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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,*
N. Iglesias-Cabeza,*U. S
A. Toledano-Díaz,*C. Castaño,*
S. P
erez-Cerezales,*A. Guti
an,*A. L
an,*P. García-Casado,
M. G. Gil,
H. Woelders,
E. Blesbois,
and J. Santiago-Moreno*
*Department of Animal Reproduction, INIA, 28040 Madrid, Spain;
Department of Reproductive Biology,
FES-Iztacala, UNAM, 54090, Tlalnepantla, M
Zoitechlab, R&D Department, 28400 Madrid, Spain;
Department of Animal Breeding, INIA, 28040 Madrid, Spain;
Wageningen University & Research, Animal
Breeding and Genomics, 6700 AH Wageningen, The Netherlands; and
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 -:--
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 (
Received March 9, 2020.
Accepted September 24, 2020.
Corresponding author:
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
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).
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
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
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
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
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
O (0.08 g), potassium acetate (0.5 g), polyvinylpyr-
rolidone (M
10,000; 0.3 g), and 100 mL H
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
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
and then from 235Cto2140Cat60
(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
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
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
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
O, 0.8 g magnesium ace-
tate tetrahydrate 4H
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
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
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).
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.
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).
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.
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.
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
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
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
-ketoacids. At
the same time, the
branched-chain ketoacid (
sovalerate, for the case of valine) (Adeva-Andany et al.,
2017) is converted into its respective amino acid.
ketoacids have demonstrated to be implicated in
decreasing oxidative stress thanks to their capacity to
reduce H
to H
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.
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.
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.
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... 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]. ...
Full-text available
Semen cryopreservation represents the main tool for preservation of biodiversity; however, in avian species, the freezing–thawing process results in a sharp reduction in sperm quality and consequently fertility. Thus, to gain a first insight into the molecular basis of the cryopreservation of turkey sperm, the NMR-assessed metabolite profiles of fresh and frozen–thawed samples were herein investigated and compared with sperm qualitative parameters. Cryopreservation decreased the sperm viability, mobility, and osmotic tolerance of frozen–thawed samples. This decrease in sperm quality was associated with the variation in the levels of some metabolites in both aqueous and lipid sperm extracts, as investigated by NMR analysis. Higher amounts of the amino acids Ala, Ile, Leu, Phe, Tyr, and Val were found in fresh than in frozen–thawed sperm; on the contrary, Gly content increased after cryopreservation. A positive correlation (p < 0.01) between the amino acid levels and all qualitative parameters was found, except in the case of Gly, the levels of which were negatively correlated (p < 0.01) with sperm quality. Other water-soluble compounds, namely formate, lactate, AMP, creatine, and carnitine, turned out to be present at higher concentrations in fresh sperm, whereas cryopreserved samples showed increased levels of citrate and acetyl-carnitine. Frozen–thawed sperm also showed decreases in cholesterol and polyunsaturated fatty acids, whereas saturated fatty acids were found to be higher in cryopreserved than in fresh sperm. Interestingly, lactate, carnitine (p < 0.01), AMP, creatine, cholesterol, and phosphatidylcholine (p < 0.05) levels were positively correlated with all sperm quality parameters, whereas citrate (p < 0.01), fumarate, acetyl- carnitine, and saturated fatty acids (p < 0.05) showed negative correlations. A detailed discussion aimed at explaining these correlations in the sperm cell context is provided, returning a clearer scenario of metabolic changes occurring in turkey sperm cryopreservation.
... Valine, for example, helps to maintain DNA integrity and has a positive effect on the viability of frozenthawed sperm (Santiago-Moreno and Blesbois, 2020). Adding valine to extenders also improves the fertilising capacity of frozen-thawed rooster sperm, with the best responses seen in those breeds with the sperm least resistant to cryopreservation (Bernal et al., 2020). To improve the cryoprotective capacity of semen extenders, several studies have been undertaken to use other additives such as antioxidants or compounds of plant origins with antioxidant activity (revised by Leão et al., 2021), such as resveratrol, lycopene, quercitin, etc. ...
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Over the last century, several reproductive biotechnologies beyond the artificial incubation of eggs were developed to improve poultry breeding stocks and conserve their genetic diversity. These include artificial insemination (AI), semen storage, diploid primordial germ cell (PGC) methodologies, and gonad tissue storage and transplantation. Currently, AI is widely used for selection purposes in the poultry industry, in the breeding of turkeys and guinea fowl, and to solve fertility problems in duck interspecies crosses for the production of mule ducklings. The decline in some wild game species has also raised interest in reproductive technologies as a means of increasing the production of fertile eggs, and ultimately the number of birds that can be raised. AI requires viable sperm to be preserved in vitro for either short (fresh) or longer periods (chilling or freezing). Since spermatozoa are the most easily accessed sex cells, they are the cell type most commonly preserved by genetic resource banks. However, the cryopreservation of sperm only preserves half of the genome, and it cannot preserve the W chromosome. For avian species, the problem of preserving oocytes and zygotes may be solved via the cryopreservation and transplantation of PGCs and gonad tissue. The present review describes all these procedures and discusses how combining these different technologies allows poultry populations to be conserved and even rapidly reconstituted.
... Results from studies of Santiago-Moreno et al. (2019) indicated there were positive associations between seminal plasma concentration of the hydrophobic amino acids such as valine, isoleucine, leucine and the charged lysine with membrane integrity as assessed by conducting the sperm viability assay after thawing of the semen sample. Accordingly, results from studies when there was addition of valine to freezing extenders for rooster sperm indicated there was a positive effect by reducing DNA fragmentation and enhancing fertilizing capacity of sperm subsequent to thawing, with there being a greater response to valine supplementation of semen extender in birds of a breed considered to be the most problematic when attempting to cryopreserve semen for AI utilization (Bernal et al., 2020). There have been inclusions of serine at optimal concentrations for modulation of antioxidant actions in spermatozoa during cryopreservation so that there is maintenance of sperm viability subsequent to thawing (Thananurak et al., 2020). ...
Active link before February 24, 2022 Semen cryopreservation is a key biotechnological strategy used to preserve and protect genetic resources, which are subject to increasingly serious reductions in some species, and to protect animal biodiversity. Assisted reproductive techniques, however, are still not utilized to the same extent in avian species to the extent that occurs in mammals. The reasons for this situation are described in this review. The content of this paper is focused on current poultry preservation systems, published since 2010, and new strategies that are very promising for preserving avian genetic resources. Two major types of storage technologies which are utilized for avian sperm preservation, liquid storage and cryopreservation, are emphasized. The issues on which there is a focus includes supplementation of avian extenders with various compounds prior to the preservation period, use of cryoprotectants and fertility results when there were in vitro sperm evaluations. Results from recent studies indicate there are opportunities to improve the quality of bird semen after preservation. It is obvious that cryo-diluent composition may be the most important factor for development of efficacious cryopreservation methods for avian semen.
... The viability of embryos obtained from the use of cryopreserved sperm is low due to DNA fragmentation [29]. Improving the composition of diluents for cryopreservation, improving the selection of cryoprotectants, and modernizing freezing methods (in straws, pellets, and fast or slow protocols) [30,31] will allow further advancements in this field of research. The properties of trehalose make it possible to reduce the molecular mobility of the vitreous matrix, and this suggests that trehalose may be effective for maintaining the protein structure during prolonged storage at low temperatures [14,23,32]. ...
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The aim of this study was to create balanced media for the cryopreservation of rooster semen in pellets to maintain the functional state of the sperm after thawing. Fructose was replaced by trehalose in experimental media in proportions of 10% (LCM-T10) and 20% (LCM-T20), while LCM was used as a control. After artificial insemination of the hens, the eggs were incubated (n = 400). To determine the functional safety of spermatozoa in the genital tract of hens after 5, 10, and 15 days from the last insemination, we used a method for assessing the interaction of sperm with the perivitelline membrane. Significantly higher rates of egg fertilization (82–86%) were obtained when using LCM-T10 and LCM-T20 compared to control (79%, p < 0.05). Egg fertility on the 5th day from the last insemination with the LCM-T20 diluent reached 100% versus 86% in the control; on the 10th day, the fertility rates were 55% versus 20%, respectively. The best results for fertility duration were obtained by freezing spermatozoa with LCM-T20 medium. The numbers of interaction points of spermatozoa with the perivitelline membrane were as follows: on the 5th day from the last insemination with LCM-T20—461.5 ± 11.5 holes/cm2 (LCM-control—13.7 ± 2.7 holes/cm2), p < 0.01; on the 10th day with LCM-T20—319.3 ± 12.9 holes/cm2 (LCM-control—14.9 ± 3.5 holes/cm2); and on the 15th day with LCM-T20—345.2 ± 11.1 holes/cm2 (LCM-control—0 holes/cm2). In conclusion, the use of trehalose in LCM diluent medium can increase the fertility of frozen/thawed sperm and the duration of their fertility in the genital tract of hens.
... Accordingly, supplementing chicken extenders with the non-coded amino acid taurine showed a positive effect in reducing sperm apoptosis and DNA damage [93]. Supplementation of valine to chicken freezing extenders showed 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 [94]. The addition of appropriate serine levels also showed a positive effect on sperm cryopreservation, partly through the antioxidant action of this amino acid [88]. ...
Full-text available
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.
1. Birchen and Blue Leonesa are two endangered chicken breeds mainly raised in Curueño Valley in North Spain. The establishment of a germplasm bank to guarantee the preservation of these breeds is needed. However, cockerels from different breeder flocks can show variance in semen cryoresistance. 2. The following work focused on the sperm characterisation and cryopreservation of Birchen and Blue Leonesa cockerels from four different breeders. A total of 30 semen pools were analysed. Besides conventional sperm analysis, including motility by computer-aided sperm analysis (CASA) and DNA fragmentation by TUNEL, the present study tested a double staining method (MitoTrackerTM Green FM/ propidium iodide). This gave simultaneous assessment of plasma, acrosomal and mitochondrial membranes, which was previously validated by SYBR-14/PI, CASA, aniline blue and TUNEL. 3. No significant differences were found among fresh semen variables between breeds or breeders. For post-thawed variables, significant differences (P<0.05) were found between breeders in sperm viability (58.0±1.90 breeder D vs. 35.2±7.41 breeder A, 37.2±4.09 breeder B and 22.3±5.92 breeder C) and DNA fragmentation (62.4±9.91 breeder C vs. 31.8±7.08 breeder B and 24.5±5.49 breeder D). The lowest DNA fragmentation values for semen from breeder D birds were coincident with higher integrity of the mitochondrial membrane. 4. The results revealed higher sperm cryoresistance in the cockerels from one of the breeders, possibly due to differences in management system (e.g., diet, housing, control of stress elements and pathogens, reproduction practices or maintenance of genetic diversity). These differences may determine the sperm freezability, and thus the effectiveness of developing a germplasm bank.
Full-text available
Chicken semen conservation is an important tool for programs of genetic diversity management and of endangered breeds’ conservation. However, the method still needs to be improved in order to be applied in a wide variety of environments and breeds. Our objective was to compare the effects of 2 external cryoprotectants saccharides (sucrose and raffinose) on the sperm freezability of a Thai local breed, Pradu Hang Dum, in which semen was frozen with a simple freezing method using nitrogen vapors and dimethyl formamide (DMF). Thirty-six males were selected on their motility vigor score for the experiments. In a first experiment, a large range of sucrose and raffinose doses were tested. Semen quality was evaluated after incubation at 5°C or after cryopreservation in straws in the saline Blumberger Hahnen Sperma Verdünner diluent + DMF (6% v/v) with or without sucrose/raffinose. The best targeted doses of sucrose and raffinose were then kept for experiment 2 that was focused on cryopreserved semen. In this experiment, semen quality was measured on frozen-thawed sperm: different objective motility data evaluated by computer-assisted sperm analysis (CASA), membrane integrity, acrosome integrity, mitochondria function evaluated using flow cytometry, lipid peroxide production assessed by the thiobarbituric acid test. Fertility obtained with frozen-thawed semen supplemented or not with sucrose or raffinose was also evaluated after artificial insemination of laying hens. The presence of sucrose at the osmotically inactive dose 1 mmol significantly increased the vigor motility, membrane integrity, acrosome integrity, and mitochondrial functions of frozen-thawed sperm (P < 0.05), and showed the highest levels of fertility after sperm cryopreservation (91% vs. control 86%, P < 0.001). Raffinose showed negative effects on in vitro semen quality from 1 to 100 mmol. Fertility was also negatively (P < 0.001) affected by raffinose (fertility rate 66 to 70%). We thus showed in the present study the high success of a simple chicken sperm cryopreservation method with an external cryoprotectant easily available and cheap, the sucrose, used at an osmotically inactive low concentration.
Full-text available
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
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Objectives To review the literature and provide an updated summary on the role of reactive oxygen species (ROS) in male infertility. Methods A review of PubMed, Cochrane review, and Web of Science databases for full-text English-language articles published between 1943 and 2017 was performed, focusing on the aetiology of ROS, physiological role of ROS on spermatic function, pathological role of ROS in infertility, evaluation of ROS, and role of antioxidants in oxidative stress. Results ROS play a role in spermatic function and fertilisation. The literature describes both a physiological and a pathological role of ROS in fertility. A delicate balance between ROS necessary for physiological activity and antioxidants to protect from cellular oxidative injury is essential for fertility. Conclusion Although elevated levels of ROS are implicated as a cause of infertility, there is no consensus on selecting patients to test for ROS, which test to perform, or if treatment for ROS can have a positive impact on infertility rates and pregnancy.
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Branched-chain amino acids (leucine, isoleucine and valine) are structurally related to branched-chain fatty acids. Leucine is 2-amino-4-methyl-pentanoic acid, isoleucine is 2-amino-3-methyl-pentanoic acid, and valine is 2-amino-3-methyl-butanoic acid. Similar to fatty acid oxidation, leucine and isoleucine produce acetyl-coA. Additionally, leucine generates acetoacetate and isoleucine yields propionyl-coA. Valine oxidation produces propionyl-coA, which is converted into methylmalonyl-coA and succinyl-coA. Branched-chain aminotransferase catalyzes the first reaction in the catabolic pathway of branched-chain amino acids, a reversible transamination that converts branched-chain amino acids into branched-chain ketoacids. Simultaneously, glutamate is converted in 2-ketoglutarate. The branched-chain ketoacid dehydrogenase complex catalyzes the irreversible oxidative decarboxylation of branched-chain ketoacids to produce branched-chain acyl-coA intermediates, which then follow separate catabolic pathways. Human tissue distribution and function of most of the enzymes involved in branched-chain amino acid catabolism is unknown. Congenital deficiencies of the enzymes involved in branched-chain amino acid metabolism are generally rare disorders. Some of them are associated with reduced pyruvate dehydrogenase complex activity and respiratory chain dysfunction that may contribute to their clinical phenotype. The biochemical phenotype is characterized by accumulation of the substrate to the deficient enzyme and its carnitine and/or glycine derivatives. It was established at the beginning of the twentieth century that the plasma level of the branched-chain amino acids is increased in conditions associated with insulin resistance such as obesity and diabetes mellitus. However, the potential clinical relevance of this elevation is uncertain.
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It is widely known that branched chain amino acids (BCAA) are not only elementary components for building muscle tissue but also participate in increasing protein synthesis in animals and humans. BCAA (isoleucine, leucine and valine) regulate many key signaling pathways, the most classic of which is the activation of the mTOR signaling pathway. This signaling pathway connects many diverse physiological and metabolic roles. Recent years have witnessed many striking developments in determining the novel functions of BCAA including: (1) Insufficient or excessive levels of BCAA in the diet enhances lipolysis. (2) BCAA, especially isoleucine, play a major role in enhancing glucose consumption and utilization by up-regulating intestinal and muscular glucose transporters. (3) Supplementation of leucine in the diet enhances meat quality in finishing pigs. (4) BCAA are beneficial for mammary health, milk quality and embryo growth. (5) BCAA enhance intestinal development, intestinal amino acid transportation and mucin production. (6) BCAA participate in up-regulating innate and adaptive immune responses. In addition, abnormally elevated BCAA levels in the blood (decreased BCAA catabolism) are a good biomarker for the early detection of obesity, diabetes and other metabolic diseases. This review will provide some insights into these novel metabolic and physiological functions of BCAA.
Asthenozoospermia is a common cause of male infertility. Due to the limitation of routine techniques, metabolic alterations associated with the disease and its pathogenesis are still unclear. Metabolic profiling is a powerful tool for studying the metabolic changes, finding potential biomarkers, and revealing the pathophysiological processes of the disease. In this study, amino acid (AA) profiling was used to explore the metabolic disturbance and discover potential biomarkers of asthenozoospermia. Gas chromatography-selective ion monitoring-mass spectrometry (GC-SIM-MS) was used to analyze AAs of seminal plasma from asthenozoospermic men (AS, n = 31) and healthy controls (HCs, n = 21). A supervised orthogonal partial least squares discriminant analysis (OPLS-DA) method was used to construct the classification model. Furthermore, canonical correlation analysis (CCA) was employed to study the correlation between AAs and clinical parameters. As a result, several amino acids including lysine (Lys), valine (Val) and glycine (Gly) were identified as potential biomarkers. The CCA results showed that rapid progressive motility (a) and vitality are the most important clinical parameters that are closely correlated with AA disorder in AS. Thus more attention should be paid to these parameters in clinical practice for monitoring AA disturbances in AS. The results have demonstrated that metabolic profiling by GC-SIM-MS combined with OPLS-DA and CCA may be a useful tool for discovering AA perturbation of seminal plasma and potential biomarkers for AS.
Aim: The study elucidates the impact of cryopreservation on lipid peroxidation (LPO), antioxidant potential, DNA integrity, and mitochondrial activity of Indian red jungle fowl sperm. Materials and methods: Semen from eight mature cocks was pooled and diluted at 37°C using a specific medium for red fowl species. The diluted semen samples were immediately refrigerated at 4°C for 2 hours (0.275°C min-1). Glycerol was then added to a final concentration of 20%, and the samples were equilibrated for 10 minutes at 5°C before loading into 0.25 mL French straws. These straws were then frozen by placing them in liquid nitrogen vapor for 10 minutes and plunged into liquid nitrogen. Motility, viability, DNA integrity, antioxidant activity, and LPO were assessed before dilution (fresh semen), after equilibration (processed semen), and post-thawing (frozen semen). Results: Sperm motility, viability, DNA integrity, and mitochondrial activity decreased (p < 0.05) in processed and frozen semen compared with fresh semen. Nevertheless, the concentration of malondialdehyde (MDA) in sperm and seminal plasma was greater (p < 0.05) in frozen-thawed and processed semen compared with fresh semen. Multivariate regression analysis showed a negative impact of MDA concentration in sperm (R2 = 0.90, Wilk's λ = 0.003, p < 0.001) and seminal plasma (R2 = 0.84, Wilk's λ = 0.02, p < 0.001) on motility, viability, DNA integrity, and mitochondrial activity. Nonetheless, total antioxidant capacity (TAC) had a positive impact on sperm variables (R2 = 0.82, Wilk's λ = 0.096, p < 0.001). Conclusions: The decrease in motility, viability, DNA integrity, and mitochondrial activity of Indian red jungle fowl sperm was associated with an increase in LPO during cryopreservation. Furthermore, TAC was reduced during the freeze-thaw process, which was insufficient in protecting the sperm against high reactive oxygen species levels.
The aim of the present study was to investigate the effects of L-glutamine on the quality of frozen-thawed rooster semen. Semen samples were collected from eight mature roosters (Ross 308). After initial semen assessments, samples of adequate quality were mixed together and diluted with modified Beltsville extender without L-glutamine (control) and supplemented with 2.5, 5, and 7.5 mM L-glutamine. Semen straws were subjected to cryopreservation and evaluated twice at 15-day intervals. After thawing, sperm viability, total and progressive sperm motilities were measured by Eosin-Nigrosine and Computer-Aided Sperm Analysis (CASA), respectively. The results showed that sperm functions decreased on day 30 compared to day 15. The extender supplemented with 5 mM glutamine improved (p < 0.05) sperm viability, total and progressive sperm motilities compared to other treatments and the control group. The best level of glutamine appeared to be 2.5 mM, as it provided the highest sperm membrane integrity and the lowest level of abnormalities. The results of this study suggest that the addition of glutamine to the diluent improves semen quality and using glutamine allows rooster sperm to be frozen for longer.