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Effect of supplementation of valine to chicken extender on sperm cryoresistance and post-thaw fertilization capacity

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Recent reports showed a positive correlation between frozen-thawed rooster sperm DNA integrity and the concentrations of valine in seminal plasma. The present study evaluated the effect of supplementing valine to semen extender for freezing sperm of 2 endangered local Spanish chicken breeds with different sperm cryoresistance: Red Villafranquina (VF) showing low sperm DNA integrity after cryopreserva-tion and Quail Castellana that shows higher DNA integrity. One pool of semen per breed was obtained twice a week for 10 wk (n 5 40, 20 per breed). Each pool was divided into 2 fractions. One of these fractions was frozen in presence of valine as additive in the extender (concentration 10 mmol), whereas the other was used as control. The evaluation of the samples before and after freezing-thawing included motility (CASA-Mot system), viability (propidium iodide and SYBR-14), DNA integrity (terminal deoxynucleotidyl transferase dUTP nick end labeling), and fertility rate (percentage of eggs with blastoderm development after artificial insemination). Supplementation of valine increased several motility variables of fresh semen. In VF breed, valine increased percentage of progressive motile sperm (P 5 0.025), curvilinear velocity (P 5 0.033), straight-line velocity (P 5 0.040), and average path velocity (P 5 0.033), whereas progressive motile sperm (P 5 0.019), curvilinear velocity (P 5 0.006), straight-line velocity (P 5 0.003) and average path velocity (P 5 0.004) were improved in the Quail Cas-tellana breed. Valine addition increased the DNA integrity of cryopreserved semen (decreased post-thaw DNA fragmentation) in both breeds, with a significant effect (P 5 0.002) in VF (36.3% VF-control vs 31%VF-valine). As expected, Quail Castellana cryopreserved sperm control showed higher fertility rate (34.4% 6 12.1) than VF cryopreserved sperm control (16.1% 1 6.2). Supplementing valine to the freezing extender doubled (P 5 0.026) the fertility rate of VF (32.6% 6 12.2) compared with the control (16.1% 1 6.2). In conclusion, supplementation of valine to chicken freezing extenders shows a positive effect on DNA fragmentation and fertilizing ability of frozen-thawed sperm, with a better response in a breed considered as the lowest freezer in our conservatory.
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Effect of supplementation of valine to chicken extender on sperm
cryoresistance and post-thaw fertilization capacity
B. Bernal,*
,x
N. Iglesias-Cabeza,*U. S
anchez-Rivera,
y
A. Toledano-Díaz,*C. Castaño,*
S. P
erez-Cerezales,*A. Guti
errez-Ad
an,*A. L
opez-Sebasti
an,*P. García-Casado,
z
M. G. Gil,
x
H. Woelders,
#
E. Blesbois,
kk
and J. Santiago-Moreno*
,1
*Department of Animal Reproduction, INIA, 28040 Madrid, Spain;
y
Department of Reproductive Biology,
FES-Iztacala, UNAM, 54090, Tlalnepantla, M
exico;
z
Zoitechlab, R&D Department, 28400 Madrid, Spain;
x
Department of Animal Breeding, INIA, 28040 Madrid, Spain;
#
Wageningen University & Research, Animal
Breeding and Genomics, 6700 AH Wageningen, The Netherlands; and
kk
INRA 0085 UMR PRC
INRA-CNRS-University François Rabelais-Haras Nationaux, 37380 Nouzilly, France
ABSTRACT Recent reports showed a positive cor-
relation between frozenthawed rooster sperm DNA
integrity and the concentrations of valine in seminal
plasma. The present study evaluated the effect of sup-
plementing valine to semen extender for freezing sperm
of 2 endangered local Spanish chicken breeds with
different sperm cryoresistance: Red Villafranquina (VF)
showing low sperm DNA integrity after cryopreserva-
tion and Quail Castellana that shows higher DNA
integrity. One pool of semen per breed was obtained
twice a week for 10 wk (n 540, 20 per breed). Each
pool was divided into 2 fractions. One of these fractions
was frozen in presence of valine as additive in the
extender (concentration 10 mmol), whereas the other
was used as control. The evaluation of the samples
before and after freezing-thawing included motility
(CASA-Mot system), viability (propidium iodide and
SYBR-14), DNA integrity (terminal deoxynucleotidyl
transferase dUTP nick end labeling), and fertility rate
(percentage of eggs with blastoderm development after
articial insemination). Supplementation of valine
increased several motility variables of fresh semen. In
VF breed, valine increased percentage of progressive
motile sperm (P50.025), curvilinear velocity (P5
0.033), straight-line velocity (P50.040), and average
path velocity (P50.033), whereas progressive motile
sperm (P50.019), curvilinear velocity (P50.006),
straight-line velocity (P50.003) and average path
velocity (P50.004) were improved in the Quail Cas-
tellana breed. Valine addition increased the DNA
integrity of cryopreserved semen (decreased post-thaw
DNA fragmentation) in both breeds, with a signicant
effect (P50.002) in VF (36.3% VF-control vs 31%VF-
valine). As expected, Quail Castellana cryopreserved
sperm control showed higher fertility rate (34.4% 6
12.1) than VF cryopreserved sperm control (16.1% 1
6.2). Supplementing valine to the freezing extender
doubled (P50.026) the fertility rate of VF (32.6% 6
12.2) compared with the control (16.1% 16.2). In
conclusion, supplementation of valine to chicken
freezing extenders shows a positive effect on DNA
fragmentation and fertilizing ability of frozenthawed
sperm, with a better response in a breed considered as
the lowest freezer in our conservatory.
Key words: valine, sperm cryoresistance, DNA integrity
2020 Poultry Science -:--
https://doi.org/10.1016/j.psj.2020.09.060
INTRODUCTION
Ex situ conservation of avian reproductive cells in gene
cryobanks results in a strategic tool to secure the genetic
diversity and give complementary support to in situ
poultry conservation programs (Blesbois et al., 2007).
Different freezing protocols have been developed for cry-
opreserving rooster semen; however, the variable and
usually low fertility rates currently obtained by articial
insemination with frozenthawed sperm remain the main
obstacle for commercial application or genetic preserva-
tion (Long, 2006). Compared with other species, the
physiology of avian spermatozoa makes them more sus-
ceptible to cell cryodamage, thus dramatically decreasing
their fertilizing capacity. For instance, they possess a
spindle form with very low cytoplasmic content and a
very long agellum making them highly susceptible to
Ó2020 Published by Elsevier Inc. on behalf of Poultry Science
Association Inc. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Received March 9, 2020.
Accepted September 24, 2020.
1
Corresponding author: moreno@inia.es
1
cryoinjury during freeze-thawing (Long, 2006;Blesbois,
2012;Thananurak et al., 2019). Along with this, the
cold shock, intracellular ice formation, and production
of reactive oxygen species (ROS) result in diverse sperm
injuries compromising the uidity and permeability of
membranes, sperm metabolism, motility, DNA integrity,
and fertilizing ability (Partyka et al., 2010;Santiago-
Moreno et al., 2019;Thananurak et al., 2019). For
instance, the mitochondrion is the main sperm metabolic
site and the main producer of ROS; the large number of
mitochondria present in avian midpiece (in rooster,
approximately 30; Etches, 1998) makes poultry sperma-
tozoa prone to oxidative and other metabolic stress. For
example, lipid peroxidation of sperm membranes induced
by ROS leads to loss of exibility and production of by-
products, such as the mutagenic and genotoxic molecule
malondialdehyde, causing indirect damage to DNA
(Wagner et al., 2018). DNA damage and lipid peroxida-
tion in mitochondrial membranes may lead to DNA frag-
mentation by a cytochrome Cmediated pathway
(apoptosis) (Ansari et al., 2019).
DNA integrity in sperms is essential for embryo devel-
opment, hence cryopreservation procedures must ensure
the intact conservation of sperm DNA (P
erez-Cerezales
et al., 2009). Although chicken spermatozoa do possess
protective antioxidant systems of cytoplasmic origin
(Surai et al., 1998), the lack of cytoplasm decreases the
capacity to counteract the oxidative stress. Contrary
to this, seminal plasma contains large amount of antiox-
idants (Henkel, 2011). Moreover, addition of antioxi-
dants such as amino acids and amino acidderived
glutathione to extenders has demonstrated to reduce
damage to spermatozoa in different mammalian species
such as goat, bull, or buffalo (Kundu et al., 2001;
Tuncer et al., 2010;Topraggaleh et al., 2014). In birds,
studies are limited, but amino acids such as glutamine
(Khiabani et al., 2017) or glycine (Lorenz and Tyler,
1951) have also been successfully used. A previous study
in our laboratory with 12 Spanish breeds of rooster
(Santiago-Moreno et al., 2019) suggested that the semi-
nal plasma amino acid prole of each breed could be rele-
vant regarding sperm cryoresistance. Results of this
study showed that concentration of certain amino acids,
such as valine, had a negative correlation with DNA
fragmentation and a positive correlation with sperm
viability in frozenthawed sperm samples. Valine is an
essential aliphatic and hydrophobic amino acid that is
involved in multiple biological processes such as protein
synthesis, energy production, lipolysis, glucose transpor-
tation, embryo development, and so on. (Zhang et al.,
2017). Besides this, valine may be metabolized/oxidized
in mitochondria. (Hutson and Hall, 1993). Considering
all these elements, in the present study, we selected
valine to be tested as a possible additive in chicken
semen freezing extenders. Two different Spanish chicken
breeds with different sperm cryoresistance were selected:
Red Villafranquina (VF) showing low sperm DNA integ-
rity after cryopreservation and Quail Castellana (QC)
that shows higher DNA integrity (Santiago-Moreno
et al., 2019).
MATERIAL AND METHODS
Experimental Birds
The birds used in this study were 40 Spanish chicken
roosters from endangered local breeds (VF and QC)
and 30 randomly chosen hens. To avoid the breed factor
in the fertility test, a third breed of hens, Black-Barred
Andaluza was included in the study together with VF
and QC. The birds, all of which were 1 yr of age at the
beginning of the experiment, were housed under natural
photoperiod and temperature conditions in 2 12 m
2
sand-oor pens with partial roof cover at the El Encín
Research Station (Madrid, Spain; 40310N). These birds
were raised as part of the INIAs genetic resources con-
servation program (Campo and Orozco, 1982;Campo,
1998). All birds were fed a commercial feed containing
16% CP, 2,700 kcal of ME/kg, 3.5% Ca, and 0.5% avail-
able P over the entire experimental period. Animals were
handled as per procedures approved by the INIA Ethics
Committee and were performed in accordance with the
Spanish Policy for Animal Protection (RD53/2,013),
which conforms to European Union Directive 86/609
regarding the protection of animals used in scientic
experiments.
Experimental Design
A pool of seminal plasma for each breed was obtained
twice a week from February to June 2018 (n 540
roosters; 20 per breed). To investigate the role of valine
on cryoresistance of rooster sperm, the pooled semen
samples were divided into 2 aliquots. One aliquot was
frozen using extender (Lake-Ravie 84) with presence of
valine (concentration 10 mmol) and the other was frozen
using the same extender without valine. Sperm variables
were analyzed prior and after freezing-thawing. Prelimi-
nary studies in our laboratory on the effect of supple-
menting different concentrations of valine (1.5, 10, and
20 mmol) to the chicken extender pointed out 10 mmol
(5 mmol when mixed with semen 1:1) as the optimal con-
centration for reducing post-thaw DNA fragmentation
without observing a negative effect on the motility vari-
ables (unpublished results). Thus, this was the concen-
tration used to supplement the sperm samples of QC
and VF roosters.
The fertilization capacity of the sperm cells from QC
(better freezer) and VF (bad freezer) was estimated in
articial insemination of 30 hens (see details in the
following sections).
Semen Collection, Management, and
Freezing
Semen was collected twice weekly over the study
period, in 15-mL graduated centrifuge tubes (Sterilin)
using the massage technique described by Burrows and
Quinn (1936). Pools of semen for each breed were
made on each occasion. Each pool was immediately
divided into 2 equal aliquots. The volume of the aliquots
BERNAL ET AL.2
was dependent on the total volume of the pool collected
per breed and ranged between 0.225 mL and 1.725 mL.
One of them was diluted 1:1 (v/v) using a Lake-Ravie
medium (Lake and Ravie, 1984) composed of sodium
glutamate (1.92 g), glucose (0.8 g), magnesium acetate
4H
2
O (0.08 g), potassium acetate (0.5 g), polyvinylpyr-
rolidone (M
r
10,000; 0.3 g), and 100 mL H
2
O(nal pH
7.08, nal osmolality 343 mOsm/kg; hereinafter referred
to as Lake-Ravie-84). The other aliquot was diluted 1:1
(v/v) using the Lake-Ravie-84 (LR) supplemented
with 10 mmol L-valine (LR-Val). The extender was
held in the hand (about 37C) previous addition to
sperm, and dilution was performed at eld temperature
(15C30C throughout the study period). The diluted,
pooled semen samples were then immediately cooled to
5C, transported to the laboratory, and sperm concen-
tration and sperm variables (sperm motility variables,
plasma membrane integrity) examined (within 45 min
of collection). Afterward, each pool was diluted as
required with LR or LR-Val media to a concentration
of 1,200 !10
6
sperm/mL. Glycerol was then added to
the diluted samples, to leave a nal 8% concentration,
and equilibrated for 10 min at 5C. After equilibration,
the samples were loaded into 0.25-mL French straws
and then frozen using a computer freezer IceCube
1,810 unit (Minit
ub; Tiefenbach, Germany) with the
following cooling rate: from 5Cto235Cat7
C/min
and then from 235Cto2140Cat60
C/min
(Santiago-Moreno et al., 2011). The frozen straws were
then plunged into and maintained in liquid nitrogen
(at 2196C) until thawing. For thawing, the straws
were warmed for 3 min in LR or LR-Val bath (both in
continuous stirring) at 5C.
Assessment of Sperm Variables
Sperm concentration and motility were assayed using
a computer-aided sperm analyses system coupled to a
phase contrast microscope (Nikon Eclipse model 50i;
Nikon Instruments Europe B.V., Izasa S.A.; negative
contrast) and using Sperm Class Analyzer (SCA; Barce-
lona, Spain) v.4.0. Software (Microptic S.L., Barcelona,
Spain). For motility analysis, sperm samples were
diluted to a concentration of approximately 40 million
sperm/mL and loaded onto warmed (38C) 20-mm Leja
8-chamber slides (Leja Products B.V., Nieuw-Vennep,
The Netherlands). The percentage of motile spermato-
zoa and the percentage showing progressive motility
were recorded. Sperm movement characteristics curvi-
linear velocity (VCL), straight-line velocity, average
path velocity (VAP), amplitude of lateral head displace-
ment were also recorded. A minimum of 3 elds and 200
sperm tracks were evaluated at a magnication of 100x
for each sample (image acquisition rate 25 frames/s).
Propidium iodide and SYBR-14 were used as uoro-
chromes in the examination of membrane integrity
(Chalah and Brillard, 1998); 200 cells were examined us-
ing an epiuorescence microscope at 400 !(wavelength:
450490 nm).
All sperm variables were measured again for each pool
after their eventual thawing. In addition, DNA integrity
was also assessed by terminal deoxynucleotidyl trans-
ferase dUTP nick end labeling (TUNEL). For this, the
kit In Situ Cell Death Detection(Roche, Basel,
Switzerland) was used following manufacturers instruc-
tions with minor changes to adapt the technique to the
analyses of rooster sperm (Santiago-Moreno et al.,
2019). Briey, each sperm sample was diluted to
12 !10
6
spermatozoa/mL in 4% formaldehyde. Subse-
quently, 10 mL of this dilution were placed on a glass
slide and left to dry. Then, the spermatozoa were per-
meabilized with 0.1% of Triton X-100 in PBS. After a
wash in PBS, fragmented DNA was nick end-labeled
with tetramethylrhodamine-conjugated dUTP by add-
ing 10 mL of the working solution provided by the kit
containing the substrates and the enzyme terminal
transferase on the sample. The reaction was conducted
incubating the slides in a humid box for 1 h at 37C. Af-
ter a wash with PBS, the nucleus were counterstained
with Hoechst at 0.1 mg/mL in PBS for 5 min in the
dark. After an additional wash with PBS, the slides
were mounted using Fluoromount (Sigma-Aldrich,
MO) previous to observation under a uorescent micro-
scope (Eclipse E200; Nikon, Japan). Percentages of pos-
itive TUNEL spermatozoa (Figure 1) per sample were
recorded by counting a minimum of 200 spermatozoa
per microscopy preparation.
Cryoprotectant Removal and Articial
Insemination
Glycerol was removed before articial insemination.
Thawed samples were progressively diluted with Lake
Centri medium at 5Ctoanal dilution of 1:4 v/v via
the following steps: 1:0.07, 1:0.18, 1:0.33, 1:0.6, 1:1.24,
and 1:1.58 (v/v) (2-min intervals). These samples were
then centrifuged at 600 !gfor 10 min, the supernatant
solution discarded, and the pellet resuspended (in the
same volume as before centrifugation) in Lake 7.1.
Briey, the Lake Centri (method adapted from the study
by Moc
e et al., 2010) medium was composed of
1,000 mL H
2
O, 1.28 g potassium citrate tribasic monohy-
drate, 19.2 g sodium L-glutamate, 6.0 g D-fructose, 5.0 g
TES, 5.1 g sodium acetate trihydrate, 0.8 g magnesium
acetate tetrahydrate, and 5.2 mL of 1N sodium hydrox-
ide (340350 mOsm/kg, pH 57.07.2). The Lake 7.1
medium (slightly modied from Lake and Ravie 1,979)
was composed of 1,000 mL H
2
O, 0.8 g magnesium ace-
tate tetrahydrate 4H
2
O, 1.28 g potassium citrate tribasic
monohydrate, 15.2 g sodium L-glutamate, 6 g glucose,
30.5 g BES, and 58 mL of 1N sodium hydroxide (370
mOsm/kg, pH 57.1).
Thirty randomly chosen hens were used for testing
fertilizing ability of frozenthawed semen of the VF
and QC, that is 15 hens were inseminated with semen
of good freezer and the remaining 15 with semen of the
bad freezer. The AI of hens was performed with
EFFECT OF VALINE ON SPERM CRYORESISTANCE 3
randomly chosen straws. Different frozenthawed
semen doses were used in each insemination session.
The fertilization capacity was estimated from the per-
centage of fertilized eggs resulting from 6 consecutive
intravaginal articial inseminations (2 inseminations
a week for 3 wk). All inseminations were performed be-
tween 15:00 h and 16:00 h. Articial insemination pro-
cedures involved 300 million sperm/female at each
insemination (250 mL). Eggs were collected from day
2aftertherst insemination until 3 d after the last
insemination. Fertility (% fertile/incubated eggs) was
determined by blastoderm development after articial
insemination.
Statistical Analyses
Data are expressed as means 6SE. The sperm vari-
ables were not normally distributed as determined by
Shapiro-Wilk test, regardless arcsin and log transforma-
tions. Thus, nonparametric analyses were used. Signi-
cant differences between the sperm variables of the
control and the samples frozen with valine within each
breed were determined by the Wilcoxons test. Signicant
differences between breeds were determined by the
Mann-Whitney U test. The association between fertility
rates and the 2 kinds of diluent (LR or LR-Val) was
assessed using the chi-square test. Correlations between
TUNEL (1) and sperm motility ratios, between viability
and motility ratios, and between TUNEL (1) and
viability were determined by the Spearman test. All sta-
tistical calculations were made using TIBCO Statistica
software v.13.3 (TIBCO Software Inc. Palo Alto, CA).
RESULTS
Breed Differences in Fresh Semen
Comparison between fresh samples of VF control and
QC control revealed signicant effect of the breed on the
percentage of total motile sperm (P,0.001) (Figure 2),
whereas no signicant difference was found on viability
(Figure 3).
Breed Differences in Control Frozen
Thawed Semen
Concerning frozenthawed semen, differences were
found between VF control and QC control in percentage
of progressive motile (P50.027) and total motile (P5
0.008) sperm (Figure 4), whereas no signicant differ-
ence was found on viability (Figure 3). Concerning
post-thawed DNA fragmentation (TUNEL 1), differ-
ences between VF control and QC control were not sig-
nicant, but the numerical differences (Figure 5)do
suggest a higher susceptibility to DNA fragmentation
in VF (36.3% 63.3) than in QC (30.9 63.2).
A number of correlations were found between the
viability values and some of the motility rates of both
breeds. Regarding VF control, viability showed positive
correlation with progressive motility (P,0.01) and to-
tal motility (P,0.001) (Table 1). About QC control, 1
correlation was found between viability and progressive
motile sperm (P,0.05) (Table 1)
With regard to fertility, comparison between the
thawed samples of VF control and QC control revealed
an effect of the breed (P50.037) on the post-thawed
fertilizing capacity of the spermatozoa. QC control
showed 2-fold higher fertility rate (34.4% 612.1) than
VF control (16.1% 66.2) (Figure 5).
Valine Effect on Motility and Viability of
Fresh Semen
The addition of valine to the freezing extender
improved several motility variables of fresh semen of
both breeds (Figure 2). With respect to VF, the variables
improved were progressive motile sperm (P50.025),
VCL (P50.033), straight-line velocity (P50.040),
and VAP (P50.033). In QC roosters, the variables
improved were progressive motile sperm (P50.019),
VCL (P50.006), straight-line velocity (P50.003),
and VAP (P50.004) (Figure 2). The addition of valine
did not improve the sperm viability values in any of the
breeds (Figure 3).
Figure 1. DNA integrity assessment by TUNEL. DNA staining by Hoechst of all rooster sperm present in the microscope eld (A) and the same
eld showing in red the sperm with DNA fragmentation, TUNEL (1) (B). Abbreviation: TUNEL, terminal deoxynucleotidyl transferase dUTP nick
end labeling.
BERNAL ET AL.4
Valine Effect on Motility and Viability of
FrozenThawed Semen
Regarding frozenthawed semen the supplementation
of valine did not improve any motility variable of any of
the breeds (Figure 4) or did it for viability (Figure 3).
Valine Effect on DNA Fragmentation of
FrozenThawed Semen
The post-thawed results of VF sperm supplemented
with valine (VF-Val) showed 14.8% lower DNA
fragmentation rate than VF control (31% vs. 36.3%,
P50.002) (Figure 5).
Negative correlations were found in VF-Val between
TUNEL (1) and progressive motile sperm (P,0.01),
total motile sperm (P,0.01), VCL (P,0.05), and
VAP (P,0.05) (Table 1). Moreover VF frozen
thawed sperm, but not QC, showed a negative correla-
tion between viability and DNA fragmentation
(TUNEL 1)(P,0.01) (Table 1).
Valine Effect on Fertilizing Ability of Frozen
Thawed Semen
Supplementing valine to the freezing extender
doubled (P50.026) the fertility rate of frozenthawed
semen of VF (32.6% 612.2) compared with the control
(16.1% 16.2). In contrast, in QC, the difference between
the fertility in QC control and QC valine was not signif-
icant, whereas the numerical results would suggest a
decrease in the fertility rate by almost a third
(24.8% 65.7) (Figure 5).
DISCUSSION
A previous work of our group with 12 different breeds
of rooster (Santiago-Moreno et al., 2019) demonstrated
that concentration of endogenous valine in rooster sem-
inal plasma was negatively correlated with post-thawed
DNA fragmentation. In the present study, we demon-
strated that supplementing valine to a chicken freezing
extender can, depending on the breed, improve motility
variables in fresh sperm as well as increase DNA
Figure 2. Motility sperm variables of fresh samples obtained from Red Villafranquina and Quail Castellana roosters, diluted in Lake Ravie 84
without (Control) or supplemented with 10 mmol valine. Different letters (a,b; A,B) indicate signicant difference (P,0.01; P,0.001, respectively)
between breeds. Appearance of * or ** on means indicates signicant difference (P,0.05; P,0.01, respectively) within each breed. n 540 (20 sam-
ples per bread). Abbreviations: ALH amplitude of lateral head displacement; QC, Quail Castellana; VAP, average path velocity; VCL, curvilinear
velocity; VF, Red Villafranquina; VSL, straight-line velocity.
Figure 3. Viability values of fresh and frozen-thawed samples ob-
tained from Red Villafranquina and Quail Castellana roosters, diluted
with Lake Ravie 84 without (Control) or supplemented with 10 mmol
valine. Different letters (a,b) indicate signicant difference between
treatments (control or valine) within the same breed (P,0.001). n 5
40 (20 samples per bread). Abbreviations: QC, Quail Castellana; VF,
Red Villafranquina.
EFFECT OF VALINE ON SPERM CRYORESISTANCE 5
cryoresistance and fertilization capacity of post-thawed
spermatozoa, mainly in the lowest fertility breed.
Comparison between VF control (bad freezer) and
QC control (better freezer) revealed superiority of
QC in fresh and frozenthawed sperm regarding
different semen parameters including, total motility
ratios, DNA integrity, and nally fertility. Addition
of valine to semen extender to dilute fresh samples
of VF and QC improved several motility values of
both breeds. This is an important issue considering
that once the chicken sperm has been inseminated in
the vagina, only 1% of the initial sperm population
reaches the sperm storage tubules from where only
1% reaches the fertilization site in the infundibulum
(Blesbois, 2018). Thus, high motility sperm is essen-
tial to obtain high fertility rates (Blesbois, 2018).
The benecial effect of valine on motility was
previously reported in a study about the composition
of free amino acids in sperm of several sh species,
where motility of perch semen was positively inu-
enced by the presence of valine (Lahnsteiner, 2010).
Regarding human, the exploration on seminal plasma
amino acid disorder in patients with asthenozoosper-
mia revealed signicantlylowervaluesofthisamino
acid than in healthy controls, pointing out valine as
a potential biomarker closely related to the clinical
parameters of this pathology (Li et al., 2019). The
mechanism by which valine exerts a benecial effect
on sperm motility is still unclear (Lahnsteiner, 2010;
Li et al., 2019); however, it has been observed that
there is a direct effect of dietary supplemented valine
on the antioxidant enzymes superoxide dismutase and
glutathione peroxidase of hypercholesterolemic rats
(Cojocaru et al., 2014).
Figure 4. Motility sperm variables of frozen-thawed semen from Red Villafranquina and Quail Castellana roosters, frozen in Lake Ravie 84 without
(Control) or with 10 mmol valine. Different letters (a,b; A,B) indicate signicant differences between breeds (P,0.05 or P,0.01, respectively). n 5
40 (20 samples per bread). Abbreviations: ALH amplitude of lateral head displacement; QC, Quail Castellana; VAP, average path velocity; VCL,
curvilinear velocity; VF, Red Villafranquina; VSL, straight-line velocity.
Figure 5. TUNEL (1) and fertility rates obtained from thawed samples of Red Villafranquina and Quail Castellana roosters, frozen without (Con-
trol) or with valine 10 mmol. Different letters (a,b; A,B) indicate signicant differences between treatments (control or valine) within the same breed
(P,0.05; P,0.01, respectively). Line over bars indicates signicant differences between breeds. n 540 (20 samples per bird). Abbreviations:
TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; VF, Red Villafranquina; QC, Quail Castellana.
BERNAL ET AL.6
Although valine signicantly increased several
motility values in fresh rooster samples of both breeds,
the corresponding differences were not signicant in
frozenthawed samples of either breed. However, a sig-
nicant benecial effect of valine was observed regarding
DNA cryoresistance and fertility rate of VF. Supplemen-
tation of valine signicantly decreased the post-thawed
DNA fragmentation rate and doubled the fertility rate
of VF-Val, a rate comparable with that of QC control.
This achievement is of special importance considering
that fertility rate of VF control was signicantly lower
than QC control. It is noteworthy that, despite the
signicantly higher post-thawed motility values of QC-
Val than those of VF-Val, this superiority was not re-
ected in the fertility rate in the present experiment.
As discussed afterward, the impact of modifying the
endogenous valine concentration may be reected in
the sperm oxidative status, affecting another sperm
functions such as the acrosome reaction or fertilizing ca-
pacity. Therefore, further study is required to conrm
the effect of valine on VF and QC fertility rate, consid-
ering assessment of features as acrosome integrity or
ROS generation.
Because mitochondria play a role in both apoptotic
DNA fragmentation and in oxidative energy metabolism
for motility, the improved DNA cryoresistance observed
in VF could also suggest an increase in post-thawed
mitochondrial integrity. At this respect, Bollwein et al.
(2008) communicated the existence of some interdepen-
dency between the DNA fragmentation index and the
loss of mitochondrial function when frozen bovine sperm
was thawed and maintained for 3 h at 38C, a relation-
ship that is expected to have an impact on motility pa-
rameters. A study on human sperm communicated a
negative correlation between DNA fragmentation and
progressive motility (Marchetti, 2002). It is not surpris-
ing then that TUNEL (1) values of VF would be nega-
tively correlated with some post-thawed motility
variables.
Reactive oxygen species can exert a negative effect on
sperm DNA, inducing the production of oxidative prod-
ucts as the 8-oxo-7,8-dihydroxyguanosine that causes
DNA fragmentation and mutagenic modications
(M
en
ezo et al., 2007). This can result in accumulation
of the protein p53, inducing mitochondria to release cy-
tochrome C and start the apoptosis program. Knowing
that redox status can either trigger or inhibit cyto-
chrome Cinduced apoptosis (Hampton and Orrenius,
1998), the decrease in the DNA fragmentation rate of
VF-Val, possibly indicating lower fraction of apoptotic
sperm cells, could suggest the involvement of valine in
maintaining the optimal oxidative status of spermatozoa
and mitochondria. At this respect, it is noteworthy that
different species/breed/individual oxidative status
might shift the kind of response to valine supplementa-
tion going from a positive effect on one species/breed/in-
dividual to a less positive or absence of effect on another.
This could explain the different response of valine sup-
plementation on DNA fragmentation and fertility rate
of VF and QC. Whether or not that is the case, the nega-
tive correlations observed between DNA fragmentation
and several motility ratios in VF-Val would further
corroborate that valine, in some way, assists in main-
taining mitochondrial integrity.
Together with leucine and isoleucine, valine forms
part of the 3 aliphatic branched-chain amino acids.
Branched-chain amino acids are some of the most hydro-
phobic amino acids which make them important in
determining the structure of globular proteins as well
as in the interaction of the transmembrane domains of
membranous proteins with phospholipid bilayers
(Brosnan and Brosnan, 2006). It is known that human
catabolism of branched-chain amino acids starts with
their transamination to branched-chain
a
-ketoacids. At
the same time, the
a
branched-chain ketoacid (
a
-ketoi-
sovalerate, for the case of valine) (Adeva-Andany et al.,
2017) is converted into its respective amino acid.
a
-
ketoacids have demonstrated to be implicated in
decreasing oxidative stress thanks to their capacity to
reduce H
2
O
2
to H
2
O(Holleman, 1904). Thus, valine
could potentially act as an antioxidant agent decreasing
or avoiding the production of harmful ROS involved
directly or indirectly in low motility, DNA fragmenta-
tion, and low fertility. The different effect of valine on
the fertility rate of VF and QC could be explained by
different needs to strengthen their antioxidant response
capacity. While in VF, supplementation of valine could
be necessary to reach enough antioxidant capacity to
decrease ROS damages involved in DNA fragmentation,
in QC, it could result irrelevant or even produce an
harmful effect, decreasing the controlled and localized
ROS that, as observed in mammals, are needed for
Table 1. Spearman correlations within each breed between different sperm assessment variables in frozenthawed
samples of Quail Castellana and Red Villafranquina.
Sperm variables TUNEL (1) (%) Progressive motile sperm (%) Total motile sperm (%) VCL (mm/s) VAP (mm/s)
VF control
Viability - 0.64** 0.73*** - -
QC control
Viability - 0.54* - - -
VF valine
Viability 20.64** - - - -
TUNEL (1)- 20.58** 20.57** 20.49* 20.50*
*P,0.05, **P,0.01, ***P,0.001.
Abbreviations: QC, Quail Castellana; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; VAP, average path
velocity; VCL, curvilinear velocity; VF, Red Villafranquina.
EFFECT OF VALINE ON SPERM CRYORESISTANCE 7
capacitation, hyperactivation, acrosome reaction, and
fertilizing capacity (Aitken, 1995;OFlaherty et al.,
2006;Wagner et al., 2018). Other possible mechanisms
suggested to date by which supplementing amino acids
to extenders can exert a protective effect are the exis-
tence of some unique targets on the plasma membrane
that are protected by selected amino acids (Kruuv and
Glofcheski, 1992) or intracellular protection against
denaturing effects of hyperosmolality in the unfrozen
fraction and within the cells during slow freezing
(Withers and King, 1979). Thus valine supplementation
can display, depending on the breed, a positive impact
on sperm motility, DNA integrity, and fertility rate,
thanks to its potential role as an antioxidant agent or
a protector agent.
In conclusion, the results of this study show that sup-
plementation with valine to chicken extenders can have
a positive effect on DNA fragmentation and fertility
rates of frozenthawed sperm, improving the current
cryopreservation protocols used for endangered Spanish
native breeds. However, as seen in this study, this effect
can be breed-specic: valine had a positive effect on VF
sperm, reducing DNA fragmentation and doubling the
fertility rate (from 16.1 to 32.6%), whereas for the case
of QC, it did not affect DNA cryoresistance or fertility
rate. This fact could be attributable to breed differences
in the seminal free amino acid prole or other seminal
components, which may also affect the antioxidant ca-
pacity. Thus, supplementation of valine to chicken
extender is recommended in these native breeds with
low fertility rates after AI using frozenthawed sperm.
ACKNOWLEDGMENTS
This paper is dedicated to the memory of our dear co-
worker, Dr. SerafínP
erez Cerezales, who recently
passed-away. Serafín took part in the DNA analyses,
and in the critical revision of the original manuscript.
This research is part of a project that received funding
from the European Unions Horizon 2020 Research and
Innovation Programme under grant agreement Nº
677353 IMAGE. Part of this research was funded by Zoi-
techlab S.L.-INIA contract CON 18-141, and INIA proj-
ect RZP2015-0002-00-00.
REFERENCES
Adeva-Andany, M. M., L. L
opez-Maside, C. Donapetry-García,
C. Fern
andez-Fern
andez, and C. Sixto-Leal. 2017. Enzymes
involved in branched-chain amino acid metabolism in humans.
Amino Acids 49:10051028.
Aitken, R. J. 1995. Free radicals, lipid peroxidation and sperm func-
tion. Reprod. Fertil. Dev. 7:659668.
Ansari, M. S., B. A. Rakha, S. Akhter, E. Blesbois, and J. Santiago-
Moreno. 2019. Effect of cryopreservation on lipid peroxidation,
antioxidant potential, chromatin integrity, and mitochondrial ac-
tivity of Indian Red Jungle fowl ( Gallus gallus murghi ) semen.
Biopreserv Biobank 17:288295.
Blesbois, E., F. Seigneurin, I. Grasseau, C. Limouzin, J. Besnard,
D. Gourichon, G. Coquerelle, P. Rault, and M. Tixier-Boi-
chard. 2007. Semen cryopreservation for ex situ management of
genetic diversity in chicken: creation of the French avian cryobank.
Poult. Sci. 86:555564.
Blesbois, E. 2012. Biological features of the avian male gamete and
their application to biotechnology of conservation. J. Poult. Sci.
49:141149.
Blesbois, E. 2018. Bird reproduction overview. Pages 579585 in
Encyclopedia of Reproduction. Elsevier.Science BV, Amsterdam,
The Netherlands.
Bollwein, H., I. Fuchs, and C. Koess. 2008. Interrelationship between
plasma membrane integrity, mitochondrial membrane potential
and dna fragmentation in cryopreserved bovine spermatozoa.
Reprod. Domest. Anim. 43:189195.
Brosnan, J. T., and M. E. Brosnan. 2006. Branched-chain amino
acids : enzyme and substrate regulation. J. Nutr. 136:207S211S.
Burrows, W. H., and J. P. Quinn. 1936. The collection of spermatozoa
from the domestic fowl and Turkey. Poult. Sci. 16:1924.
Campo, J. L. 1998. Conservation and genetical study of Spanish
chicken breeds. Pages 155158 in Proceedings of the 6th
World Congress on Genetic Applied to Livestock Production.
University of New England, Armidale.
Campo, J. L., and F. Orozco. 1982. Conservation and genetical study
of Spanish chicken breeds. Pages 8893 in Proceedings of the
Second World Congress on Genetics Applied to Livestock Pro-
duction. Editorial Garsi, Madrid.
Chalah, T., and J. P. Brillard. 1998. Comparison of assessment of fowl
sperm viability by eosin-nigrosin and dual uorescence (SYBR-14/
PI). Theriogenology 50:487493.
Cojocaru, E., N. Filip, C. Ungureanu, C. Filip, and M. Danciu. 2014.
Effects of valine and leucine on some antioxidant enzymes in hy-
percholesterolemic rats. Health 06:23132321.
Etches, R. J. 1998. Reproducci
on Aviar. Page 236 in Acribia,
Zaragoza.
Hampton, M. B., and S. Orrenius. 1998. Redox Regulation of
apoptotic cell death in the immune system. Toxicol. Lett. 102
103:355358.
Henkel, R. R. 2011. Leukocytes and oxidative stress: dilemma for
sperm function and male fertility. Asian J. Androl. 13:4352.
Holleman, M. A. F. 1904. Notice Sur laction de leau Oxyg
en
ee Sur
Les Acides Alpha-C
etoniques at Sur Les Dic
etones 1.2. Recl. Trav.
Chim.23:169171.
Hutson, S. M., and T. R. Hall. 1993. Identication of the mitochon-
drial branched chain aminotransferase as a branched chain
a
-keto
acid transport protein. J. Biol.Chem.268:30843091.
Khiabani, A. B., G. Moghaddam, and H. D. Kia. 2017. Effects of
adding different levels of glutamine to modied beltsville extender
on the survival of frozen rooster semen. Anim. Reprod. Sci.
184:172177.
Kruuv, J., and D. J. Glofcheski. 1992. Protective effects of amino acids
against freeze-thaw damage in mammalian cells. Cryobiology
29:291295.
Kundu, C. N., K. Das, and G. C. Majumder. 2001. Effect of amino
acids on goat cauda epididymal sperm cryopreservation using a
chemically dened model system. Cryobiology 42:2127.
Lahnsteiner, F. 2010. A Comparative study on the composition and
importance of free amino acids in semen of gilthead sea bream,
sparus aurata, and perch, perca uviatilis. Fish Physiol. Biochem.
36:12971305.
Lake, P. E., and O. Ravie. 1984. Effect of age and sex on the chemical
composition of edible offal and blood from broilers. Br. Poult. Sci.
25:145150.
Li, M. J., Z. M. Zhang, F. Fan, P. Ma, Y. Wang, and H. M. Lu. 2019.
Exploring asthenozoospermia seminal plasma amino acid disorder
based on GC-SIM-MS combined with chemometrics methods.
Anal. Methods 11:28952902.
Long, J. A. 2006. Avian semen cryopreservation: what are the bio-
logical challenges? Poult. Sci.85:232236.
Lorenz, F. W., and A. Tyler. 1951. Extension of motile life span of
spermatozoa on the domestic fowl by amino acids and proteins.
Proc. Soc. Exp. Biol. Med. 78:5762.
DISCLOSURES
The authors declare no conicts of interest. 2015-
00002-00-00.
BERNAL ET AL.8
Marchetti, C. G Obert, A. Deffosez, P. Formstecher, and
P. Marchetti. 2002. Study of mitochondrial membrane poten-
tial, reactive oxygen species, DNA fragmentation and cell
viability by ow cytometry in human sperm. Hum. Reprod.
17:12571265.
M
en
ezo, Y. J. R.,A. Hazout,G. Panteix,F. Robert, J. Rollet, P. Cohen-
Bacrie,F.Chapuis,P.Cl
ement, and M. Benkhalifa. 2007. Antioxi-
dants to reduce sperm DNA fragmentation: an unexpected adverse
effect. Reprod. Biomed. Online 14:418421.
Moc
e, E., I. Grasseau, and E. Blesbois. 2010. Cryoprotectant and
freezing-process alter the ability of chicken sperm to acrosome
react. Anim. Reprod. Sci. 122:359366.
OFlaherty, C., E. de Lamirande, and C. Gagnon. 2006. Positive role
of reactive oxygen species in mammalian sperm capacitation:
triggering and modulation of phosphorylation events. Free Radic.
Biol. Med. 41:528540.
Partyka, A., W. Niza
nski, and E. qukaszewicz. 2010. Evaluation of
fresh and frozen-thawed fowl semen by ow cytometry. Ther-
iogenology 74:10191027.
P
erez-Cerezales, S., S. Martínez-P
aramo, E. Cabrita, F. Martínez-
Pastor, P. de Paz, and M. P. Herr
aez. 2009. Evaluation of oxidative
dna damage promoted by storage in sperm from sex-reversed
rainbow trout. Theriogenology 71:605613.
Santiago-Moreno, J., B. Bernal, S. P
erez-Cerezales, C. Castaño,
A. Toledano-Diaz, M. C. Esteso, A. Guti
errez-Ad
an, A. L
opez-
Sebasti
an, M. G. Gil, H. Woelders, and E. Blesbois. 2019. Seminal
plasma amino acid prole in different breeds of chicken: role of
seminal plasma on sperm cryoresistance. PLoS One 14:119.
Santiago-Moreno, J., C. Castaño, A. Toledano-Díaz, M. A. Coloma,
A. L
opez-Sebasti
an, M. T. Prieto, and J. L. Campo. 2011. Semen
cryopreservation for the creation of a Spanish poultry breeds cry-
obank: Optimization of freezing rate and equilibration time. Poult.
Sci. 90:20472053.
Surai, P. F., S. Cerolini, G. J. Wishart, B. K. Speake, R. C. Noble, and
N. H. C. Sparks. 1998. Lipid and antioxidant composition of
chicken semen and its susceptibility to peroxidation. Avian Pout.
Biol. Rev.9:1123.
Thananurak, P., N. Chuaychu-noo, A. Th
elie, Y. Phasuk,
T. Vongpralub, and E. Blesbois. 2019. Sucrose Increases the
quality and fertilizing ability of cryopreserved chicken sperms in
contrast to rafnose. Poult. Sci. 98:41614171.
Topraggaleh, T. R., A. Shahverdi, A. Rastegarnia, B. Ebrahimi,
V. Shaepour, M. Sharbatoghli, V. Esmaeili, and
E. Janzamin. 2014. Effect of cysteine and glutamine added to
extender on post-thaw sperm functional parameters of buffalo bull.
Andrologia 46:777783.
Tuncer, P. B., M. N. Bucak, S. B
uy
ukleblebici, S. Sari
ozkan, D. Yeni,
A. Eken, P. P. Akalin, H. Kinet, F. Avdatek, A. F. Fidan, and
M. G
undog
an. 2010. The effect of cysteine and glutathione on
sperm and oxidative stress parameters of post-thawed bull semen.
Cryobiology 61:303307.
Wagner, H., J. W. Cheng, and E. Y. Ko. 2018. Role of reactive oxygen
species in male infertility: an updated review of literature. Arab J.
Urol. 16:3543.
Withers, L. A., and P. J. King. 1979. Proline: a novel cryoprotectant
for the freeze preservation of cultured cells of Zea mays L. Plant
Physiol. 64:675678.
Zhang, S., X. Zeng, M. Ren, X. Mao, and S. Qiao. 2017. Novel meta-
bolic and physiological functions of branched chain amino acids: a
review. J. Anim. Sci. Biotechno.8:415.
EFFECT OF VALINE ON SPERM CRYORESISTANCE 9
... This is also consistent with previous research involving both human [40] and other mammalian species [57][58][59][60][61][62] in which the supplementation of amino acids was successfully used to improve post-thawing sperm quality. More recently, amino acid supplementation was successfully checked in chicken [63][64][65][66]. ...
... In addition, the supplementation of amino acids to semen diluents can lead to a reduction in the concentration of toxic solutes to levels associated with lesser toxicity; moreover, some amino acids can protect sperm cells against the denaturing effects of hyperosmolality during cryopreservation process [2,67]. In accordance with our results, in a recent study, the supplementation of valine to chicken freezing extenders resulted in a decrease in DNA fragmentation and a positive effect on the fertilizing ability of frozen-thawed sperm, with a better response in a breed that is considered to have the lowest semen freezability [64]. ...
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