted
with Lake-Ravie medium to a concentration of 800 million spermatozoa/mL. The samples
were centrifuged to 700 g10 min. The pellet of one aliquot was resuspended with its
own seminal plasma. In the other one, the seminal plasma was removed and the pellet
resuspended with the same volume of Lake Ravie medium. The aliquots were incubated 5
C for 1 h. Afterwards, the Me
2
SO was added resulting in final concentrations of 8 vol%.
We decided to use this cryoprotectant agent because in the first experiment there were not
significates differences among both cryoprotectants, and the DMA has an effect very toxic
in rooster sperm at high concentration [
29
]. The samples were equilibrated 10 min with the
cryoprotectant and frozen as described in the experiment 1.
2.4. Semen Thawing
The straws were thawed in a bath at 5
C for 3 min and they were kept at this
temperature in a refrigerated display case until sperm variables were analyzed [2].
2.5. Assessment of Semen Variables
2.5.1. Sperm Motility
First, all semen was examined using a phase contrast microscope to confirm the
presence of spermatozoa and the subjective motility (percentage of motile spermatozoa and
score of 0 to 5 depend of quality of movement) [
3
]. Afterwards, sperm concentrations and
motility were assessed using a computer-aided sperm analysis (CASA) system coupled to a
Nikon Eclipse model 50i phase contrast microscope (Nikon Instruments Europe B.V., Izasa
S.A., Barcelona, Spain; negative contrast mode) and a Sperm Class Analyzer v.4.0. Software
(Microptic S.L., Barcelona, Spain). The samples were diluted in Lake-Ravie medium and
loaded onto warmed (37
C) 20
µ
m Leja
®
8-chamber slides (Leja Products B.V., Nieuw-
Vennep, The Netherlands). The program determined the percentage of motile spermatozoa,
the kind and characteristics of sperm movement—curvilinear velocity (VCL), straight-
line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement
(ALH), and beat-cross frequency (BCF). Three progression ratios, expressed as percentages,
were calculated for the program: Linearity (LIN = VSL/VCL
×
100), straightness (STR =
VSL/VAP
×
100), and wobble (WOB = VAP/VCL
×
100). CASA settings adjusted to detect
avian spermatozoa (A
2
= 5
µ
m
2
). CASA settings for motility were: VCL: 10–100
µ
m/s;
progressive motility >75% STR, circular movement <50% LIN. According to their velocity,
the spermatozoa were classified as slow (<10
µ
m/s), medium (10–50
µ
m/s) or rapid (>50
µ
m/s). A minimum of three fields and 500 sperm tracks at a magnification of 100
×
for
each sample were evaluated (image acquisition rate 25 frames/s).
The motility was analyzed as much in fresh samples as freezing–thawed samples of
the two experiments. The frozen–thawed samples were diluted in ASG medium [
30
,
31
]
(64.7 mM Sodium-L-glutamate1H
2
O, 3.1 mM tri-potassium-citrate1H
2
O, 3 mM magnesium
acetate4H
2
O, 26.5 mM D-(+)-Glucose monohydrate, 114 mM BES (N,N-Bis(2-hydroxyethyl)-
2-aminoethanesulfonic acid, pH 7.1, osmolality 325 mOsm/kg), 46.2 mM NaOH) with BSA
10 mg/mL before the CASA analysis.
Animals 2021,11, 203 5 of 13
2.5.2. Membrane Integrity
Propidium iodide (PI) and SYBR-14 were used as fluorochromes in the examination of
membrane integrity with 200 cells being examined [
32
]. When conducting this procedure
2
µ
L of SYBR-14 (1 mM in Me
2
SO diluted 1:19 in Me
2
SO) and 5
µ
L of the semen sample
were added to an Eppendorf tube containing 100
µ
L of HEPES medium (20 mM Hepes,
197 mM NaCl, 2.5 mM KOH, and 10 mM glucose), and incubated at 5
C for 10 min in
darkness. Afterwards, 1
µ
L of PI (stock solution: 2.4 mM in water) was added to the
mix, and incubated for 2 min at 5
C in darkness. The samples were then examined
using an epifluorescence microscope at 400
×
(wavelength: 450–490 nm) with fluorescent
microscopy (Eclipse E200, Nikon, Japan). Two spermatozoa population were distinguished:
Spermatozoa stained green (no PI staining) were considered to be alive, while red colored
spermatozoa (PI-positive) and spermatozoa with red and green colors were considered to
be dead.
These parameters were analyzed as much in fresh samples as freezing–thawed samples
of the two experiments.
2.5.3. DNA Fragmentation
DNA integrity was measured by terminal deoxynucleotidyl transferase dUTP nick
end labelling (TUNEL) as previously described procedures using the kit “In Situ Cell Death
Detection” (Roche, Basel, Switzerland) [
5
]. The samples were diluted in 4% paraformalde-
hyde, and 10
µ
L of this dilution were placed on a glass slide and left to dry. Subsequently,
the spermatozoa were permeabilized with 0.1% of Triton X-100 in PBS and washed in PBS.
After the DNA fragmentation was nick end-labelled with the kit work solution, which
content the substrates and the enzyme terminal transferase. The samples were incubated
1 h in humid box at 37
C. Then the samples were washed in PBS and counterstained
with Hoechst at 0.1 mg/mL in PBS for 5 min in the dark. Afterwards, the samples were
washed in PBS and mounted using Fluoromount (Sigma-Aldrich, St. Louis, MO, USA).
Samples were observed using a fluorescent microscopy (Eclipse E200, Nikon, Japan), and
200 spermatozoa were counted at 400×(wavelength: 510–560 nm).
This parameter was measured only in fresh samples of the second experiment and all
freezing–thawed samples of the two experiments.
2.5.4. Mitochondrial and Acrosomal Status
Mitochondrial function and acrosome state were analyzed by Mitotracker Green
FM
®
(MITO, Invitrogen M7514) in the experiment 2, for freezing–thawed samples, as
described by Bernal et al., with some modification [
33
,
34
]. For this, 5
µ
L of the semen
sample were added to an Eppendorf tube that contains 100
µ
L of HEPES medium. After,
2
µ
L of antifade and 0.2
µ
L of Mitotraker Green were added too. The Eppendorf tubes
were incubated 23 min at 5
C in darkness. The samples were then examined using an
epifluorescence microscope at 1000
×
with fluorescent microscopy (Eclipse E200, Nikon,
Japan). Four subpopulations were distinguished: (1) Spermatozoa with intact acrosome
and high mitochondrial function, (2) spermatozoa with damaged acrosome and high
mitochondrial function, (3) spermatozoa with intact acrosome and low mitochondrial
function, and (4) spermatozoa with damaged acrosome and low mitochondrial function.
The percentage of spermatozoa with an intact acrosome was also determined by
examining 200 aniline blue-stained cells by phase-contrast microscopy (magnification
1000
×
), following the procedure of Santiago-Moreno et al. [
35
]. The dry smears were fixed
at room temperature during 30 min in buffered 2% glutaraldehyde in PBS and air-dried.
The slides were then stained for 5 min with 5% aqueous aniline blue mixed with 2% acetic
acid (pH = 3.5) (Merck, Germany), washed with distilled water and air-dried again [
35
].
For the aniline blue smears was used a phase-contrast microscope (Zeiss, Oberkochen,
Germany) (magnification 1000×) and 200 spermatozoa were analyzed by smear.
Animals 2021,11, 203 6 of 13
2.5.5. Sperm Head Morphometric Analysis
Head morphometry was examined as previously described by Villaverde-Morcillo
et al. [
36
]. Smears were prepared by spreading 5
µ
L of diluted semen samples onto glass
slides and air-dried. Smears were then fixed and stained with Hemacolor
®
during 2 min
each step with corresponding kit’s acid and basic stains, according to the manufacturer
´
s
recommendations [
37
]. After, all slides were sealed with Eukitt mounting medium (Panreac
Quimica S.L.U., Barcelona, Spain) and a coverslip. The Motic Image Advanced V.3.0
software (Motic Spain, S.L.U., Barcelona, Spain) and Motic BA 210 optical microscope
(Motic Spain, S.L.U.) were used for the analysis. The captures were done with a Moticam
3+ (Motic Spain S.L.) camera [
36
]; 25 spermatozoa were captured and their head were
measured with acrosome (Figure 1).
Animals 2021, 11, 203 6 of 13
mitochondrial function, (3) spermatozoa with intact acrosome and low mitochondrial
function, and (4) spermatozoa with damaged acrosome and low mitochondrial function.
The percentage of spermatozoa with an intact acrosome was also determined by
examining 200 aniline blue-stained cells by phase-contrast microscopy (magnification
1000×), following the procedure of Santiago-Moreno et al. [35]. The dry smears were fixed
at room temperature during 30 min in buffered 2% glutaraldehyde in PBS and air-dried.
The slides were then stained for 5 min with 5% aqueous aniline blue mixed with 2% acetic
acid (pH = 3.5) (Merck, Germany), washed with distilled water and air-dried again [35].
For the aniline blue smears was used a phase-contrast microscope (Zeiss, Oberkochen,
Germany) (magnification 1000×) and 200 spermatozoa were analyzed by smear.
2.5.5. Sperm Head Morphometric Analysis
Head morphometry was examined as previously described by Villaverde-Morcillo
et al. [36]. Smears were prepared by spreading 5 µL of diluted semen samples onto glass
slides and air-dried. Smears were then fixed and stained with Hemacolor® during 2 min
each step with corresponding kit’s acid and basic stains, according to the manufacturer´s
recommendations [37]. After, all slides were sealed with Eukitt mounting medium
(Panreac Quimica S.L.U., Barcelona, Spain) and a coverslip. The Motic Image Advanced
V.3.0 software (Motic Spain, S.L.U., Barcelona, Spain) and Motic BA 210 optical
microscope (Motic Spain, S.L.U.) were used for the analysis. The captures were done
with a Moticam 3+ (Motic Spain S.L.) camera [36]; 25 spermatozoa were captured and
their head were measured with acrosome (Figure 1).
Figure 1. Flamingo sperm cell stained with Hemacolor® (A) Image of capture of flamingo sperm,
(B) image of analyzed capture of flamingo sperm with Motic Image Advance V 3.0 software
(Motic Spain, S.L.U., Barcelona, Spain)).
2.6. Statistical Analysis
Values for semen characteristics were reported as means ± SE (Standard Error),
except the sperm head measurements that were expressed as means ± SD (Standard
Deviations). For several variables, there was not a normal distribution when there was
assessment using the Shapiro–Wilk’s test. The Mann–Whitney test was used to compare
the effect within frozen–thawed samples between the two cryoprotectant agents, and the
Wilcoxon test for matched pairs was used to compare the effect within frozen–thawed
samples with presence or absence of seminal plasma. The Mann–Whitney test was used
to compare the percentage of sperm with intact acrosome according aniline blue assay.
To assess the response to freezing-thawing in both experiments, a cryoresistance ratio
[38,39] was determined for each of the semen variables as follows:
Cryoresistance ratio (CR) = (value after thawing (post)/value before thawing (pre)) × 100.
Differences in the CRs for the first experiment were compared using the Mann–
Whitney test and for the second experiment were compared using the Wilcoxon test. All
Figure 1.
Flamingo sperm cell stained with Hemacolor
®
(
A
) Image of capture of flamingo sperm,
(
B
) image of analyzed capture of flamingo sperm with Motic Image Advance V 3.0 software (Motic
Spain, S.L.U., Barcelona, Spain)).
2.6. Statistical Analysis
Values for semen characteristics were reported as means
±
SE (Standard Error), except
the sperm head measurements that were expressed as means
±
SD (Standard Deviations).
For several variables, there was not a normal distribution when there was assessment using
the Shapiro–Wilk’s test. The Mann–Whitney test was used to compare the effect within
frozen–thawed samples between the two cryoprotectant agents, and the Wilcoxon test for
matched pairs was used to compare the effect within frozen–thawed samples with presence
or absence of seminal plasma. The Mann–Whitney test was used to compare the percentage
of sperm with intact acrosome according aniline blue assay. To assess the response to
freezing-thawing in both experiments, a cryoresistance ratio [
38
,
39
] was determined for
each of the semen variables as follows:
Cryoresistance ratio (CR)=(value after thawing (post)/value before thawing (pre)) ×100.
Differences in the CRs for the first experiment were compared using the Mann–
Whitney test and for the second experiment were compared using the Wilcoxon test.
All calculations were performed using Statistica software v.13 (Dell Statistica, StatSoft Inc.,
Tulsa, OK, USA).
3. Results
The sperm head measurements (mean
±
SD) reported by computerized morphometric
analysis were: 11.3
±
1.2
µ
m length, 2.3
±
0.5
µ
m width, 27.4
±
3.1
µ
m perimeter, and 21.6
±4.6 µm2area. Sperm characteristics of fresh samples are shown in Tables 1and 2.
Animals 2021,11, 203 7 of 13
Table 1.
American flamingo sperm variables in fresh and froze-thawed samples using two cryopro-
tectant agents (8% Me2SO and 6% DMA).
Sperm Variables 8% Me2SO 6% DMA
Fresh Thawed Fresh Thawed
Motility (%) 37.5 ±9.9 10.0 ±3.1 42.5 ±9.0 17.0 ±7.0
Score 2.2 ±0.6 1.7 ±0.3 2.5 ±0.5 1.4 ±0.4
Viability (%) 78.6 ±2.3 43.6 ±3.5 80.1 ±3.1 48.2 ±3.8
DNA damage (Tunel + %) - 17.2 ±3.5 - 16.5 ±5.7
Static (%) 67.6 ±9.2 82.7 ±4.2 61.2 ±9.2 85.1 ±5.2
No progressive motility (%) 23.2 ±6.2 12.7 ±2.8 27.7 ±6.3 9.5 ±3.2
Progressive motility (%) 9.2 ±3.9 4.6 ±1.5 11.1 ±3.9 5.4 ±3.4
VCL (µm/s) 36.3 ±8.0 33.1 ±5.9 40.0 ±7.7 29.4 ±11.2
VSL (µm/s) 22.5 ±6.4 22.1 ±4.6 26.1 ±6.1 22.1 ±10.0
VAP (µm/s) 28.2 ±7.3 25.9 ±5.0 31.8 ±6.9 24.8 ±10.4
LIN (%) 47.8 ±8.0 58.7 ±7.5 53.4 ±7.7 48.5 ±11.0
STR (%) 64.7 ±8.6 75.7 ±8.7 69.6 ±8.5 61.1 ±12.2
WOB (%) 64.0 ±8.4 69.2 ±8.1 67.7 ±8.3 59.2 ±11.3
ALH (µm) 1.8 ±0.4 1.6 ±0.3 2.0 ±0.4 1.1 ±0.4
BCF (Hz) 5.9 ±1.3 6.3 ±1.2 6.9 ±1.2 4.5 ±1.5
Acrosome integrity (%) 91.8 ±2.0 73.9 ±3.6 91.8 ±2.0 63.0 ±3.0
Curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN),
straightness (STR), wobble (WOB), amplitude of lateral head (ALH), beat-cross frequency (BCF).
Results are expressed as mean ±SE.
Table 2.
American flamingo sperm variables in fresh and frozen–thawed samples with or without
seminal plasma.
Semen Variables Fresh Thawed
With Seminal
Plasma
Without Seminal
Plasma
Motility (%) 51.4 ±7.5 11.4 ±3.4 9.0 ±3.1
Score 2.2 ±0.2 2.2 ±0.2 1.9 ±0.3
Viability (%) 78.0 ±4.5 44.0 ±5.0 45.4 ±2.8
DNA damage (Tunel + %) 12.1 ±3.4 32.9 ±3.8 29.1 ±4.0
Mito+, Acro+ (%) - 29.6 ±4.1 35.9 ±4.3
Mito+, Acro(%) - 49.5 ±3.8 45.9 ±4.5
Mito, Acro+ (%) - 4.3 ±1.4 3.9 ±1.9
Mito, Acro(%) - 16.6 ±2.3 14.3 ±2.8
Static (%) 56.5 ±8.3 87.6 ±2.0 88.9 ±2.8
No progressive motility (%) 34.5 ±6.3 9.3 ±1.4 9.0 ±2.1
Progressive motility (%) 9.0 ±3.9 3.1 ±0.7 2.1 ±0.8
VCL (µm/s) 39.3 ±5.7 35.0 ±3.0 29.5 ±4.1
VSL (µm/s) 20.1 ±4.0 22.4 ±2.5 a16.6 ±3.3 b
VAP (µm/s) 28.2 ±5.0 26.9 ±2.7 21.6 ±3.8
LIN (%) 47.0 ±3.6 62.7 ±2.7 A52.0 ±4.4 B
STR (%) 67.6 ±3.1 82.4 ±1.6 A73.8 ±3.2 B
WOB (%) 68.4 ±2.7 75.8 ±1.9 a69.4 ±3.6 b
ALH (µm) 2.5 ±0.3 1.5 ±0.3 1.3 ±0.3
BCF (Hz) 6.5 ±1.0 6.0 ±1.1 5.1 ±1.3
Acrosome integrity (%) - 55.6 ±2.7 b66.9 ±3.8 a
Different letters indicate significant differences (lower case p< 0.05; capital letter p< 0.01) within
each sperm variable, between samples with or without seminal plasma. Curvilinear velocity (VCL),
straight line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR), wobble
(WOB), amplitude of lateral head (ALH), beat-cross frequency (BCF). Results are expressed as mean
±SE.
Animals 2021,11, 203 8 of 13
3.1. Experiment 1: Effect of DMA 6% and Me2SO 8% in the Cryopreservation
The mean semen volume was 20.9
±
5.1
µ
L, and the mean sperm concentration was
1228.2
×
10
6±
348
×
10
6
spermatozoa/mL. The total number of sperm per ejaculate was
27.1 ×106±11.6 ×106spermatozoa.
There were no significant differences for any sperm variable between samples frozen
with 6% DMA and those frozen in 8% Me
2
SO (Table 1). There were also no differences in
any of the values for the cryoresistance ratios (Table 3).
Table 3.
Cryoresistance ratio of American flamingo sperm variables frozen with two cryoprotectant
agents (8% Me2SO and 6% DMA).
Cryoresistance Ratio 8% Me2SO 6% DMA
Motility 17.0 ±6.1 40.2 ±19.1
Score 49.7 ±13.7 51.5 ±19.2
Viability 56.3 ±5.0 56.2 ±8.0
Static 153.3 ±31.7 183.8 ±40.3
No progressive motility 71.3 ±25.4 94.5 ±55.7
Progressive motility 70.8 ±27.7 122.5 ±72.6
VCL 70.6 ±16.5 106.5 ±44.1
VSL 96.3 ±28.1 170.6 ±73.5
VAP 80.1 ±21.0 129.4 ±52.8
LIN 102.9 ±22.2 89.0 ±24.8
STR 92.1 ±16.8 76.2 ±19.6
WOB 86.8 ±15.5 75.0 ±18.5
ALH 48.5 ±12.8 32.4 ±13.5
BCF 63.7 ±16.6 44.9 ±18.5
Acrosome integrity 82.2 ±6.0 70.3 ±1.5
Curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN),
straightness (STR), wobble (WOB), amplitude of lateral head (ALH), beat-cross frequency (BCF).
Results are expressed as mean ±SE.
3.2. Experiment 2: Effect of Seminal Plasma in the Cryopreservation
The mean semen volume was 40.3
±
6.6
µ
L, and the mean sperm concentration was
1912.2
×
10
6±
713.9
×
10
6
spermatozoa/mL. The total number of sperm per ejaculate was
43.3
×
10
6±
10.7
×
10
6
spermatozoa. Significant differences were found for the parameters
VSL, WOB, intact acrosome percentage (aniline blue-stained) (p< 0.05), LIN and STR (p<
0.01). These parameters were higher in samples frozen with seminal plasma than samples
frozen without seminal plasma, except the percentage of spermatozoa with intact acrosome
(Table 2).
Different letters indicate significant differences (lower case p< 0.05; capital letter p
< 0.01) within each sperm variable, between samples with or without seminal plasma.
Mitotraker (MITO), Acrosome (ACRO), Curvilinear velocity (VCL), straight line velocity
(VSL), average path velocity (VAP), linearity (LIN), straightness (STR), wobble (WOB),
amplitude of lateral head (ALH), beat-cross frequency (BCF). Results are expressed as
mean ±SE.
CR values were higher for samples in presence of seminal plasma that samples in
absence of seminal plasma for the following parameters: Progressive motility, VCL, VSL,
VAP, WOB (p< 0.05), LIN, and STR (p< 0.01) (Table 4).
Animals 2021,11, 203 9 of 13
Table 4.
Cryoresistance ratio of American flamingo sperm variables frozen with or without seminal
plasma.
Cryoresistance Ratio With Seminal Plasma Without Seminal Plasma
Motility 27.4 ±9.0 26.3 ±13.2
Score 112.7 ±19.9 85.3 ±9.6
Viability 56.1 ±6.0 60.6 ±5.7
Tunel+ 472.7 ±166.8 599.7 ±265.9
Static 244.4 ±84.3 241.6 ±78.4
No progressive motility 46.9 ±12.8 51.1 ±21.5
Progressive motility 138.7 ±87.6 a54.9 ±30.7 b
VCL 106.0 ±18.0 a89.3 ±20.4 b
VSL 152.8 ±32.2 a112.6 ±37.7 b
VAP 122.5 ±25.7 a98.1 ±30.0 b
LIN 139.4 ±8.2 A113.2 ±8.2 B
STR 124.0 ±4.8 A110.0 ±3.5 B
WOB 111.7 ±3.1 a102.0 ±5.1 b
ALH 74.2 ±28.6 64.3 ±27.7
BCF 103.2 ±27.6 87.5 ±32.2
Different letters indicate significant differences (lower case p< 0.05; capital letter p< 0.01) within
each sperm variable, between samples with or without seminal plasma. Curvilinear velocity (VCL),
straight line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR), wobble
(WOB), amplitude of lateral head (ALH), beat-cross frequency (BCF). Results are expressed as mean
±SE.
4. Discussion
This study is the first describing the sperm characteristics and sperm head morpho-
metric variables in flamingos. The length of head (11.3
µ
m) is similar to turkey and emu,
but the width (2.3
µ
m) is similar to gyrfalcon, which makes its area largest (21.6
µ
m
2
) [
40
].
Head morphometric data provide relevant information in future studies about sperm
cryobiology in these species because the head size is related with the ability of sperm to
survive the freeze/thawed process [40].
There were no apparent differences among cryoprotectants regarding its effects on
frozen–thawed sperm variables. However, the wide individual differences could explain
the lack of significance for many variables. The removal of the seminal plasma provided
lower results after thawing than in samples with seminal plasma.
DMA and Me
2
SO are permeating cryoprotectant which act intra- and extracellularly
inducing spermatozoa dehydration [
41
], osmotic stress [
42
], and even membrane fusion at
high concentrations [
43
]. Despite this, the use of these cryoprotectants at concentrations
of 6% of DMA and 8% of Me
2
SO resulted in high post-thaw plasma membrane integrity,
viability, acrosomal integrity, motility and fertility in sperm from chicken, crane or Indian
red jungle fowl [
3
,
10
,
13
]. The findings in the present study did not reveal differences
between the two cryoprotectants. This fact may be related to the similar cryoprotective
mechanism of both cryoprotectants. They share physical-chemical properties as two
hydrophobic methyl groups that can create three hydrogen bonds with water [
44
]. Even
though there were no differences, we decided to use Me
2
SO in the experiment 2 because
the toxic effect of DMA when high concentrations are used [
29
]. Our findings showed
poor motility values after thawing, which may be explained in part by the low motility
values in fresh (10% progressive motility). Despite the low initial motility, the reduction
of progressive motility after freezing-thawing process was 50%. These values agree with
previous freeze–thawing procedures in chicken rooster sperm with fertilization capacity [
6
].
The seminal plasma is a complex mixture of proteins, free amino acids, lipids, carbo-
hydrates, and hormones [
5
,
20
24
]. In the second experiment, it was decided to remove the
seminal plasma because other studies reported a deleterious effect in the storage of semen
samples of chicken and turkey [
25
27
]. Our findings revealed better motility results in
presence of seminal plasma. This may be due to the presence of certain proteins in the sem-
inal plasma that might contribute to the cryoprotectant permeability [
45
]. Moreover, free
Animals 2021,11, 203 10 of 13
amino acids content of flamingo’s seminal plasma might improve sperm kinetic activity;
for instance, glutamine and proline improve sperm motility variables in goat sperm [
5
,
46
].
Also, it has been shown that certain amino acids might stabilize the membranes when
interacting with the lipid bilayer [
47
], protect spermatozoa against toxic solutes [
48
], and
induce cell dehydration during freezing process [
49
]. Blesbois and Reviers’s studies [
50
]
showed that the high molecular weight fractions of seminal plasma appeared to enhance
fertilizing ability in fowl, which might be related to best values of motility as we see in this
research. Some seminal plasma components, as reduced glutathione (GSH), glutathione
peroxidase (GPx), phospholipid hydroperoxide glutathione peroxidase (PHGPx), and su-
peroxide dismutase (SOD) [
51
,
52
] might contribute to reduce the concentration of reactive
oxygen species, and thus improve the sperm motility [53,54].
Other hypothesis is the seminal plasma action on spermatozoa through exosomes, as
it has been described in mammals. These exosomes are membranes vesicles of 30–120 nm
coming from organs of male genital tract that may regulate some physiologic process of the
spermatozoa as capacitation, acrosome reaction and anti-oxidation [
55
,
56
]. Some works
have described that the human, canine, equine and bovine prostasomes are able to produce
extracellular ATP thanks of content glycolytic enzymes of their inside [
57
,
58
]. Some studies
have shown that extracellular ATP improves sperm motility [
59
61
]. Extracellular ATP
might be used by some membrane calcium ATPase, which presence has been described in
murine sperm, and this might produce a calcium flow in the spermatozoa [
62
]. In addition,
the prostasomes can induce a calcium flow through the transfer of progesterone receptors,
cyclic adenosine diphosphoribose (cADPR)-synthesizing enzymes, ryanodine receptors
(RyRs), and other Ca
2+
signaling tools by their fusion with the sperm [
56
]. Calcium flow
improves the motility variables and induces acrosome reaction [
63
]. This may explain
our findings related to a higher VSL, WOB, LIN, and STR but lower acrosome integrity in
frozen/thawed samples in presence of seminal plasma.
The low percentage of motile sperm determines that a high number of sperm should
be used in each dose for artificial insemination (AI). Taking into account the sperm quality
after freezing-thawing (about 45% viability and 15 % motility) along with the criteria for AI
in wild poultry [
10
], we estimate that at least two consecutive intravaginal AI procedures (3
days apart) involving 400 million sperm (two straws)/female at each insemination should
be required for a successful fertility. More effective overall cryopreservation protocols
(e.g., optimizing cooling rates and researching new additives) and eventually fertility trials
using intravaginal insemination techniques should be approached in future studies.
5. Conclusions
This is the first report describing sperm characteristics and cryopreservation proce-
dures in a flamingo species. Our findings revealed that the use of DMA 6% or Me
2
SO 8%
did not show differences; we recommend the use of Me
2
SO because the cell toxic effect of
DMA. On the other hand, the removal of seminal plasma did not contribute a benefit in the
cryopreservation method.
Author Contributions:
Conceptualization J.S.-M.; methodology M.G.M.d.l.B., E.M.-N., C.C., J.G.,
B.B., A.T.-D., P.B., L.M.-F., A.L.-S., and J.S.-M.; formal analysis J.S.-M., M.G.M.d.l.B., and C.C.; investi-
gation J.S.-M., E.M.-N., and M.C.E.; resources E.M.-N. and J.G.; writing-original draft preparation
M.G.M.d.l.B.; writing review and editing C.C., B.B., and J.S.-M.; supervision J.S.-M.; funding acquisi-
tion J.S.-M. All authors have read and agreed to the published version of the manuscript.
Funding:
This research was funded by the Fundación Parques Reunidos—INIA agreement CC19-096.
Institutional Review Board Statement:
The study was conducted according to the guidelines of the
Declaration of Helsinki, and approved by the INIA Ethics Committee (Reference number ORCEEA
2016/001).
Informed Consent Statement: Not applicable.
Animals 2021,11, 203 11 of 13
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Acknowledgments:
The authors would like to thank the staff of Madrid Zoo-Aquarium (Madrid,
Spain) for providing the semen samples.
Conflicts of Interest: The authors declare no conflict of interest.
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... Nevertheless, another study concludes that the sandhill crane (Antigone canadensis) sperm had greater results with DMA than with DMSO (Blanco et al., 2012). In the American flamingo there were no differences between the same two cryoprotectants (Millán de la Blanca et al., 2021). In Magellanic penguin, DMSO and EG were equally effective in maintaining penguin sperm quality parameters during the cryopreservation and thawing process (O'Brien et al., 1999). ...
... Our results showed no differences with both cryoprotectants in the percentage of sperm with DNA damage. In falcons (Cardoso et al., 2020) and American flamingos (Millán de la Blanca et al., 2021) there were no differences with the use of DMSO and DMA on this parameter. These results suggest that the type of cryoprotectant seems to have no relevant impact on DNA fragmentation. ...
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Black-footed penguins (Spheniscus demersus) are classified as endangered, and the populations of gentoo penguins (Pygoscelis papua) are rapidly decreasing. The optimization of semen cryopreservation in these species, for preserving their genetic diversity in genome resource banks, is essential for the success of captive breeding programs. This study compares the effectiveness of two permeating cryoprotectants, dimethylacetamide (DMA) and dimethylsulfoxide (DMSO), on frozen–thawed sperm characteristics. Semen samples were collected during each breeding season once a week during two consecutive years. Semen samples were packaged in 0.25 ml straws and frozen by placing them in nitrogen vapors. After thawing, sperm motility characteristics were examined by computer-assisted sperm analysis. Propidium iodide and SYBR-14 were used as fluorochromes for the examination of membrane integrity. DNA integrity was evaluated by TUNEL assay. Gentoo sperm characteristics after freeze-thawing did not show any differences when using DMSO or DMA. In black-footed samples, progressive motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and straightness (STR) were greater using 8% DMSO (P < 0.05) than 6% DMA. The cryoresistance ratio (CR) using 8% DMSO was greater (P < 0.05) in gentoo than black-footed samples for CR-VCL and CR-VAP, and 6% DMA returned greater CR values (P < 0.05) than in black-footed samples for all characteristics evaluated. No differences were found in DNA fragmentation. In conclusion, the present results highlight the benefits of using 8% DMSO compared to 6% DMA in penguins. Sperm from black-footed showed a higher sensitivity to freezing-thawing process than gentoo sperm.
... However, the success and efficiency of cryopreservation of bird semen differ among species and breeds or selection lines (117). Recent reports on sperm freezing of American flamingos (118) and peregrine falcons (119) are promising examples. In addition, because bird spermatozoa are stored in sperm storage tubules, possibly for several weeks, artificial insemination could be more successful than in mammals, as there is less need for absolute synchrony with ovulation. ...
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There is a remarkable diversity in the animal kingdom regarding mechanisms underlying the production, maturation, structure, and function of sperm cells. Spermatology studies contribute to the knowledge of species diversity and also provide information about individual or population fitness. Furthermore, this fundamental research is required before collected spermatozoa can be used for conservation breeding, including assisted reproduction and cryobanking. This article aims to ( a) review the most recent knowledge on sperm morphology and function in wild animal species, ( b) analyze how this knowledge can be used to save species in their natural habitat or ex situ, and ( c) propose future scientific directions in wildlife spermatology that could positively impact animal conservation. Variations in sperm structure and performance within and between species have multiple origins and significance. This collective body of knowledge enables the design and implementation of conservation strategies and action plans that integrate several disciplines. Expected final online publication date for the Annual Review of Animal Biosciences, Volume 10 is February 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... However, the success and efficiency of cryopreservation of bird semen differ among species and breeds or selection lines (117). Recent reports on sperm freezing of American flamingos (118) and peregrine falcons (119) are promising examples. In addition, because bird spermatozoa are stored in sperm storage tubules, possibly for several weeks, artificial insemination could be more successful than in mammals, as there is less need for absolute synchrony with ovulation. ...
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There is a remarkable diversity in the animal kingdom regarding mechanisms underlying the production, maturation, structure, and function of sperm cells. Spermatology studies contribute to the knowledge of species diversity and also provide information about individual or population fitness. Furthermore, this fundamental research is required before collected spermatozoa can be used for conservation breeding, including assisted reproduction and cryobanking. This article aims to (a) review the most recent knowledge on sperm morphology and function in wild animal species, (b) analyze how this knowledge can be used to save species in their natural habitat or ex situ, and (c) propose future scientific directions in wildlife spermatology that could positively impact animal conservation. Variations in sperm structure and performance within and between species have multiple origins and significance. This collective body of knowledge enables the design and implementation of conservation strategies and action plans that integrate several disciplines.
... Alternatively, the development of selective washing techniques (e.g., density-gradient centrifugation) in falcon sperm might be a useful strategy to eliminate immature cells and to decrease ROS production. Conversely, in other wild species studied in our lab, such as American flamingo (Phoenicopterus ruber), seminal plasma removal does not provide a benefit for sperm cryopreservation [104]. ...
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This review provides an updated overview of the seminal plasma composition, and the role of metabolic and protein components on the sperm function of avian species. In addition, the implication of seminal plasma on assisted reproductive techniques of birds was discussed. The semen of birds usually has exceptionally high sperm concentration with relatively little seminal plasma, but this contributes to very fast changes in sperm metabolism and function. The biochemical characteristics and physiological roles of the various seminal plasma components in birds (carbohydrates, lipids, amino acids, hormones, and proteins) are poorly understood. Seminal plasma content of proteins has an action on most cellular functions: metabolism, immunity, oxido-reduction regulation, proteolysis, apoptosis, ion homeostasis, and antimicrobial defenses. The variable amount of many proteins is related to a different fertility capacity of poultry sperm. The role of seminal plasma on semen conservation (chilling and freezing) remains largely a matter of speculation, as both inhibitory and stimulating effects have been found. Whereas the presence of seminal plasma did not seem to affect the sperm survival after freezing–thawing, DNA fragmentation is lower in the absence of seminal plasma. The molecular basis of the influence of seminal plasma on sperm cryo-resistance was also discussed in the present review.
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This review provides an updated overview of the seminal plasma composition, and the role of metabolic and protein components on the sperm function of avian species. In addition, the implication of seminal plasma on assisted reproductive techniques of birds was discussed. The semen of birds usually has exceptionally high sperm concentration with relatively little seminal plasma, but this contributes to very fast changes in sperm metabolism and function. The biochemical characteristics and physiological roles of the various seminal plasma components in birds (carbohydrates, lipids, amino acids, hormones, and proteins) are poorly understood. Seminal plasma content of proteins has an action on most cellular functions: metabolism, immunity, oxido-reduction regulation, proteolysis, apoptosis, ion homeostasis, and antimicrobial defenses. The variable amount of many proteins is related to a different fertility capacity of poultry sperm. The role of seminal plasma on semen conservation (chilling and freezing) remains largely a matter of speculation, as both inhibitory and stimulating effects have been found. Whereas the presence of seminal plasma did not seem to affect the sperm survival after freezing–thawing, DNA fragmentation is lower in the absence of seminal plasma. The molecular basis of the influence of seminal plasma on sperm cryo-resistance was also discussed in the present review.
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Substantial concentrations of testosterone are not only present in a male’s circulation, but also in its ejaculate. Surprisingly, the regulation of ejaculate T and its effects on females and their offspring, potentially a cryptic paternal trait, are not known. We found lower circulating and higher ejaculate T concentrations in subordinate red junglefowl (Gallus gallus gallus) males compared to dominant males, suggestive of an adaptive trade-off in T allocation to circulation and their ejaculate. Subsequently, we artificially inseminated females with either testosterone enriched (TE) or control ejaculates (CE) in a cross-over design. TE females produced heavier eggs than CE females. Offspring growth and tonic immobility were affected in a sex-specific way by TE. TE sons were heavier with shorter TI duration than CE sons, and TE daughters were lighter than CE daughters but daughters did not differ in TI score. However, the chicks competitiveness was not influenced by the TE nor CE. This indicates a previously unknown function of ejaculate testosterone as well as a new form of interaction between a cryptic paternal trait and a maternal effect that may be widespread in the animal kingdom.
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The poor fertility of ram semen stored chilled for long periods has encouraged the development of protocols designed to improve the kinetic vigour and cervical barrier-crossing capacity of sperm. The present work evaluated the effect of sperm selection with Sephadex filtration and the supplementation of 2% glycerol (GLY) to extenders based on ultra-heat-treated skimmed milk (UHT) or Tris-Tes-Glucose (TEST) on ram sperm kinetic parameters, plasma membrane integrity, acrosome integrity, mitochondrial function and fertilizing ability, over long chilling times. The results showed that for non-filtered semen, values for progressive sperm motility (%PSM), straight line velocity (VSL, μm/s) and the percentage of sperm with an intact plasma membrane/intact acrosome/a high mitochondrial function index (%IPIAHM) at all times up to 96 h of chilling were higher when the UHT extender (P < 0.01) was used compared to TEST extender irrespective of the presence of GLY. When semen was previously filtered with Sephadex, the addition of GLY to the UHT extender improved total motility (%TM), the %PSM and the VSL at 96 h compared to all other treatments (P < 0.01). The best results of all were obtained with non-filtered semen and UHT either with or without GLY. Heterologous IVF using zona-intact bovine oocytes was used to assess the fertilizing capacity of non-filtered fresh (FS0), chilled-for-24 h (CS24) or chilled-for-48 h (CS48) ram semen diluted in UHT extender (GLY-free). Heterologous IVF showed that ram sperm, either FS0, CS24 or CS48, were equally capable of penetrating zona pellucida intact bovine oocytes, leading to pronuclear formation and hybrid embryo cleavage (46.3 ± 3.2; 48.8 ± 3.2; and 43.3 ± 3.5, respectively). No differences were seen with respect to fresh sperm in terms of sperm binding, penetration, polyspermy, pronucleus formation or cleavage rates (P > 0.05). In conclusion, neither Sephadex filtration nor addition of glycerol provided extra benefits to ram sperm chilled up to 96 h. Chilled, non-filtered sperm extended with UHT without GLY showed better sperm functionality than did similar sperm extended with TEST extenders. Indeed, sperm diluted in UHT extender, maintained fertilizing ability up to 48 h.
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Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P
Chapter
Cryopreservation protocols for semen exist for bird species used in animal production, fancy and hobby species, and wild bird species. Freezing of bird oocytes or embryos is not possible. Cryopreservation of avian semen is used for preserving (genetic diversity of) endangered species or breeds. Freezing semen can also be used in the breeding industry for maintaining breeding lines, as a cost-effective alternative to holding live birds. Success and efficiency of cryopreservation of bird semen differs among species and breeds or selection lines. This chapter describes important variables of methods for collecting, diluting, cold storage, and freezing and thawing of bird semen, notably the medium composition, cryoprotectant used and its concentration, cooling rate, freezing method, and warming method. Media and methods are described for freezing semen using either glycerol or DMA as cryoprotectant, which both are known in chicken and a number of other bird species to render adequate post-thaw fertility rates.
Conference Paper
A repeatable equine in vitro fertilization (IVF) protocol has not yet been developed and could be due to suboptimal composition of IVF media. In bovine, metabolites like glucose can inhibit sperm capacitation resulting in IVF failure. Our aim was to assess the metabolomics of equine oviductal fluid (OF). OF was recovered from 2 mare oviducts contralateral to the ovulation site by pipetting immediately after slaughter; OF was centrifuged for 2 min., the supernatant was recovered and maintained in dry ice. At the laboratory, OF was centrifuged at 16000 g for 20 min. at 4°C, and 40 µl were recovered and frozen at −80°C. For its analysis 20 µl of OF were resuspended in 280 µl of a buffer containing 40 mM of PO4 3-, 12.5% of deuterated water (v/v) and 2 mM of maleic acid as internal standard for quantification (pH 7.4). The sample was analyzed by proton nuclear magnetic resonance spectroscopy (1H- NMR) using a presaturated CPMG pulse sequence. Thirteen metabolites were quantified, being the most abundant lactate (28.3 mM), myoinositol (26.7 mM), glutamate (9.5 mM), creatine (5 mM), glycerol (1.6 mM) and alanine (1.2 mM). Other metabolites, such as glutamine, ascorbate, 1- phosphoglucose and 6- phosphoglucose were tentatively identified but not quantified due to low intensity or overlapped signals in the spectrum. Our results show that OF composition greatly differs compared to the classical equine IVF media. Even if these are still preliminary results, our data provide relevant information to develop better in vitro fertilization protocols, as they strongly suggest that the composition of equine IVF media can be further improved. Funding: AGL2017- 84681, AGL2015- 73249- JIN & RYC2017- 21545.
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This study compares the effectiveness of the ultra-rapid and conventional freezing of sperm from captive bovids, giraffids, cervids, ursids, a cercopithecid, a delphinid and a phascolarctid. The relationship between sperm head dimensions and cryosurvival was also examined. Compared to conventional freezing, the ultra-rapid freezing of epididymal sperm from the dama gazelle, giraffe and brown bear returned higher cryoresistance ratios (CR, the ratio, in percentage, between the value of the variable after thawing/value before thawing) for sperm viability and motility. In the remaining species, the conventional freezing of epididymal sperm returned better CR values. The conventional freezing method also returned better CR values for ejaculated samples from all species. The head dimensions of both fresh epididymal and ejaculated sperm differed widely among species: for epididymal sperm, dolphin sperm heads were the smallest (7.189 ± 0.049 μm ² ) and dama gazelle sperm heads the largest (43.746 ± 0.291 μm ² ), while for ejaculated sperm, giant panda sperm heads were the smallest (15.926 ± 0.150 μm ² ) and mouflon sperm heads the largest (38.258 ± 0.104 μm ² ). However, no significant correlations were detected between the CR for motility, viability, membrane functional integrity or acrosome integrity and the sperm head area, either for epididymal or ejaculated sperm. In conclusion, ultra-rapid freezing is especially recommended for the cryopreservation of dama gazelle, giraffe and brown bear epididymal sperm. Sperm head dimensions appear not to be useful predictors of how well sperm might survive freezing.
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In this study, there was an examination of the effect on the characteristics of cryopreserved black-footed (Spheniscus demersus) and gentoo (Pygoscelis papua) penguin semen, of thawing at 37 and 5 °C. For two consecutive years, semen was collected and frozen during the April-June period from six gentoo penguins, and during the October-November period from 13 black-footed penguins. After thawing, sperm motility variables were examined by computer-assisted sperm analysis. Propidium iodide and SYBR-14 were used as fluorochromes for the examination of membrane integrity. For the gentoo penguins, no differences were detected in the values of frozen-thawed semen characteristics after thawing at 37 or 5 °C. For the black-footed penguins, however, thawing at 5 °C resulted in greater values (P < 0.05) for straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), straightness (STR), and wobble (WOB) as compared with thawing at 37 °C. After thawing at 37 ºC, there were greater values with gentoo penguin sperm for percentage motile sperm, progressive motility, curvilinear velocity (VCL), VSL VAP, LIN, STR, WOB and beat-cross frequency (BCF; P < 0.05) than that for black-footed penguin sperm. After thawing at 5 ºC, there were no differences in values for any variables between the two species. In conclusion, thawing temperature affects semen characteristics in a species-specific manner. The present data strongly suggest that cryopreservation procedures should be adapted for use with each penguin species. Cryopreserved black-footed penguin semen should be thawed after cryopreservation at 5 ºC, while that of gentoo penguins can be thawed at either 5 or 37 ºC.
Article
Glycerol is a least toxic and most effective cryoprotectant for cryopreservation of poultry semen, but due to its contraceptive properties removal of glycerol is usually needed prior to artificial insemination. Dimethylsulfoxide (DMSO), a small amphiphilic molecule used as penetrating cryoprotectant for biological cells, has been recognized as an adequate alternative for cryopreservation of sperm from several species. This study was designed to evaluate the efficacy of different concentrations of DMSO as cryoprotectant for Indian red jungle fowl (Gallus gallus murghi) sperm. Semen was collected from Indian red jungle fowl cocks, pooled and divided into five aliquots. Different concentrations of DMSO (0%, 4%, 6%, 8% and 10%) were compared. Diluted semen was cooled from 37 °C to 4 °C (-0.275 °C min-1), 20% glycerol added to control and equilibrated for 10 min. After equilibration, semen was filled in 0.5 mL French straws, kept over liquid nitrogen vapors for 10 min and plunged into liquid nitrogen. Semen samples were thawed at 37 °C for 30 s. Cryo-survival of Indian red jungle fowl sperm was affected by cryopreservation stages and different concentrations of cryoprotectant used. Highest sperm motility (85.0 ± 2.9; 80.0 ± 3.5; 71.3 ± 4.3; 60.0 ± 1.3), plasma membrane integrity (79.5 ± 3.8; 75.3 ± 2.4; 72.8 ± 3.3; 60.3 ± 2.8), viability (80.8 ± 4.6; 75.5 ± 2.9; 71.0 ± 7.6; 58.8 ± 1.3) and acrosomal integrity (76.3 ± 2.4; 72.0 ± 6.0; 62.5 ± 4.3; 55.0 ± 3.2) were recorded in a diluent having 8% DMSO at post-dilution, cooling, equilibration and freeze-thawing. Highest fertility results were obtained after artificial insemination with 8% DMSO compared to 20% glycerol (73.0 ± 4.4 vs 53.1 ± 4.3, P < 0.05). It is concluded that 8% DMSO as a permeable cryoprotectant improves the post thaw semen quality and fertility in Indian red jungle fowl and can be used effectively to avoid the contraceptive effects of glycerol.
Article
Seminal plasma (SP) contains proteins that may influence cryosurvival and prevent capacitation-like changes due to freezing and thawing. The objective of this study was to investigate the effect of adding pooled SP from “good” (GF) or “bad” (BF) freezer stallions on sperms cells´ fertilizing ability. “Good freezers” refers to stallions that usually produce ejaculates which can withstand cryopreservation, whilst “bad freezer” stallions produce ejaculates which cannot tolerate the freezing process. A heterologous zona binding assay with in vitro matured bovine oocytes was used to assess the binding ability of equine sperm cells as a possible alternative to artificial insemination trials. The effect of adding SP i) prior to cryopreservation; ii) after thawing of sperm cells selected by single layer centrifugation (SLC); iii) to capacitation medium, was evaluated. Adding SP from GF stallions prior to cryopreservation reduced the mean number of sperm cells bound to the zona pellucida (ZP) compared to control (P = 0.0003), SP-free sperm cells and group received SP from BF stallions (P ≤ 0.0001 for both). After thawing SLC-selected sperm cells treated with 5% SP showed a decrease in binding ability compared with SP-free sperm cells (P ≤ 0.0001). The binding affinity of sperm cells was higher in the group treated with SP from GF than with SP from BF stallions (P ≤ 0.05). Prolonged exposure to SP impaired the ability of stallion sperm cells to undergo capacitation and bind to ZP, regardless of the source of SP (P ≤ 0.0001). The response of equine sperm cells to SP is influenced by the ability of the sperm cells to withstand cryopreservation and is affected by the timing of exposure and the origin of SP. Customization of the protocol for individual stallions is recommended to optimize the effect.