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Seminal plasma is a key biological fluid that modulates sperm function in the reproduction process. However, its role in sperm biotechnologies is scarce in poultry. The aims of the present study were to study the amino acids profile and total proteins of seminal plasma in 12 Spanish chicken breeds and to investigate the role of seminal plasma on cryoresistance of rooster sperm. To investigate the role of seminal plasma on cryoresistance, diluted pooled semen samples were cryopreserved in the presence and absence of seminal plasma. Glutamic acid was the most abundant free amino acid in seminal plasma, followed by alanine, serine, valine, and glycine. There was an influence of breed (P
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RESEARCH ARTICLE
Seminal plasma amino acid profile in different
breeds of chicken: Role of seminal plasma on
sperm cryoresistance
Julia
´n Santiago-MorenoID
1
*, Berenice Bernal
1
, Serafı
´n Pe
´rez-Cerezales
1
,
Cristina Castaño
1
, Adolfo Toledano-Dı
´az
1
, Milagros C. Esteso
1
, Alfonso Gutie
´rrez-Ada
´n
1
,
Antonio Lo
´pez-Sebastia
´n
1
, Marı
´a G. Gil
2
, Henri Woelders
3
, Elisabeth Blesbois
4
1Departamento de Reproduccio
´n Animal, INIA, Madrid, Spain, 2Departamento de Mejora Gene
´tica Animal,
INIA, Madrid, Spain, 3Wageningen University and Research, Animal Breeding and Genomics, Wageningen,
the Netherlands, 4UMR Physiologie de la Reproduction et des Comportements, INRA-CNRS-Universite
´
Franc¸ois Rabelais-Haras Nationaux, Nouzilly, France
*moreno@inia.es
Abstract
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<0.05) on the per-
centage of viable sperm after freezing-thawing of samples with seminal plasma. Cluster
analysis revealed that White Prat, Black Castellana, Blue Andaluza, Quail Castellana, and
Red-Barred Vasca returned the best freezing-thawing response (good freezers). There was
a positive correlation between seminal plasma concentrations of valine, isoleucine lysine,
leucine and post thaw viability. The evaluation of fertilization capacity of frozen-thawed
semen from the breeds White Prat (‘good freezer’) and Black-Red Andaluza (‘bad freezer’)
showed that good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed
(14/76, 18.4%), even if the difference was not significant (P = 0.08). The TUNEL assay
revealed that freezing/thawing procedures in presence of seminal plasma provoked higher
DNA fragmentation in most of the breeds, with a positive correlation between seminal ala-
nine, valine, isoleucine, methionine, leucine, tyrosine, phenylalanine concentrations and
DNA integrity. DNA fragmentation was lower in absence of seminal plasma and the breed
effect on sperm viability was highly reduced. It is concluded that specific seminal plasma
amino acids were associated with post-thaw percentage of viable sperm and DNA integrity.
The removal of seminal plasma decreases the variability of the results and DNA fragmenta-
tion damages.
PLOS ONE | https://doi.org/10.1371/journal.pone.0209910 January 4, 2019 1 / 19
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OPEN ACCESS
Citation: Santiago-Moreno J, Bernal B, Pe
´rez-
Cerezales S, Castaño C, Toledano-Dı
´az A, Esteso
MC, et al. (2019) Seminal plasma amino acid
profile in different breeds of chicken: Role of
seminal plasma on sperm cryoresistance. PLoS
ONE 14(1): e0209910. https://doi.org/10.1371/
journal.pone.0209910
Editor: Gerardo M. Nava, Universidad Autonoma
de Queretaro, MEXICO
Received: June 12, 2018
Accepted: December 13, 2018
Published: January 4, 2019
Copyright: ©2019 Santiago-Moreno et al. This is
an open access article distributed under the terms
of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: The study reported here was part of a
project that has received funding from the
European Union’s Horizon 2020 Research and
Innovation Programme under grant agreement
Nº677353.
Competing interests: The authors have declared
that no competing interests exist.
Introduction
The influence of seminal plasma on sperm storage may vary among species. Its removal is
recommended in the majority of semen cryopreservation protocols of species such as cap-
rine in order to ensure maximal sperm viability [1], but it isn’t entirely recommended in
other mammals (e.g. ovine; [2]). Mammalian seminal plasma may contain factors that
influence resistance of sperm to cold-shock damage and may prevent cryoinjury [35].
Conversely, detrimental effects of seminal plasma on sperm variables after freezing have
also been reported [1,6,7]. In birds, early preliminary studies showed contrasted effects of
seminal plasma fractions on refrigerated rooster sperm [8], and a global deleterious effect
in chickens and turkeys [9,10], but its effects on frozen semen have never been studied.
Rooster semen is usually frozen complete, i.e with presence of seminal plasma. However,
the effects (advantages or disadvantages) of seminal plasma during cryopreservation of
rooster semen are not clear. Seminal plasma provides metabolic support, as energy
sources for the sperm cells, and influences sperm functionality in a not completely under-
stood way.
The components of rooster seminal plasma derive from the proximal efferent ducts,
epididymides and deferent ducts [11]. During natural mating, the transparent fluid from
the paracloacal vascular bodies joins to the deferent duct fluid [12]. Besides inorganics
ions (Na
+
, K
+
, Ca
+
) [13], a characteristic biochemical feature of the seminal plasma is the
occurrence of a wide range of organic constituents such as carbohydrates, lipids, lipopro-
tein complexes, proteins, peptides, and amino acids [8,14]. The functional significance of
free amino acids is diverse: scavenge free radicals, act as a solute protecting the cell
against the denaturing effects of hyperosmolality, provide buffers with protective influ-
ence on sperm cells, and serve as oxidizable substrates for spermatozoa, [15]. The role of
seminal plasma amino acids during cryopreservation process is not clear, but it has been
described that a number of amino acids have a cryoprotective effect during freezing and
thawing of mammalian sperm [16,17], or of isolated enzymes, such as calcium ATPase
[18] or phosphofructokinase [19]. Moreover, some organisms accumulate amino acids in
response to cold temperatures [20]. Several studies have focused on their use as additives
in extenders. Glutamine, proline, histidine, glycine, alanine have been used for cryopres-
ervation of ram, stallion, goat and human semen [2124]. Although the cryoprotectant
mechanism of amino acids is not well known, some hypotheses regarding possible mecha-
nisms have been provided, such as anti-oxidative activity [25] and protection against
denaturing effects of low water potential during freezing [26]. Carpenter and Crowe [19]
noted that amino acid might stabilize proteins, thus avoiding denaturation and dissocia-
tion that would lead to a greater contact surface between proteins and solutes during
freeze-thaw process.
The seminal plasma amino acid profile may vary among genotypes [11], and differ-
ences between chicken breeds may be expected. Individual semen donors, or breeds may
also differ in ‘freezability’ of the semen, and be categorized as ‘‘good” or ‘‘bad freezers”.
The mechanisms underlying differences in cryosensitivity between different individuals
have yet to be elucidated. It has been demonstrated that consistent inter-individual varia-
tions in sperm freezability are genetically determined [27]. Considering the above men-
tioned cryoprotective properties of amino acids, we may suggest that a possible variability
among chicken breeds to sustain sperm cryopreservation could be related to differences
in seminal plasma amino acid compositions and protein content. Therefore, the aims of
the present study were to investigate the role of seminal plasma and seminal amino acids
profiles of different chicken breeds on sperm cryoresistance variability.
Seminal plasma and sperm freezability
PLOS ONE | https://doi.org/10.1371/journal.pone.0209910 January 4, 2019 2 / 19
Material and methods
Experimental birds
The birds used in this study were of 12 Spanish chicken breeds (Black-Barred Andaluza,
Black-Red Andaluza, Blue Andaluza, Black Castellana, Buff Prat, White Prat, Red-Barred
Vasca, Red Villafranquina, Birchen Leonesa, White-Faced Spanish, Quail Castellana and Quail
Silver Castellana). One hundred forty four roosters (12 of each breed), all of which were one
year old at the beginning of the experiment were used for the collection of semen. In addition,
hens of White Prat and Black-Red Andaluza breeds (30 hens per breed) were later used for
insemination experiments. All animals were housed under natural photoperiod and tempera-
ture conditions in two 12 m
2
sand-floor pens with partial roof cover at the El Encı
´n Research
Station (Madrid, Spain, 40˚ 31’ N). These birds were raised as part of the INIA’s genetic
resources conservation program [28,29]. All birds were fed a commercial feed containing 16%
CP, 2700 kcal of ME/kg, 3.5% Ca and 0.5% available P over the entire experimental period.
Animals were handled according to procedures approved by the INIA Ethics Committee
(O
´rgano Regulador de los Comite
´s de E
´tica de Experimentacio
´n Animal, reference number
ORCEEA 2016/001) and were performed in accordance with the Spanish Policy for Animal
Protection (RD53/2013), which conforms to European Union Directive 86/609 regarding the
protection of animals used in scientific experiments.
Experimental design
The amino acid profile and total proteins in seminal plasma were studied within each breed. A
pool of seminal plasma for each breed was obtained every month, from August to November,
and amino acid and total protein analysed in them (n = 48; 4 per breed). Mean sperm concen-
trations were also evaluated within each breed.
To investigate the role of seminal plasma on cryoresistance of rooster sperm, diluted pooled
semen samples (n = 168; 14 replicates per breed x 12 breeds) collected from June to November,
were divided into two aliquots. One aliquot was frozen with presence of seminal plasma, and
the other one was frozen after removal of seminal plasma by centrifugation. Sperm variables
were analysed before and after freezing-thawing.
The fertilization capacity of frozen semen from one ‘good freezer’ and one ‘bad freezer’
breed, as judged from the obtained in vitro post-thaw sperm assessments, was estimated in an
artificial insemination (AI) experiment from the percentage fertile eggs resulting from two
consecutive intravaginal AI, three days apart, of a total of 60 hens (30 per breed). Hens of each
breed were used for testing fertilizing ability of frozen-thawed semen of the two mentioned
breeds; i.e. 15 hens belonging to good freezer breed were inseminated with semen of good
freezer, and the remaining 15 with semen of bad freezer; the same criterion was used for the
hens of bad freezer breed.
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 [30]. Pools of semen
for each breed were made on each occasion. Samples were managed differently, depending on
whether they were used for seminal plasma amino acid analysis, or for freezing. Seminal
plasma for amino acid assay was obtained by centrifugation of pooled raw semen at 1400g for
30 min. The plasma was evaluated by microscopy to ensure the absence of cells. If any cell
were seen, a second centrifugation was made. The pellet was discarded. When semen samples
were used in freezing experiments, each pool of semen was immediately diluted 1:1 (v/v) at
Seminal plasma and sperm freezability
PLOS ONE | https://doi.org/10.1371/journal.pone.0209910 January 4, 2019 3 / 19
field temperature using a Lake-Ravie medium [31] composed of sodium glutamate (1.92 g),
glucose (0.8 g), magnesium acetate 4H
2
O (0.08 g), potassium acetate (0.5 g), polyvinylpyrroli-
done (M
r
10 000; 0.3 g) and 100 mL H
2
O (final pH 7.08, final osmolality 343 mOsm/kg; here-
inafter referred to as Lake and Ravie medium). This diluted, pooled semen was then
immediately placed at 5˚C, transported to the laboratory, and sperm concentration and sperm
variables (sperm motility variables, plasma membrane integrity) examined (within 45 min of
collection). Afterward, each pool was divided into two aliquots. One aliquot, diluted as
required with Lake and Ravie medium to a concentration of 1200 ×10
6
sperm/mL (aliquot
with presence of seminal plasma). In the other one (aliquot without seminal plasma) the semi-
nal plasma was removed by dilution with Lake-Centri diluent (1:4 v/v) and centrifugation at
600 g during 20 min prior to freezing. Briefly, the Lake-Centri medium was composed of 1000
ml H
2
O, 1.28 g potassium citrate tribasic monohydrate, 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 hydroxide (340–350 mOsm/kg, pH = 7.0–7.2). The pellet obtained was
reconstituted with Lake-Ravie medium. Both aliquot with and without seminal plasma were
diluted with Lake-Ravie medium to a final concentration of 1200 ×10
6
sperm/mL. Pure
(99%) glycerol (GLY) was then added to the diluted samples, to leave a final 8% concentra-
tion (vol/vol), and equilibrated for 10 min at 5˚C. After equilibration, the samples were loaded
into 0.25 mL French straws and then frozen in two steps, i.e., from 5˚C to 35˚C at 7˚C/min,
and then from 35˚C to 140˚C at 60˚C/min [32]. Freezing was performed using a Computer
Freezer-Icetube 1810 freezer unit (Minitu¨b, Tiefenbach, Germany). The frozen straws were
then plunged into and maintained in liquid nitrogen (at -196˚C) until thawing. For thawing,
the straws were warmed for 3 min in a water bath at 5˚C.
Amino acid and total protein assay
Seminal plasma obtained for amino acid assay (see above) was immediately stored at -20˚C
until determination of the seminal plasma free amino acid composition. Separation and deter-
mination of amino acid was made by ion exchange column chromatography [33,34]. Briefly,
the samples were initially precipitated with three volumes of ice-cold acetone and incubated
for 2h at—20˚C. After centrifugation, the supernatant was removed and freeze dried in a speed
vac. The pellet was redissolved in citrate buffer and applied to an ion exchange chromatogra-
phy amino acid analyzer (Biochrom 30) using post column derivatization with ninhydrin. The
ninhydrin reacts with amino acids forming a dye complex. Total protein was assessed by the
Coomassie (Bradford) Protein Assay Kit (Thermo Scientific). The seminal plasma was diluted
10 times with Milli-Q water, then, 0.03 mL of the diluted plasma was mixed with 1.5 mL of the
Coomassie reagent. The samples were incubated 10 min at room temperature and were ana-
lysed by measuring the absorbance at 595 nm (Agilent 8453 Spectrophotometer). The protein
concentration was determined by a BSA standard curve with a linear working range of 25–
500 μg/mL.
Assessment of sperm variables
Sperm concentration and motility were assayed using a computer-aided sperm analyses
(CASA) system coupled to a phase contrast microscope (Nikon Eclipse model 50i; Nikon
Instruments Europe B.V., Izasa S.A.; negative contrast) and employing Sperm Class Analyzer
(SCA, Barcelona, Spain) v.4.0. software (Microptic S.L., Barcelona, Spain) [35]. For motility
analysis, sperm samples were diluted to a concentration of approximately 40 million sperm/ml
and loaded onto warmed (38˚C) 20 μm Leja 8-chamber slides (Leja Products B.V., Nieuw-
Vennep, The Netherlands). The percentage of motile spermatozoa and the percentage showing
Seminal plasma and sperm freezability
PLOS ONE | https://doi.org/10.1371/journal.pone.0209910 January 4, 2019 4 / 19
progressive motility were recorded. Sperm movement characteristics—curvilinear velocity
(VCL), straight-line velocity (VSL), average path velocity (VAP), amplitude of lateral head dis-
placement (ALH), and beat-cross frequency (BCF)—were also recorded. Three progression
ratios, expressed as percentages, were calculated from the velocity measurements described
above: linearity (LIN = VSL/VCL x 100), straightness (STR = VSL/VAP x 100), and wobble
(WOB = VAP/VCL x 100). A minimum of three fields and 500 sperm tracks were evaluated at
a magnification of 100x for each sample (image acquisition rate 25 frames/s).
Propidium iodide (PI) and SYBR-14 were used as fluorochromes in the examination of
membrane integrity [36]; 200 cells were examined using an epifluorescence microscope at
400×(wavelength: 450–490 nm).
All sperm variables were measured again for each pool after their eventual thawing. In addi-
tion, DNA integrity was also assessed in fresh sperm and after freezing-thawing by terminal
deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). For this, the kit “In Situ Cell
Death Detection” (Roche, Basel, Switzerland) was used following manufacturer’s instructions
with minor changes in order to adapt the technique to the analyses of rooster sperm. Briefly,
each sperm sample was diluted to 12 x 10
6
spermatozoa/mL in 4% paraformaldehyde. Subse-
quently 10 μL of this dilution were placed on a glass slide and left to dry. Then, the spermato-
zoa were permeabilized with 0.1% of Triton X-100 in PBS. After a wash in PBS, fragmented
DNA was nick end-labelled with tetramethylrhodamine-conjugated dUTP by adding 10 μL 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 37˚C. After a wash with PBS the nucleus were counterstained with Hoechst at
0.1mg/mL in PBS for 5 min in the dark. Following an additional wash with PBS the slides were
mounted using Fluoromount (Sigma-Aldrich, MO, USA) and observed under fluorescent
microscopy (Eclipse E200, Nikon, Japan). Percentages of positive TUNEL spermatozoa
(TUNEL+) per sample were recorded by counting a minimum of 200 spermatozoa per micros-
copy preparation, using an epifluorescence microscope at 400×(wavelength: 510–560 nm).
Cryoprotectant removal and artificial insemination
White Prat and Black-Red Andaluza breeds were chosen for the insemination trial as examples
of ‘good freezers’ and ‘bad freezers’, respectively on the basis of the post-thaw in vitro sperm
assessments. Glycerol was removed prior to AI. Straws of semen frozen were thawed, and the
thawed semen was progressively diluted with four volume parts of Lake Centri medium at 5˚C
by successively adding 0.07, 0.18, 0.33, 0.6, 1.24, and 1.58 volumes of medium to one volume
of semen (2 min intervals). These samples were then centrifuged at 600xg for 10 min, the
supernatant solution discarded, and the pellet resuspended (to the original volume of the
thawed semen) in Lake and Ravie medium (method adapted from Moce
´et al. [37]). All insem-
inations (see above) were performed between 12:00 h and 14:00 h. AI procedures involved 300
million sperm /female at each insemination. Eggs were collected from day two after the first AI
until 3 days after the second AI. Fertility (% fertile/incubated eggs) was determined by can-
dling the eggs (n = 144) on day 7 of incubation.
Statistical analyses
Clustering by the amino acid content in seminal plasma of each breed was performed using
the iterative k-technique to classify the amino acids into three clusters. Statistica software
(TIBCO Software Inc. Palo Alto, CA, USA) specifically uses Lloyd’s method to implement the
k-Means algorithm [38]. The right number of clusters was determined by a v-fold cross-valida-
tion algorithm included in the Statistica package. Briefly, this method divides the overall
Seminal plasma and sperm freezability
PLOS ONE | https://doi.org/10.1371/journal.pone.0209910 January 4, 2019 5 / 19
sample into a number of v folds (v value: The default value is 10, the minimum is 2, and the
maximum is 999). The same type of analysis is then successively applied to the observations
belonging to the v-1 folds (training sample), and the results of the analyses are applied to sam-
ple v (testing sample) to compute an index of "predictive validity". Variables with a skewed dis-
tribution were arcsine-transformed (sperm variables), log-transformed (proteins) or
submitted to box-cox transformation (amino acids) before statistical analysis. The influence of
breed on amino acid and total protein were analysed by one way ANOVA, following the statis-
tical model xij = m + Ai + eij, where xij = the measured variable (amino acid or total protein),
m = the overall mean of x, Ai = the effect of breed (i = 1–12), and eij = the residual (j = 1–4). A
Tukey post hoc analysis was performed to compare the differences between means of amino
acids. Correlations between amino acids and sperm TUNEL+ and between amino acids and
sperm viability were determined by the Spearman test; data of all breeds were included in the
correlation analysis. The influence of breed and seminal plasma on frozen-thawed sperm vari-
ables were analysed by ANOVA, following the statistical model xijk = m + Ai + Bj + ABij
+ eijk, where xijk = the measured sperm variable, m = the overall mean of variable x, Ai = the
effect of breed (i = 1–12), Bj = the effect of seminal plasma (j = 1–2), ABij = the interaction
between A and B, and eijk = the residual (k = 1–14). A post hoc Newman-Keuls analysis was
performed to compare the differences in mean sperm variable values between breeds and
treatments (with and w/o seminal plasma). Comparisons between fresh and frozen-thawed
sperm variables were made using a paired t-test. Identification of good and bad freezer was
made by clustering (k-means cluster analysis; see above) the differences between percentage of
sperm viability before and after freezing of each breed. The association among fertility rate and
semen from good and bad freezers was assessed using the Chi-squared test. Data were
expressed as means ±S.E. All statistical calculations were made using TIBCO Statistica soft-
ware v.13.3 (TIBCO Software Inc.).
Results
Seminal plasma amino acids, total protein content and sperm
concentration in different Spanish local chicken breeds
Seminal plasma content in amino acid in each breed is shown in Table 1. Glutamic acid was by
far the most abundant free amino acid in seminal plasma, accounting on average for more
than 80% of the total free amino acid molar content, its concentration being the highest
(P<0.001). Next to glutamine, the most abundant amino acids present in all breeds were ala-
nine, serine, valine, and glycine. Proline was relatively abundant in some breeds, but was
below detection limits in other breeds. Tryptophan was absent, or was present only in trace
quantities. The highest (P<0.05) concentration of alanine, proline, cysteine and arginine were
observed in Black-red Andaluza, Birchen Leonesa, White-Faced Spanish and Quail Silver Cas-
tellana, respectively. Buff Prat breed showed the lowest concentrations in these amino acids.
Grouping the amino acid in three clusters, within each breed, revealed that some breeds
showed the same pattern (Table 2). There was an effect of breed (P<0.05) on the seminal
plasma concentrations of total proteins. Significant differences (P<0.05) were found between
Buff Prat (the highest value) and Blue Andaluza, Black Castellana, Black-Barred Andaluza,
White Prat breeds (the lowest values) (Fig 1). Statistical analysis on sperm concentration
revealed significant differences (P<0.05) between Red-Barred Vasca (3556.3 x 10
6
spermato-
zoa/mL) and Quail Silver Castellana (1515.8 x 10
6
spermatozoa/mL). The overall mean sperm
concentration was 2204.9 (±107.03) x 10
6
spermatozoa/mL. No correlation was found
between plasma total protein concentration and sperm concentration.
Seminal plasma and sperm freezability
PLOS ONE | https://doi.org/10.1371/journal.pone.0209910 January 4, 2019 6 / 19
Table 1. Comparison of seminal plasma free amino acid concentrations (mean, range) between different breeds of chicken.
Free
amino
acids
(mM)
Black-Red
Andaluza
White-Faced
Spanish
Quail
Castellana
Quail Silver
Castellana
Black-Barred
Andaluza
Buff Prat White Prat Birchen
Leonesa
Red-Barred
Vasca
Black
Castellana
Red
Villafranquina
Blue
Andaluza
Asp 0.55
(0.40–0.82)
0.63
(0.46–0.94)
0.56
(0.28–0.80)
0.56
(0.48–0.62)
0.55
(0.18–0.94)
0.34
(0.26–0.50)
0.34
(0.28–0.42)
0.63
(0.40–0.90)
0.47
(0.34–0.56)
0.52
(0.39–0.70)
0.43
(0.27–0.56)
0.74
(0.30–1.36)
Thr 0.68
(0.58–0.92)
1.03
(0.80–1.50)
0.71
(0.42–0.98)
0.77
(0.38–1.42)
0.96
(0.26–1.68)
0.45
(0.23–0.56)
0.61
(0.42–0.96)
0.80
(0.62–1.02)
0.61
(0.22–1.02)
0.44
(0.20–0.58)
0.51
(0.17–1.08)
0.59
(0.48–0.70)
Ser 1.41
(1.28–1.64)
1.43
(1.36–1.54)
1.48
(1.04–1.84)
1.55
(1.42–1.68)
1.14
(0.58–1.40)
0.86
(0.40–1.14)
1.36
(1.18–1.60)
1.40
(1.22–1.64)
1.14
(0.42–1.48)
0.82
(0.35–1.18)
0.80
(0.32–1.28)
1.23
(0.66–1.62)
Glu 49,70
(39.14–66.54)
51.17
(41.52–58.90)
45.13
(38.66–49.54)
36.48
(21.24–44.82)
61.12
(47.6–94.64)
40.03
(29.15–55.58)
32.60
(21.38–39.72)
43.84
(23.92–66.54)
37.81
(27.55–44.14)
47.36
(29.55–66.10)
39.02
(27.39–52.08)
54.46
(28.22–85.80)
Gly 1.01
(0.88–1.18)
1.05
(0.96–1.18)
0.95
(0.72–1.22)
0.97
(0.78–1.28)
0.88
(0.36–1.18)
0.58
(0.28–0.80)
0.99
(0.76–1.34)
0.99
(0.82–1.22)
0.77
(0.31–1.00)
0.57
(0.25–0.74)
0.66
(0.30–1.02)
0.91
(0.84–1.00)
Ala 1.73
a
(1.54–1.90)
1.59
(1.42–1.72)
1.37
(1.00–1.62)
1.40
(1.34–1.52)
1.23
(0.72–1.52)
0.99
b
(0.42–1.38)
1.34
(1.20–1.52)
1.53
(1.24–1.72)
1.17
(0.43–1.54)
0.90
b
(0.39–1.24)
0.82
b
(0.33–1.36)
1.32
(0.60–1.65)
Cys 0.13
(0.00–0.28)
0.32
a
(0.28–0.40)
0.26
ab
(0.20–0.32)
0.26
ab
(0.22–0.32)
0.25
ab
(0.18–0.30)
0.00
c
(0.00–0.00)
0.27
ab
(0.24–0.28)
0.26
ab
(0.24–0.30)
0.06
bc
(0.00–0.24)
0.17
(0.00–0.26)
0.10
bc
(0.00–0.28)
0.27
ab
(0.24–0.36)
Val 1.22
(0.90–1.44)
1.07
(0.98–1.12)
1.06
(0.68–1.42)
1.26
(1.00–1.44)
0.65
(0.28–0.96)
0.89
(0.40–1.20)
1.04
(0.94–1.20)
0.96
(0.66–1.20)
0.89
(0.45–1.16)
0.70
(0.45–0.90)
0.74
(0.31–1.18)
0.84
(0.48–1.12)
Met 0.30
(0.22–0.38)
0.30
(0.16–0.38)
0.25
(0.08–0.34)
0.34
(0.30–0.38)
0.14
(0.04–0.30)
0.19
(0.02–0.36)
0.32
(0.30–0.36)
0.24
(0.12–0.32)
0.18
(0.12–0.26)
0.20
(0.08–0.28)
0.19
(0.10–0.28)
0.14
(0.00–0.28)
Ile 0.32
(0.20–0.48)
0.36
(0.26–0.46)
0.37
(0.18–0.58)
0.41
(0.32–0.56)
0.26
(0.12–0.40)
0.15
(0.04–0.32)
0.33
(0.22–0.38)
0.26
(0.18–0.36)
0.19
(0.08–0.30)
0.13
(0.04–0.20)
0.22
(0.04–0.46)
0.23
(0.06–0.34)
Leu 0.53
(0.44–0.68)
0.54
(0.46–0.64)
0.61
(0.42–0.88)
0.66
(0.46–0.90)
0.40
(0.18–0.50)
0.30
(0.14–0.42)
0.52
(0.38–0.64)
0.51
(0.44–0.56)
0.37
(0.12–0.52)
0.27
(0.12–0.38)
0.30
(0.09–0.56)
0.43
(0.14–0.62)
Tyr 0.22
(0.18–0.28)
0.25
(0.22–0.30)
0.27
(0.18–0.36)
0.33
(0.24–0.42)
0.21
(0.10–0.30)
0.15
(0.06–0.20)
0.27
(0.22–0.30)
0.28
(0.24–0.34)
0.18
(0.06–0.22)
0.12
(0.05–0.20)
0.13
(0.03–0.26)
0.26
(0.06–0.42)
Phe 0.34
(0.26–0.40)
0.34
(0.30–0.38)
0.34
(0.24–0.44)
0.40
(0.32–0.58)
0.32
(0.26–0.38)
0.19
(0.12–0.22)
0.32
(0.24–0.44)
0.35
(0.30–0.38)
0.27
(0.10–0.36)
0.24
(0.11–0.34)
0.20
(0.00–0.36)
0.25
(0.00–0.40)
His 0.33
(0.26–0.38)
0.41
(0.34–0.50)
0.27
(0.20–0.30)
0.35
(0.32–0.36)
0.26
(0.14–0.34)
0.22
(0.08–0.30)
0.34
(0.32–0.34)
0.38
(0.28–0.42)
0.31
(0.08–0.42)
0.19
(0.06–0.28)
0.21
(0.07–0.36)
0.32
(0.08–0.46)
Lys 0.17
(0.12–0.22)
0.32
(0.20–0.62)
0.23
(0.10–0.36)
0.30
(0.14–0.42)
0.23
(0.04–0.42)
0.14
(0.06–0.20)
0.30
(0.26–0.38)
0.25
(0.20–0.38)
0.14
(0.06–0.20)
0.17
(0.10–0.20)
0.21
(0.06–0.48)
0.32
(0.10–0.66)
Arg 0.37
(0.28–0.44)
0.59
(0.50–0.64)
0.63
(0.30–1.00)
0.73
a
(0.44–0.90)
0.32
c
(0.10–0.42)
0.29
c
(0.26–0.32)
0.61
(0.40–0.76)
0.55
(0.44–0.62)
0.37
(0.20–0.46)
0.30
bc
(0.19–0.36)
0.26
c
(0.11–0.44)
0.70
ab
(0.48–0.84)
Pro 0.91
(0.00–1.31)
0.32
(0.00–1.28)
0.72
(0.00–1.19)
1.21
(0.00–2.50)
0.00
b
(0.00–0.00)
0.01
b
(0.00–0.04)
1.13
(0.91–1.60)
1.36
a
(0.99–2.20)
0.62
(0.00–1.33)
0.00
b
(0.00–0.00)
0.00
b
(0.00–0.00)
0.67
(0.00–1.47)
Total 59.88
(47.06–77.97)
61.68
(50.76–70.62)
55.19
(45.66–60.83)
47.94
(36.18–53.02)
68.86
(56.60–103.06)
45.76
(32.17–62.80)
42.66
(33.66–48.84)
54.54
(32.63–78.98)
45.52
(30.54–54.01)
53.12
(32.33–73.06)
44.78
(29.97–60.32)
63.643
(37.81–92.42)
Aspartic acid (Asp), Threonine (Thr), Serine (Ser), Glutamic acid (Glu), Glycine (Gly), Alanine (Ala), Cysteine (Cys), Valine (Val), Methionine (Met), Isoleucine (Ile), Leucine (Leu), Tyrosine
(Tyr), Phenylalanine (Phe), Histidine (His), Lysine (Lys), Arginine (Arg), Proline (Pro). N = 48 (4 samples/breed).
a,b,c
Different letters within rows indicate significant differences between breeds.
https://doi.org/10.1371/journal.pone.0209910.t001
Seminal plasma and sperm freezability
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Sperm viability
There were no significant differences in fresh sperm viability between breeds. The removal of
the seminal plasma by centrifugation reduced the sperm viability in seven of the 12 breeds
(Black-Red Andaluza, White-Faced Spanish, Buff Prat, Birchen Leonesa, Red-Barred Vasca,
Black Castellana, and Red Villafranquina; Fig 2). Despite the negative effect of centrifugation
in seven breeds, the post-thaw sperm viability was not significantly affected by the removal of
seminal plasma in all breeds, with the only exception of Red Villafranquina breed (P<0.05)
(Fig 2). There were no significant differences between breeds in the percentage of viable sperm
after freezing-thawing in samples without seminal plasma. In contrast, a significant effect of
the breed was found (P<0.05) on the percentage of viable sperm after freezing-thawing in
samples with seminal plasma. Cluster analysis in these last samples, carried out with the values
obtained from the differences between the percentages of sperm viability before freezing and
after thawing of each breed, revealed that White Prat, Black Castellana, Blue Andaluza, Quail
Castellana, and Red-Barred Vasca returned the best freezing-thawing response (good freezers:
mean sperm viability after freezing-thawing in samples with seminal plasma: 63.4±1.5%; dif-
ference between fresh and post-thawed sperm viability with plasma: 14.7±0.9%), whereas the
worst response was found in Black-Red Andaluza, Black-Barred Andaluza, White-Faced Span-
ish and Buff Prat (bad freezers: mean sperm viability after freezing-thawing in samples with
seminal plasma: 54.8±2.5%; difference between fresh and post-thawed sperm viability with
plasma: 25.6±2.3%).
The differences in sperm freezing-thawing response were not correlated with protein con-
centrations. In frozen-thawed semen samples with seminal plasma, there was a positive corre-
lation between sperm viability and the concentrations of valine, isoleucine, lysine and leucine
(Fig 3).
Sperm motility
The removal of seminal plasma did not significantly affected sperm motion parameters mea-
sured in frozen-thawed samples in the majority of breeds. In particular, the removal of seminal
plasma significantly decreased progressive motility (P<0.05) and VCL (P<0.05) in Black-
Table 2. Clusters of seminal plasma free amino acid concentrations in different breeds of chicken. Amino acid concentrations in cluster 1 >cluster 2 >cluster 3. Sim-
ilar number of asterisks for each breed indicates the same amino acids in the three clusters.
Breed Cluster 1 Cluster 2 Cluster 3
Black-Red AndaluzaGlu Ser, Gly, Ala, Val, Pro Asp, Thr, Cys, Met, Ile, Leu, Tyr, Phe, His, Lis, Arg
Quail Silver CastellanaGlu Ser, Gly, Ala, Val, Pro Asp, Thr, Cys, Met, Ile, Leu, Tyr, Phe, His, Lys, Arg
White PratGlu Ser, Gly, Ala, Val, Pro Asp, Thr, Cys, Met, Ile, Leu, Tyr, Phe, His, Lys, Arg
Birchen LeonesaGlu Ser, Gly, Ala, Val, Pro Asp, Thr,Cys, Met, Ile, Leu, Tyr, Phe, His, Lys, Arg
Blue AndaluzaGlu Ser, Gly, Ala, Val, Pro Asp, Thr, Cys, Met, Ile, Leu, Tyr, Phe, His, Lys, Arg
Quail Castellana Glu Ser, Gly, Ala, Val Asp, Thr, Cys, Met, Ile, Leu, Tyr, Phe, His, Lys, Arg, Pro
Buff Prat Glu Ser, Gly, Ala, Val Asp, Thr, Cys, Met, Ile, leu, Tyr, Phe, His, Lys, Arg, Pro
White-Faced SpanishGlu Thr, Ser, Gly, Ala, Val Asp, Cys, Met, Ile, Leu, Tyr, Phe, His, Lys, Arg, Pro
Red VillafranquinaGlu Thr, Ser, Gly, Ala, Val Asp, Cys, Met, Ile, Leu, Tyr, Phe, His, Lys, Arg, Pro
Black Castellana Glu Asp, Thr, Ser, Gly, Ala, Val Cys, Met, Ile, Leu, Tyr, Phe, His, Lys, Arg, Pro
Black-Barred Andaluza Glu Thr, Ser, Gly, Ala Asp, Cys, Val, Met, Ile, Leu,Tyr, Phe, His, Lys, Arg, Pro
Red-Barred Vasca Glu Thr, Ser, Gly, Ala, Val, Pro Asp, Cys, Met, Ile, leu, Tyr, Phe, His, Lys, Arg
Aspartic acid (Asp), Threonine (Thr), Serine (Ser), Glutamic acid (Glu), Glycine (Gly), Alanine (Ala), Cysteine (Cys), Valine (Val), Methionine (Met), Isoleucine (Ile),
Leucine (Leu), Tyrosine (Tyr), Phenylalanine (Phe), Histidine (His), Lysine (Lys), Arginine (Arg), Proline (Pro). N = 48 (4samples/breed).
https://doi.org/10.1371/journal.pone.0209910.t002
Seminal plasma and sperm freezability
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Barred Andaluza, and total (P<0.05) and progressive motility (P<0.05) in Quail Silver Castel-
lana; in contrast, the removal of seminal plasma significantly increased VSL (P<0.05) in Red
Villafranquina (Fig 4). Significant correlations between amino acid concentrations and motil-
ity sperm variables were not found.
Sperm DNA fragmentation
The TUNEL assay revealed that fresh sperm from White-Faced Spanish had the highest degree
of DNA fragmentation being statistically higher (P<0.05) than most of the breeds (Fig 5). We
found that the presence of seminal plasma increased DNA fragmentation during freezing/
thawing in most of the breeds (Fig 5). Only in Quail Castellana breed (with low DNA fragmen-
tation in fresh sperm) and White-Faced Spanish (with high DNA fragmentation in fresh
sperm) there were no differences in the percentage of TUNEL + sperm between fresh and fro-
zen samples with seminal plasma (Fig 5). DNA fragmentation in Blue Andaluza was less
(P<0.05) than in White-Faced Spanish and Birchen Leonesa for samples cryopreserved with-
out seminal plasma.
In fresh samples, cysteine was the only amino acid with a positive correlation with %
TUNEL+ sperm (i.e. DNA damaged sperm; rs = 0.43, P<0.05). In frozen-thawed semen sam-
ples with plasma, there was a negative correlation between the values of post-thaw TUNEL
+ and certain amino acids such as alanine, valine, isoleucine, methionine, leucine, tyrosine,
phenylalanine, serine (i.e. a positive correlation of the concentration of these amino acids with
integrity of DNA) (Fig 6).
Fig 1. Comparison of seminal plasma protein concentrations (mean ±SE) between different breeds of chicken.
Different letters (a,b,c) indicate significant differences (p<0.05). N = 48 (4 samples/breed).
https://doi.org/10.1371/journal.pone.0209910.g001
Seminal plasma and sperm freezability
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Fertility
The evaluation of fertilization capacity of frozen-thawed semen from the breeds White Prat
and Black-Red Andaluza, as examples of ‘good freezers’ and ‘bad freezers’, respectively,
showed that the good freezer had higher fertility (20/68, 29.4%) compared to bad freezer breed
(14/76, 18.4%), even if the difference was not significant (P = 0.08).
Discussion
In the present study, we showed that seminal plasma and breed differences in seminal amino
acids contents affect the results of chicken sperm cryopreservation. There was a relationship
between concentrations of some amino acids (i.e. valine and leucine that are common for all
criteria) and sperm viability and DNA integrity after freezing-thawing irrespective of the
breed. Although the deleterious effect of freezing-thawing on the DNA integrity of chicken
sperm has been previously reported [39,40,41], herein we show for the first time that there is
differential susceptibility of the DNA to the cryoinjury depending on the chicken breed.
Despite a frequent harmful effect of centrifugation, the removal of seminal plasma before
freezing allowed to decrease the DNA fragmentation damages induced by cryopreservation
and allowed to reduce the breed variability effect on post-thaw sperm viability.
Differences between breeds were found with respect to seminal free amino acid concentra-
tions, and some of these differences were relevant regarding sperm cryoprotection. Glutamic
acid was the main amino acid in seminal plasma, accounting for 76–89% (on molar basis) of
Fig 2. Viable sperm in fresh samples, after centrifugation and frozen-thawed samples without (w/o/p) or with (w/p) seminal plasma. Different
letters (a,b) within each breed, indicates significant differences (p<0,05). Lines over bars (Frozen w/p) indicate significant differences between breeds
(p<0,05; p<0,01). N = 168 (14 samples/breed).
https://doi.org/10.1371/journal.pone.0209910.g002
Seminal plasma and sperm freezability
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the total amino acid content. This is in agreement with earlier observations in cock semen
[42]. Glutamate is thought to serve as the main anion in place of Cl- [43]. In addition, gluta-
mate may act as a motility agonist when sperm is co-incubated with Ca2+ under aerobic con-
ditions [44]; it has been suggested that rooster sperm express glutamate channels that mediate
the flux of Ca2+ and K+ at the mitochondria membrane levels, and hence contributing to
sperm kinetics [44,45]. We didn’t find any relationship between glutamate and motility vari-
ables between breeds. The other amino acids were present in much lower concentrations.
Excluding glutamic acid, Ahluwalia and Graham [46] reported that arginine, asparagine, thre-
onine, and glycine are the most prevalent in chicken seminal plasma of mixed breed. Aspartic
acid is the second most abundant amino acid in Delaware and New Hampshire rooster [47],
and in Brown Leghorn [48]. Our data showed that apart from glutamine, the most abundant
amino acids present in all breeds studied here were alanine, serine, valine, and glycine, but that
there were different clusters of specific seminal plasma amino acids levels depending on the
Fig 3. Correlation between sperm viability and amino acids concentrations. Sperm viability corresponds to the percentage of sperm with intact membrane in frozen-
thawed samples with seminal plasma. The amino acids concetration includes all chicken breeds. rs, Spearman rank correlation coefficient.
https://doi.org/10.1371/journal.pone.0209910.g003
Seminal plasma and sperm freezability
PLOS ONE | https://doi.org/10.1371/journal.pone.0209910 January 4, 2019 11 / 19
breed. This highlights the role of genetic components (breed differences) on the free amino
acid concentrations of rooster seminal plasma.
The use of glutamine as a component of the freezing medium resulted in a higher
post-thaw motility in human [23] and rooster [49] sperm. Glutamine, glycine and cyste-
ine as additives in conventional freezing medium enhanced post-thaw motility and
improved membrane and acrosome integrity of buffalo bull semen [50]. Addition of
Fig 4. Motility sperm variables in fresh and frozen-thawed samples without (w/o/p) or with (w/p) seminal plasma. Different letters (a,b) within each
breed indicates significant differences (p<0,05). N = 168 (14 samples/breed).
https://doi.org/10.1371/journal.pone.0209910.g004
Seminal plasma and sperm freezability
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glutamine and proline improved sperm motility variables, membrane and acrosome
integrity in frozen-thawed goat sperm [51]. Although the cryoprotectant mechanism of
amino acids is not well known, some hypotheses have been provided in this way. Amino
acids might interact with phospholipids bilayers during freezing [52] allowing stabilizing
the cell membrane. In addition, they might protect sperm during freezing by colligative
action through their unspecific ability to reduce the concentration of toxic solutes below
the limit of toxicity [53]. Some amino acids, such as proline, might act as a solute protect-
ing the cell against the denaturing effects of hyperosmolality induced by dehydration
during slow freezing [26].
We found a positive association between the seminal plasma concentration of the hydro-
phobic amino acids valine, isoleucine and leucine and the charged lysine with membrane
integrity as measured by the viability assay. It remains unknown whether these associations
point to a casual effect, i.e. a cryoprotective effect of these amino acids. This could be tested in
future experiments by including these amino acids in varying concentrations in the freezing
medium.
There were no significant differences between breeds in the percentage of sperm viability
after freezing-thawing of samples without seminal plasma, whereas there was an influence of
breed on the percentage of viable sperm after freezing-thawing of samples with seminal
plasma. This suggests a role of seminal plasma to prevent or to favor cryoinjury during
Fig 5. TUNEL + in fresh and frozen-thawed samples without (w/o/p) or with (w/p) seminal plasma. Different
letters (a,b) within each breed indicate significant differences (P<0,05). Different letters (A,B) between breeds indicate
significant differences (P<0,05) in fresh samples. Lines over bars (Frozen w/o/p) indicate significant differences
between breeds. N = 168 (14 samples/breed).
https://doi.org/10.1371/journal.pone.0209910.g005
Seminal plasma and sperm freezability
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freezing-thawing process. The influence of factors that may vary between breeds other than
seminal plasma amino acids should not be ruled out. For instance, Blesbois and de Reviers [8],
reported that low molecular weight seminal plasma fractions can reduce the fertilizing ability
of sperm during storage at 4˚C, whereas high molecular weight fractions appeared to enhance
fertilizing ability. Seminal plasma is involved in degradation of sperm phospholipids, possibly
by phospholipase activity, accelerating the sperm damage of turkey sperm during in vitro stor-
age [10]. Genetic variations in seminal plasma proteins expression [14,54] or other plasma
components, may thus affect the freezability of the semen of a breed.
The decreasing of sperm viability in samples frozen without plasma seems mainly due to
damage during centrifugation. Interestingly, we found that, whereas the presence of seminal
plasma did not seem to affect the sperm survival (% viable), as the relative decrease of viability,
i.e. post-thaw compared with pre-freeze, was not different between semen frozen with and with-
out plasma, the DNA was less damaged in the latter. This could reflect different mechanisms of
Fig 6. Correlation between sperm TUNEL + and amino acids concentrations. TUNEL + corresponds to the percentage of sperm with DNA fragmentation in frozen-
thawed samples with seminal plasma. The amino acids concetration includes all chicken breeds. rs, Spearman rank correlation coefficient.
https://doi.org/10.1371/journal.pone.0209910.g006
Seminal plasma and sperm freezability
PLOS ONE | https://doi.org/10.1371/journal.pone.0209910 January 4, 2019 14 / 19
damage; plasma membrane integrity may predominantly be damaged by temperature- and
dehydration-dependent membrane phase transitions [55], along with mechanical forces associ-
ated with ice formation and shrinking of the cells during freezing, while DNA might be more
sensitive to the generation of reactive oxygen species (ROS) [56]. Possibly, this latter mechanism
is stimulated by the presence of seminal plasma. The overproduction of ROS which exceeds the
seminal plasma antioxidant capacity disturbs the balance between seminal ROS and antioxidant
capacity, and results in oxidative stress. Oxidative damage may originate from several potential
resources from seminal plasma, such as leukocytes [57] and presence of immature sperm. In
addition, our findings suggest that others non-identified components of the seminal plasma of
chickens might stimulate excessive generation of ROS by sperm and/or strongly decrease the
levels of antioxidant defenses [58] during freezing-thawing process.
Negative correlations were seen between post-thaw %TUNEL+ sperm frozen with seminal
plasma and the concentrations in seminal plasma of specific amino acids: alanine, valine, isoleu-
cine, methionine, leucine, tyrosine, phenylalanine, serine, most of which are of hydrophobic
nature. Thus, it could be expected that incorporating some of these amino acids in freezing
media could decrease DNA damage during freeze-thawing. It was indeed reported that the
incorporation of methionine to bovine [59] and fish [60] freezing media reduced the loss of
DNA integrity during freezing and thawing. Accordingly, supplementing chicken extenders with
the non-coded amino acid taurine, showed also a positive effect in reducing sperm apoptosis and
DNA damage [61]. Although cysteine concentrations weren’t correlated with DNA integrity in
frozen-thawed sperm with seminal plasma, we found that cysteine was the only amino acid posi-
tively correlated with DNA damage in fresh samples, and White-Faced Spanish, the breed with
the highest value of cysteine, showed the highest values of damage in DNA of samples in fresh
plasma. However, supplementation of extenders with L-cysteine for the cryopreservation of carp
sperm reported a decrease in DNA damage in post-thawed sperm [62] and incorporating cyste-
ine to buffalo extenders did not significantly affect the DNA damage [63]. Thus, the protective
effect of the amino acids on DNA during cryopreservation might vary among species, possibly
related to the different packaging and ultrastructure of the chromatin. For example, carp [64]
and other fish species [65] maintain a nucleosome organization of the sperm chromatin whereas
in mammals and birds [65,66] there is a substitution of the histones for protamines during sper-
matogenesis in order to increase the chromatin compaction and the protection of the DNA. In
addition, the chromatin of rooster sperm contains no cysteine residues [65] and lacks the poten-
tial stabilizing effect of S-S bonds of mammalian sperm chromatin.
The seminal plasma protein concentrations differed between breeds, in agreement with
previous studies in other chicken breeds [47]. While significant differences were found
between sperm concentration of two of the twelve breeds studied, these sperm concentrations
did not seem related to the respective total plasma protein concentration, and over all breeds
there was no correlation between both variables. However, these differences did not seem
related with the observed differences in sperm cryodamage.
In conclusion, the results suggest that the decreasing of sperm viability in samples frozen
without seminal plasma is largely due to damage during centrifugation. Our findings indicate
that removal of seminal plasma did not seem to affect sperm survival during freezing and
thawing, but did clearly reduce DNA damage of sperm. Specific amino acids were associated
with post-thaw percentage of viable sperm and DNA integrity.
Supporting information
S1 Dataset. Amino acids profile in seminal plasma of 12 Spanish rooster breeds.
(PDF)
Seminal plasma and sperm freezability
PLOS ONE | https://doi.org/10.1371/journal.pone.0209910 January 4, 2019 15 / 19
S2 Dataset. Protein concentrations in seminal plasma of 12 Spanish rooster breeds.
(PDF)
S3 Dataset. Viability and concentration of fresh sperm. Sperm viability of frozen sperm with
plasma and without plasma of 12 Spanish rooster breeds.
(PDF)
S4 Dataset. Motility variables of sperm of 12 Spanish rooster breeds (fresh, frozen with
plasma and frozen without plasma).
(PDF)
S5 Dataset. Tunel + of sperm of 12 Spanish rooster breeds (fresh, frozen without plasma
and frozen with plasma).
(PDF)
Author Contributions
Conceptualization: Elisabeth Blesbois.
Data curation: Cristina Castaño.
Formal analysis: Julia
´n Santiago-Moreno, Berenice Bernal, Serafı
´n Pe
´rez-Cerezales, Cristina
Castaño, Henri Woelders, Elisabeth Blesbois.
Funding acquisition: Julia
´n Santiago-Moreno, Milagros C. Esteso, Antonio Lo
´pez-Sebastia
´n,
Elisabeth Blesbois.
Investigation: Julia
´n Santiago-Moreno, Adolfo Toledano-Dı
´az, Milagros C. Esteso, Alfonso
Gutie
´rrez-Ada
´n, Antonio Lo
´pez-Sebastia
´n, Marı
´a G. Gil, Henri Woelders, Elisabeth
Blesbois.
Methodology: Julia
´n Santiago-Moreno, Berenice Bernal, Serafı
´n Pe
´rez-Cerezales, Cristina
Castaño, Adolfo Toledano-Dı
´az, Alfonso Gutie
´rrez-Ada
´n, Marı
´a G. Gil.
Project administration: Julia
´n Santiago-Moreno.
Supervision: Julia
´n Santiago-Moreno.
Writing – original draft: Julia
´n Santiago-Moreno, Berenice Bernal, Serafı
´n Pe
´rez-Cerezales,
Cristina Castaño, Milagros C. Esteso, Alfonso Gutie
´rrez-Ada
´n, Antonio Lo
´pez-Sebastia
´n,
Marı
´a G. Gil, Henri Woelders, Elisabeth Blesbois.
Writing – review & editing: Julia
´n Santiago-Moreno.
References
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PMID: 20092881
2. Lo
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´rez A, Pe
´rez-Clariget R. Ram seminal plasma improves pregnancy rates in ewes cervically
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Seminal plasma and sperm freezability
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... However, glycerol, the commonly sperm cryoprotectant used in many mammal species for sperm freezing appears to have contraceptive effects on most avian species (Hammerstedt and Graham, 1992). Glycerol has been successfully used for cryopreservation of black-footed and gentoo penguins (Santiago-Moreno et al., 2019a). Although the use of glycerol in sperm diluents is usually associated with high frozen-thawed sperm motility and viability, it leads a low fertility because its contraceptive effects caused by alteration of sperm storage at the at the uterovaginal junction and/or alteration of transport through the genital tract (Abouelezz et al., 2015a) Thus, when using glycerol, the semen cannot be inseminated right from the straw. ...
... Semen samples were collected during each breeding season once a week during two consecutive years, between April and June from gentoo penguins and between October and December from black-footed penguins. The Burrows and Quinn technique (Burrows and Quinn, 1937) adapted to this species was used to collect semen samples (Santiago-Moreno et al., 2019a). Briefly, an individual skilled in handling penguins held the animal in a wooden U-shaped cradle, holding the legs with one hand, and softly immobilizing body and wings with the other. ...
... Even though there were no differences in fresh sperm, both cryoprotectants returned better results for many sperm characteristics in gentoo than in black-footed penguins. Our results support previous results (Santiago-Moreno et al., 2019a) showing the black-footed penguin sperm to be more sensitive to the freezing-thawing process than gentoo sperm. The susceptibility of sperm to damage during cryopreservation differs among penguin species and many factors might explain this finding, such as interspecies differences in sperm head morphometric and morphological traits (Santiago-Moreno et al., 2016), variations the composition phospholipid content in plasma membranes (Ushiyama et al., 2016) and in the components of seminal plasma amino acids (Santiago-Moreno and Blesbois, 2020). ...
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Black-footed penguins (Spheniscus demersus) are classified as endangered, and the populations of gentoo penguins (Pygoscelis papua) are rapidly decreasing. The optimization of semen cryopreservation in these species, for preserving their genetic diversity in genome resource banks, is essential for the success of captive breeding programs. This study compares the effectiveness of two permeating cryoprotectants, dimethylacetamide (DMA) and dimethylsulfoxide (DMSO), on frozen–thawed sperm characteristics. Semen samples were collected during each breeding season once a week during two consecutive years. Semen samples were packaged in 0.25 ml straws and frozen by placing them in nitrogen vapors. After thawing, sperm motility characteristics were examined by computer-assisted sperm analysis. Propidium iodide and SYBR-14 were used as fluorochromes for the examination of membrane integrity. DNA integrity was evaluated by TUNEL assay. Gentoo sperm characteristics after freeze-thawing did not show any differences when using DMSO or DMA. In black-footed samples, progressive motility, curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and straightness (STR) were greater using 8% DMSO (P < 0.05) than 6% DMA. The cryoresistance ratio (CR) using 8% DMSO was greater (P < 0.05) in gentoo than black-footed samples for CR-VCL and CR-VAP, and 6% DMA returned greater CR values (P < 0.05) than in black-footed samples for all characteristics evaluated. No differences were found in DNA fragmentation. In conclusion, the present results highlight the benefits of using 8% DMSO compared to 6% DMA in penguins. Sperm from black-footed showed a higher sensitivity to freezing-thawing process than gentoo sperm.
... Seminal plasma of human and other species contain large numbers of AAs (6,95). Velho et al. (95) identified 63 seminal plasma metabolites (of which 21 were AAs), demonstrating the relation of their profiles with low and high fertility seminal plasma of bulls. ...
... Oxidation of the AAs in semen, leads to energy supply, causing biochemical activities and reactions in semen (97). Glutamic acid (usually with an active glutamic oxaloacetic transaminase) is the most abundant AAs in seminal plasma (95). Differences between the concentrations of L-leucine and ornithine were found between the fertility groups, as well as the concentration of fructose was correlated with glutamic acid and amino-butyrolactone content (96). ...
... Despite that AAs has an influence on physiological processes in vivo, it was reported that addition of glutamine to human semen as a cryoprotectant agent increased post-thaw motility in sperm (101). It was reported that supplementation of extender solutions with glutamine, glycine, and cysteine AAs, increases acrosome and membrane integrity of bualo bull semen (102), and a positive correlation between the concentrations of valine, isoleucine, leucine, and lysine with membrane integrity were established (95). In addition, AAs plays a significant role in protection against oxidative stress (81,103). ...
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Male infertility is one of the many problems currently faced by science and medicine. Despite intensive research in this area conducted in recent years, the reasons for the lack of the desired pregnancy are often unrecognized. The current standards and general recommendations, including the World Health Organization (WHO) guidelines for diagnostic testing of male reproductive organs and sperm quality analysis, seem to be insufficient. Hence, it has been postulated for years that it is necessary to search for and identify new, unknown factors that significantly affect male fertility, and to define modern indicators/biomarkers that would enable precise determination of male reproductive potential. Among the many interesting recently published data, the information on the identification and expression analysis of aquaporins (AQPs) in the male reproductive system and metabolomic semen analysis is of particular interest. In this review, we will try to solve the question whether AQPs and metabolomic sperm analysis can be the answer to the current needs and whether their measurements may become a useful parameter in the future for determining male reproductive potential.
... Significant enrichment of unique proteins in poor quality spermatozoa was also observed in buffalo to isoleucine and leucine degradation pathways (Binsila et al., 2020). There was a positive correlation between membrane integrity and the concentrations of isoleucine (Santiago-Moreno et al., 2019). This suggests that isoleucine may also have a positive impact on sperm motility and structural integrity after thawing, but further research was need on isoleucine supplementation on sperm performance. ...
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Trehalose is commonly used as an impermeable cryoprotectant for cryopreservation of cells, but its cryoprotective mechanism has now not but been determined. This study investigated the cryopreservation impact of trehalose on buck semen cryopreservation and finished metabolic profiling of freeze-thawed media by way of the GC–MS-based metabolomics for the first time. Metabolic pattern recognition and metabolite identification by means of principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA) and metabolic pathway topology analysis revealed the results of trehalose on buck sperm metabolism at some point of cryopreservation. The results confirmed that trehalose drastically progressed sperm motility parameters and structural integrity after thawing. PCA and PLS-DA analysis discovered that the metabolic patterns of the freezing-thawing media of buck semen cryopreserved with trehalose (T group) or without trehalose (G group, Control) were certainly separated. Using screening conditions of VIP >1.5 and p vaule <0.05, a total of 48 differential metabolites have been recognized, whithin l -isoleucine, L-leucine, L-threonine, and dihydroxyacetone were notably enriched in valine, leucine and isoleucine biosynthesis, glycerolipid metabolism, and aminoacyl-tRNA biosynthesis pathways. In brief, trehalose can efficiently improve membrane structural integrity and motion parameters in buck sperm after thawing, and it exerts a cryoprotective impact with the aid of changing sperm amino acid synthesis and the glycerol metabolism pathway.
... In addition, amino acids could play a role in avian sperm function as shown for mammals: some amino acids participate in many metabolic processes involved in motility, acrosome reaction, and capacitation of human and other mammalian spermatozoa [35,36]. Amino acids also have antioxidant properties able to protect sperm cells from cold shock [37,38]; consistently, plasma amino acids seem to play a role in chicken sperm cryoresistance [39]. In mammals, it has been demonstrated that amino acids act at the extracellular level and improve sperm motility, acrosome integrity, and fertilizing potential after the freezing-thawing process [40][41][42][43][44]. ...
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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.
Chapter
Bull fertility is an important economic trait in sustainable cattle production, as infertile or subfertile bulls give rise to large economic losses. Current methods to assess bull fertility are tedious and not totally accurate. The massive collection of functional data analyses, including genomics, proteomics, metabolomics, transcriptomics, and epigenomics, helps researchers generate extensive knowledge to better understand the unraveling physiological mechanisms underlying subpar male fertility. This review focuses on the sperm phenomes of the functional genome and epigenome that are associated with bull fertility. Findings from multiple sources were integrated to generate new knowledge that is transferable to applied andrology. Diverse methods encompassing analyses of molecular and cellular dynamics in the fertility-associated molecules and conventional sperm parameters can be considered an effective approach to determine bull fertility for efficient and sustainable cattle production. In addition to gene expression information, we also provide methodological information, which is important for the rigor and reliability of the studies. Fertility is a complex trait influenced by several factors and has low heritability, although heritability of scrotal circumference is high and that it is a known fertility maker. There is a need for new knowledge on the expression levels and functions of sperm RNA, proteins, and metabolites. The new knowledge can shed light on additional fertility markers that can be used in combination with scrotal circumference to predict the fertility of breeding bulls. This review provides a comprehensive review of sperm functional characteristics or phenotypes associated with bull fertility.
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Background: The Cantabrian capercaillie (Tetrao urogallus cantabricus) is critically endangered. This subspecies has the lowest genetic variability and it is in regression. It belongs to Phasianidae family; therefore, the domestic chicken (Gallus gallus domesticus) could be a good model for developing reproductive technologies for use in capercaillie populations with low availability of animals. Objectives: In this study, we analyzed the response of capercaillie sperm to the freezing-thawing process for contributing to the development of a semen cryobank of Cantabrian capercaillie. Methods: We used domestic chicken as the animal model in order to obtain the freezing protocol before applying on capercaillie. In the first experiment, two different extenders (EK and LR84) and different concentrations [4% and 6% dimethyl-acetamide (DMA) v:v] of cryoprotectants were evaluated using in-straw freezing method in domestic chickens. A pilot study in capercaillie males, using the same conditions evaluated in chicken, was performed. Results: In chicken, we found that the LR84-4% DMA media provided the best results for freezing semen. In capercaillie study, LR84 extender seemed to be the most appropriate diluent and 4% was the better dose of DMA cryoprotectant agent. Further, based on previous studies carried out in rooster samples, we also tested the glycerol (8% v/v) as a cryoprotectant for capercaillie semen cryopreservation. Conclusions: Our results suggest that sperm from both domestic and wild species had a similar response to freezing-thawing processes. Mediterranean chickens may be used as a suitable model for developing sperm freezing protocols that can be extrapolated to threatened capercaillie populations. In addition, LR84 media with glycerol was the most efficient extender to freeze capercaillie sperm native.
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