G C A T

T A C G
G C A T
genes
Article
Effects of Heat Stress on Motion Characteristics and
Metabolomic Profiles of Boar Spermatozoa
Heming Sui 1,† , Shiqi Wang 2,† , Gang Liu 1 , Fei Meng 1 , Zubing Cao 2 and Yunhai Zhang 2, *

1 National Animal Husbandry Service, Beijing 100125, China

2 Anhui Province Key Laboratory of Local Livestock and Poultry, Genetical Resource Conservation and
Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China
* Correspondence: yunhaizhang@ahau.edu.cn; Tel.: +86-551-6578-6537
† These authors contributed equally to this work.

Abstract: Heat stress (HS) commonly causes boar infertility and economic loss in the swine indus-
try. The heat tolerance of boar semen presents obvious differences among individuals. However,
whether heat stress affects motion characteristics and the metabolome profile in boar sperm remains
unclear. In this study, the kinetic features of sperm from HS and non-HS (NHS) groups were de-
tected by computer-assisted sperm analysis, and metabolomic profiling was performed by liquid
chromatography–mass spectrometry. The results showed that heat stress significantly reduced sperm
motility, average path distance (APD), straight-line velocity (VSL), straightness (STR), and linearity
(LIN) (p < 0.05). A total of 528 and 194 metabolites in sperm were identified in the positive and
negative ion modes, respectively. Lipids and lipid-like molecules, and organic acids and derivatives
were major metabolic classes in the two modes. Furthermore, we separately identified 163 and
171 differential metabolites in the two modes between HS and NHS groups. Clustering analysis
further revealed significant metabolic changes in sperm after heat stress. The Kyoto Encyclopedia of
Genes and Genomes (KEGG) analysis showed that differential metabolites in the two modes were en-
riched in glycerophospholipid, choline, and alanine, aspartate, and glutamate and lysine metabolism.
Citation: Sui, H.; Wang, S.; Liu, G.; Taken together, these results demonstrate that heat stress can alter the motion characteristics and
Meng, F.; Cao, Z.; Zhang, Y. Effects of metabolomic profiles of boar sperm.
Heat Stress on Motion Characteristics
and Metabolomic Profiles of Boar Keywords: heat stress; pig; sperm; motion characteristics; metabolome
Spermatozoa. Genes 2022, 13, 1647.
https://doi.org/10.3390/
genes13091647

Academic Editor: Shensong Xie 1. Introduction

Received: 2 August 2022

Heat stress occurs when environmental temperatures during the summer period
Accepted: 9 September 2022
exceed boars’ physiological range. Heat-stressed boars commonly suffer from low repro-
Published: 14 September 2022
ductive performance, which leads to significant economic loss for the swine industry [1,2].
Previous studies showed that the infertility of heat-stressed boars is characterized by
Publisher’s Note: MDPI stays neutral
reduced sperm motility, concentration, and volume, as well as abnormal sperm mor-
with regard to jurisdictional claims in
phology [3,4]. Spermatogenesis is highly susceptible to heat stress, but the physiological
published maps and institutional affil-
response to heat stress varies between boar individuals. It was reported that the heat toler-
iations.
ance of sperm presents visible differences among individuals [5]. Thus, the pre-selection of
heat-tolerant boar semen could enhance utilization efficiency in artificial insemination pro-
grams. Recently, an in vitro heat stress experimental model for boar semen was established
Copyright: © 2022 by the authors.
to provide a valuable tool to screen for reliable sperm biomarkers of heat stress [6].
Licensee MDPI, Basel, Switzerland. The capacity of sperm to endure heat is recognized as a critical genetic feature in breed-
This article is an open access article ing [5]. Heat stress, thus, inevitably alters sperm molecular compositions at the genetic and
distributed under the terms and epigenetic levels. The identification of molecular markers associated with sperm heat stress
conditions of the Creative Commons is a prerequisite to develop abatement strategies for thermal stress. Currently, multiple
Attribution (CC BY) license (https:// OMICS technologies were applied to seek heat stress markers in animal reproductive
creativecommons.org/licenses/by/ organs. At the genomic level, tropical summer could induce DNA damage in boar sperma-
4.0/). tozoa [7]. Transcriptome analysis revealed that the expression of multiple genes was altered

Genes 2022, 13, 1647. https://doi.org/10.3390/genes13091647 https://www.mdpi.com/journal/genes

Genes 2022, 13, 1647 2 of 12

in boars exposed to heat stress, and some differentially expressed genes were also identified
between heat-tolerant and heat-susceptible boars [8]. Proteomic profiling showed that heat
stress caused the differential expression of 60 and 85 proteins in human [9] and boar [10]
sperm, respectively. Moreover, heat stress also severely perturbed the proteomic profiles of
seminal plasma in rams [11] and Brangus bulls [12]. It was noted that subtle changes in
transcriptome and proteome are eventually manifested in metabolome. Following exposure
to high-temperatures, Holstein bull semen exhibited aberrant concentrations of fatty acids
and cholesterol [13]. High environmental temperatures resulted in up-regulation of metabo-
lites in rat epididymis [14]. A recent study showed that seminal plasma metabolites related
to hormone secretion, energy metabolism, and fatty acid oxidation were associated with
heat stress in Mediterranean buffalo bulls [15]. Although heat-stress-induced metabolic
changes are clarified in some male species, whether heat stress affects metabolome in boar
sperm remains unclear.
In the present study, the motion features and metabolomic profiles of boar sperm
in vitro exposed to high temperatures were deeply analyzed. We found that heat stress
altered the kinematic parameters of sperm and caused significant changes in metabolites
associated with fatty acid and amino acid metabolism in sperm. Furthermore, differential
metabolite-enriched pathways are mainly associated with sperm quality. Our results
provide potential markers for screening heat-tolerant semen and developing strategies to
mitigate heat stress.

2. Materials and Methods

2.1. Heat Stress Treatment for Boar Sperm
Twenty Huoshou black boars, a Chinese native pig breed, were raised under the same
management conditions and fed with the same diets. The boars were healthy and had no
testicular disorders. The quality of the fresh boar semen met the national criteria in China.
Fresh semen from Huoshou black boars was collected using the gloved hand method. The
semen was diluted in an extender at 35 ◦ C. Following dilution, the semen was divided into
two groups, in which one was incubated for 1 h at 35 ◦ C (NHS group), and the other one
was incubated for 1 h at 41 ◦ C (HS group).

2.2. Evaluation of Sperm Quality

Semen from several boars was collected and pooled. The pooled semen was separated
into NHS and HS groups. Semen from each group was used to analyze the kinetic parame-
ters. One µL of semen was mixed with nine µL of diluent, and a ten-µL sample was then
placed on the glass slide. The sperm motility and kinetic parameters were detected using a
CASA system (IMV, AB2625S, Guangzhou, China).

2.3. Purification of Spermatozoa

The sperm samples were purified using a 90–45% discontinuous Percoll gradient
centrifugation. The semen was carefully layered over the top of a prepared Percoll gradient
fraction. The sperm samples were then pelleted by centrifugation at 950× g for 15 min at
room temperature, and the sperm pellet was washed three times using phosphate-buffered
saline.

2.4. Metabolite Extraction

Two-hundred µL of water was added to the samples. After 30 s of vortexing, the
samples were frozen and thawed with liquid nitrogen for 3 times. The samples were then
sonicated for 10 min in an ice-water bath. Fifty µL of homogenate was used to measure
the protein concentration. Then, 600 µL acetonitrile: methanol = 1:1 was added to the
rest part and transferred to 2 mL EP tube. Following the 30 s vortex, the samples were
incubated at 40 ◦ C for 1 h and centrifuged at 12,000 (RCF = 13,800× g), R = 8.6 cm) rpm for
15 min at 4 ◦ C. Seven-hundred µL of supernatant was transferred to an EP tube and dried
in a vacuum concentrator. Acetonitrile: methanol: water = 2:2:1, with isotopically-labelled
Genes 2022, 13, 1647 3 of 12

internal standard mixture, was added in proportion. After 30 s of vortexing, the samples
were sonicated for 10 min in ice-water bath. The samples were then centrifuged at 12,000
(RCF = 13,800× g), R = 8.6 cm) rpm for 15 min at 4 ◦ C. The resulting supernatant was
transferred to a fresh glass vial for analysis. The quality control (QC) sample was prepared
by mixing an equal aliquot of the supernatants from all of the samples.

2.5. Liquid Chromatography–Mass Spectrometry (LC-MS) Analysis

LC-MS/MS analyses were performed using an UHPLC system (Vanquish, Thermo
Fisher Scientific, Waltham, MA, USA) with a UPLC BEH Amide column (2.1 mm ×
100 mm, 1.7 µm) coupled to a Orbitrap Exploris 120 mass spectrometer (Orbitrap MS,
Thermo, Waltham, MA, USA). The mobile phase consisted of 25 mmol/L ammonium
acetate and 25 ammonia hydroxide in water (pH = 9.75) (A) and acetonitrile (B). The auto-
sampler temperature was 4 ◦ C, and the injection volume was 4 µL. The Orbitrap Exploris
120 mass spectrometer was used for its ability to acquire MS/MS spectra on information-
dependent acquisition (IDA) mode in the control of the acquisition software (Xcalibur,
Thermo, Waltham, MA, USA). In this mode, the acquisition software continuously eval-
uates the full-scan MS spectrum. The ESI source conditions were set as follows: sheath
gas flow rate as 50 Arb, Aux gas flow rate as 15 Arb, capillary temperature as 320 ◦ C, full
MS resolution as 60,000, MS/MS resolution as 15,000, collision energy as 10/30/60 in NCE
mode, spray voltage as 3.8 kV (positive) or −3.4 Kv (negative), respectively.

2.6. Data Preprocessing and Annotation

The raw data were converted to the mzXML format using ProteoWizard, and pro-
cessed with an in-house program, which was developed using R and based on XCMS, for
peak detection, extraction, alignment, and integration. Then, an in-house MS2 database
(BiotreeDB, Shanghai, China) was applied in metabolite annotation. The cutoff for annota-
tion was set at 0.3.

2.7. Statistical Analysis

The results were presented as mean ± standard error of mean (mean ± S.E.M). All data
were logarithmically transformed using SIMCA software (v16.2, Umea, Sweden) to normal-
ize distributions. The independent-sample t-test and the bivariate correlation analysis were
carried out using SPSS 17.0 (Armonk, New York, USA) to determine significant difference
levels. Orthonormal partial least squares discriminant analysis (OPLS-DA) of metabolomic
data was performed by using SIMCA-P 14.0 software (Umetrics, Umea, Sweden). Those
variables with variable importance in the projection (VIP) > 1.0, p-value < 0.05, and fold
change (FC) > 1.50 or <0.66 were identified as differentiated metabolites. Based on the
differentiated metabolites, metabolic pathway analysis was performed by using MetaboAn-
alyst 3.0 (Baiqu, Shanghai, China). The volcano plots and heat map were generated using
Origin 9.0 software (Hampton, Waltham, MA, USA).

3. Results
3.1. Effect of Elevated Ambient Temperature on Motion Characteristics in Boar Spermatozoa
To determine whether high temperatures impaired boar sperm quality, kinetic pa-
rameters were analyzed using the CASA system. The results showed that HS treatment
significantly reduced sperm motility (Figure 1A) (p < 0.05). The average path distance
(APD) (Figure 1B), straight-line velocity (VSL) (Figure 1D), straightness (STR) (Figure 1F),
and linearity (LIN) (Figure 1G) of sperm in the HS group were also decreased compared to
the non-heat-stress (NHS) group (p < 0.05). However, the HS treatment did not significantly
affect the following kinetic parameters: average path velocity (VAP) (Figure 1C), curvilinear
velocity (VCL) (Figure 1E), and amplitude of lateral head (ALH) (Figure 1H). Therefore,
these results indicate that HS impairs the kinematic features of boar sperm.
 Genes 2022, 13, x FOR PEER REVIEW 4 of 13

1F), and linearity (LIN) (Figure 1G) of sperm in the HS group were also decreased com-
pared to the non-heat-stress (NHS) group (p < 0.05). However, the HS treatment did not
significantly affect the following kinetic parameters: average path velocity (VAP) (Figure
Genes 2022, 13, 1647 1C), curvilinear velocity (VCL) (Figure 1E), and amplitude of lateral head (ALH) (Figure 4 of 12
1H). Therefore, these results indicate that HS impairs the kinematic features of boar
sperm.

Figure
Figure 1. Effect of heatof
1. Effect stress
heatonstress
kinetic on
parameters
kineticofparameters
boar sperm. (A)of Sperm
boarmotility
sperm. in NHS
(A) andSperm motility in NHS and
HS groups. Kinetic parameters of sperm, including average path distance (B), average path veloc-
ity (C), straight line velocity (D), curvilinear velocity (E), straightness (F), linearity (G), and am-(B), average path velocity
HS groups. Kinetic parameters of sperm, including average path distance
plitude of lateralline
(C), straight headvelocity
(H), were (D),
detected in NHS andvelocity
curvilinear HS group.(E),
NHS: non-heat-stress,
straightness (F),HS:linearity
heat (G), and amplitude of
stress. All data are shown as mean ± S.E.M, and different letters on the bars indicate significant
lateral head
differences (H), were detected in NHS and HS group. NHS: non-heat-stress, HS: heat stress. All data
(p < 0.05).
are shown as mean ± S.E.M, and different letters on the bars indicate significant differences (p < 0.05).
3.2. Changes of Metabolomics Features in Boar Spermatozoa
3.2.ToChanges of Metabolomics
investigate whether heat Features in Boar
stress altered the Spermatozoa
metabolism of boar sperm,
non-targeted metabolomics sequencing was performed to identify metabolic changes.
To investigate
The OPLS-DA whether
models in the positiveheat stress altered
and negative ion modes the
weremetabolism
established toof boar sperm, non-targeted
com-
metabolomics
pare sequencing
sperm metabolites between the was
HS and performed
NHS groups.to Asidentify metabolic
shown in Figure changes. The OPLS-DA
2A,C, the
HS and NHS
models groups
in the could and
positive be clearly segregated,
negative indicating
ion modes werethatestablished
certain metabolic
to compare sperm metabo-
changes occurred in the sperm after heat stress. In addition, permutation tests were per-
lites between the HS and NHS groups. As shown in Figure 2A,C, the HS and NHS groups
formed to confirm the reliability of the OPLS-DA models (Figure 2B,D). R2 and Q2 rep-
could
resent thebe clearly segregated,
interpretability indicating
and predictability thatrespectively.
of the models, certain metabolic changes occurred in the
R2 and Q2 in the
positive and negative ion modes were near to 1 or less than
sperm after heat stress. In addition, permutation tests were zero, respectively, indicating
performed to confirm the
that there was no overfitting, and the OPLS-DA models were stable and reliable.
reliability of the OPLS-DA models (Figure 2B,D). R2 and Q2 represent the interpretability
and predictability of the models, respectively. R2 and Q2 in the positive and negative ion
Genes 2022, modes were
13, x FOR PEER near to 1 or less than zero, respectively, indicating that there was no5 overfitting,
REVIEW of 13

and the OPLS-DA models were stable and reliable.

Figure 2. The scatter and permutation plot of OPLS-DA model. (A,C) OPLS-DA score scatter plots
Figure 2. The scatter and permutation plot of OPLS-DA model. (A,C) OPLS-DA score scatter plots
of sperm metabolites in the positive and negative ion modes. The plots were used to analyze the
of sperm metabolites in the
segregation positive
of sperm and
samples negative
between NHS andionHS
modes. Themarks
groups. Red plotsNHSwere used
group, blueto analyze the
denotes
HS group. (B,D) OPLS-DA permutation plots of sperm metabolites in the positive and negative
segregation of sperm samples between NHS and HS groups. Red marks NHS group, blue denotes
ion modes. The slope of R2 is >0 and the Y-intercept of Q2 is <0.05, indicating a valid model.
HS group. (B,D) OPLS-DA permutation plots of sperm metabolites in the positive and negative ion
modes. The slope3.3.
ofClassification
R2 is >0 and of Sperm Metabolites inof
the Y-intercept theQ2
Positive and Negative
is <0.05, Ion Modes
indicating a valid model.
In the positive ion mode, a total of 528 metabolites were identified in the sperm (data
shown in the online repository). According to the chemical properties of the metabolites,
the metabolites were classified into 16 categories. The main metabolites in the positive
ion mode were lipids and lipid-like molecules, which contained 248 metabolites, ac-
counting for 45.01% of all metabolites (Figure 3A) (Supplementary Table S1). The re-
maining metabolites were organic acids and derivatives (20.41%), organoheterocyclic
 Genes 2022, 13, 1647 5 of 12

3.3. Classification of Sperm Metabolites in the Positive and Negative Ion Modes
In the positive ion mode, a total of 528 metabolites were identified in the sperm (data
shown in the online repository). According to the chemical properties of the metabolites,
the metabolites were classified into 16 categories. The main metabolites in the positive ion
mode were lipids and lipid-like molecules, which contained 248 metabolites, accounting for
45.01% of all metabolites (Figure 3A) (Supplementary Table S1). The remaining metabolites
were organic acids and derivatives (20.41%), organoheterocyclic compounds (11.74%),
and the other 13 super classes (Figure 3A). In the negative ion mode, 194 metabolites in
total were identified in the sperm (data shown in the online repository). The metabolites
were classified into nine categories. Organic acids and derivatives were major metabolic
classes and contained 61 metabolites, accounting for 33.63% of all metabolites (Figure 3B)
(Supplementary Table S2). The remaining metabolites were separately distributed into the
Genes 2022, 13, x FOR PEER REVIEW 6 of 13
following classes: lipids and lipid-like molecules (51 metabolites, 26.45%), organic oxygen
compounds (27 metabolites, 13.45%), and the other six classes (Figure 3B). Therefore, these
data show that lipids and organic acids are the main metabolites in boar sperm.

B
Figure
Figure3.3.Classification
Classificationofofmetabolites
metabolites identified in boar
identified in boarsperm.
sperm.(A)
(A)Major
Majorclasses
classes
ofof sperm
sperm metab-
metabolites
olites are identified
are identified in theinpositive
the positive ion mode.
ion mode. According
According to properties
to properties of metabolites,
of metabolites, spermsperm me-
metabolites
tabolites identified in the positive ion mode were classified into different classes. The percentage
identified in the positive ion mode were classified into different classes. The percentage of each class
of each class is shown in the pie chart. The colors represent each class of metabolite. (B) Major
is shown in the pie chart. The colors represent each class of metabolite. (B) Major classes of sperm
classes of sperm metabolites are identified in the negative ion mode. Sperm metabolites identified
metabolites are identified in the negative ion mode. Sperm metabolites identified in the negative ion
in the negative ion mode were classified into different classes. The percentage of each class is
modeinwere
shown classified
the pie intocolors
chart. The different classes.each
represent Theclass
percentage of each class is shown in the pie chart.
of metabolite.
The colors represent each class of metabolite.
3.4. Identification and Clustering Analysis of Differential Metabolites in Sperm
The metabolites with variable importance in projection (VIP) > 1.0 and p < 0.05 were
considered to be significantly changed. In the positive ion mode, we found that 163 me-
tabolites in the HS group were significantly different from those in the NHS group, in-
Genes 2022, 13, 1647 6 of 12

3.4. Identification and Clustering Analysis of Differential Metabolites in Sperm

Genes 2022, 13, x FOR PEER REVIEW The metabolites with variable importance in projection (VIP) > 1.0 and 7p of< 130.05
were considered to be significantly changed. In the positive ion mode, we found that
163 metabolites in the HS group were significantly different from those in the NHS group,
including 130 up-regulated
down-regulated
and 33 down-regulated
metabolite,
metabolitesisopalmitic
including
(Figure 4A). Among these,
acid,
the chemical names for only six upregulated
2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3, metabolites that contained 3-aminobutanoic
4-dihydro-2H-1-benzopyran-4-one,
acid, biotin sulfone, campestanol,
L-2-Hydroxyglutaric acid, LysoPC N-Acetyl-L-aspartic
(18_3(6Z, 9Z, 12Z)), LysoPCacid,(22_5(4Z, 7Z, and10Z,
13Z, 16Z)), and N, O-didesmethylvenlafaxine were determined. The heatmap
N-Carboxyethyl-g-aminobutyric acid, were matched. The heatmap of five differential of six differ-
ential metabolites
metabolites is displayed
is displayed in Figure
in Figure 4B.addition,
4D. In As showntheinquantitative
the heatmap, samples
analysis in there-
further same
group
vealedclustered together,
the differential and the levels
expression of fiveofmetabolites
each metabolite
betweenexhibited
the HSaand
differential
NHS groupschange
between the HS
(Figure 5B). and these
Hence, NHSresults
groupsdemonstrate
(Figure 4B).that
Furthermore, the differential
certain metabolites expression of
exhibit differential
six metabolites was visible between HS
expression between the HS and NHS groups. and NHS groups (Figure 5A).

Figure4.4.Differential
Figure Differential expression
expression and
and cluster
clusteranalysis
analysisofofmetabolites
metabolitesbetween
between HSHS
andand
NHSNHSgroup.
group.
(A,C) Volcano plot of sperm metabolites identified in the positive and negative ion modes. The
(A,C) Volcano plot of sperm metabolites identified in the positive and negative ion modes. The plots
plots were used to display changes of metabolites between HS and NHS groups. The red circles
were used to
represent display changes
up-regulated of metabolites
metabolites, the blue between HS anddown-regulated
circles represent NHS groups. The red circlesand
metabolites, represent
the
up-regulated metabolites,
gray circles represent no the blue circles
changes represent(B,D)
of metabolites. down-regulated
Heat map ofmetabolites, and theidentified
sperm metabolites gray circles
represent no changes
in the positive of metabolites.
and negative (B,D)
ion modes. TheHeat
heatmap
mapsofwere
sperm metabolites
used identified
to visualize in the
the dynamic positive
chang-
es negative
and of metabolite content The
ion modes. between HS and
heat maps NHS
were groups.
used The pink
to visualize therepresents HS group,
dynamic changes of and the
metabolite
purplebetween
content represents
HSNHS group.
and NHS The color
groups. Thescale
pink of the heatmap
represents represents
HS group, the purple
and the gradualrepresents
decrease ofNHS
metabolite content from red to blue.
group. The color scale of the heatmap represents the gradual decrease of metabolite content from red
to blue.

In the negative ion mode, we identified 171 differential metabolites in the HS group
compared to the NHS group, including 131 up-regulated and 40 down-regulated metabo-
lites (Figure 4C). Among them, the chemical names for only four up-regulated and one
down-regulated metabolite, including isopalmitic acid, 2-(3,4-dihydroxyphenyl)-3,5,7-
trihydroxy-3, 4-dihydro-2H-1-benzopyran-4-one, L-2-Hydroxyglutaric acid, N-Acetyl-L-
aspartic acid, and N-Carboxyethyl-g-aminobutyric acid, were matched. The heatmap of
five differential metabolites is displayed in Figure 4D. In addition, the quantitative anal-
Genes 2022, 13, 1647 7 of 12

ysis further revealed the differential expression of five metabolites between the HS and
Genes 2022, 13, x FOR PEER REVIEW 8 of 13
NHS groups (Figure 5B). Hence, these results demonstrate that certain metabolites exhibit
differential expression between the HS and NHS groups.

Figure5.5.Verification
Figure Verification of
of differential
differential metabolites
metabolites between
between HSHS
andand
NHS groups.
NHS (A) Abundance
groups. (A) Abundanceof
sperm metabolites in the positive ion mode between HS and NHS groups. The blue bars denote
of sperm metabolites in the positive ion mode between HS and NHS groups. The blue bars
HS group, the red bars indicate NHS group. The metabolites identified in the positive ion mode
denote HS group, the red bars indicate NHS group. The metabolites identified in the positive
included 3-aminobutanoic acid, biotin sulfone, campestanol, lysoPC(18_3(6Z,9Z,12Z)),
ion mode included 3-aminobutanoic
lysoPC(22_5(4Z,7Z,10Z,13Z,16Z)), andacid, biotin sulfone, campestanol,
N,O-Didesmethylvenlafaxine. lysoPC(18_3(6Z,9Z,12Z)),
The data are shown as mean
lysoPC(22_5(4Z,7Z,10Z,13Z,16Z)),
± S.E.M, and different letters on the bars indicate significant differences (p <data
and N,O-Didesmethylvenlafaxine. The 0.05).are
(B)shown as mean
Abundance
±of spermand
S.E.M, metabolites
differentinletters
the negative ion mode
on the bars between
indicate HS anddifferences
significant NHS groups. (p <The blue(B)
0.05). bars denote
Abundance
ofHS group,
sperm the red barsinindicate
metabolites NHS group.
the negative The metabolites
ion mode between HS identified
and NHS in the negative
groups. Theionblue
mode bars
included isopalmitic acid,
denote HS group, the red bars indicate NHS group. The metabolites identified in the negative
2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-1-benzopyran-4-one,
ion mode included isopalmitic acid, 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-3,4-dihydro-2H-1-
L-2-Hydroxyglutaric acid, N-Acetyl-L-aspartic acid, and N-Carboxyethyl-g-aminobutyric acid.
benzopyran-4-one,
The data are shownL-2-Hydroxyglutaric
as mean ± S.E.M, andacid, N-Acetyl-L-aspartic
different letters on the barsacid, and significant
indicate N-Carboxyethyl-g-
differ-
aminobutyric acid. The data are shown as mean ± S.E.M, and different letters on the bars indicate
ences (p < 0.05).
significant differences (p < 0.05).
3.5. Metabolic Pathway Analysis for Differential Metabolites in Sperm
3.5. Metabolic Pathway Analysis for Differential Metabolites in Sperm
All differential metabolites were subjected to metabolic pathway analysis using the
KEGGAll database.
differential
In metabolites were
the positive ion subjected
mode, to metabolic
differential pathway
metabolites analysis
were mainly using the
enriched
KEGG
in twodatabase. Inmetabolic
significant the positive ion mode,
pathways, differential
involving metabolites
choline were
metabolism andmainly enriched
glycerophos-
inpholipid
two significant
metabolismmetabolic
(Figure pathways, involving
6A). In the negative ioncholine metabolismmetabolites
mode, differential and glycerophos-
were
mainly involved in three significant metabolic pathways, including common metabolism
 Genes 2022, 13, 1647 8 of 12

Genes 2022, 13, x FOR PEER REVIEW 9 of 13

pholipid metabolism (Figure 6A). In the negative ion mode, differential metabolites were
mainly involved in three significant metabolic pathways, including common metabolism
pathways,
pathways,alanine,
alanine, aspartate glutamatemetabolism,
aspartate and glutamate metabolism,and
andlysine
lysine metabolism
metabolism (Figure
(Figure 6B).
6B). In summary,
In summary, differential
differential metabolites
metabolites were enriched
were enriched in metabolic
in metabolic pathways
pathways importantim-
for
sperm for
portant quality.
sperm quality.

Figure 6. KEGG analysis of differential metabolites between HS and NHS groups. (A) Illustration
Figure 6. KEGG analysis of differential metabolites between HS and NHS groups. (A) Illustration of
of differential metabolite-enriched pathways in the positive ion mode. The color scale in the right
differential metabolite-enriched pathways in the positive ion mode. The color scale in the right of
of the bubble image represents gradual decrease of p-value from blue to red. Metabolic pathways
thedisplayed
are bubble image represents
in the gradual
left of the bubbledecrease
image. of(B)p-value from of
Illustration blue to red. Metabolic
differential pathways are
metabolite-enriched
displayedin
pathways in the
the left of theion
positive bubble image.
mode. The (B) Illustration
color scale in of
thedifferential metabolite-enriched
right of the pathways
bubble image represents
in the positive
gradual decrease ionofmode.
p-valueThe color
from scale
blue toin theMetabolic
red. right of thepathways
bubble image representsingradual
are displayed the leftdecrease
of the
bubble image.
of p-value from blue to red. Metabolic pathways are displayed in the left of the bubble image.

4. Discussion
Genes 2022, 13, 1647 9 of 12

4. Discussion
HS significantly reduces semen quality and fertility in boars [4]. The ability of sperm to
tolerate heat stress exhibits visible variations among individuals [5]. However, it is unclear
if HS causes metabolic changes in swine sperm, and whether metabolic indicators for sperm
thermal stress exist. In this study, we investigated the effects of HS on the kinetic features
and metabolomic profiles of boar sperm, and further analyzed the potential functions of
differential metabolites in sperm heat stress.
The kinematic parameters of sperm can not only be used to evaluate semen quality,
but also can crudely predict male fertility [16]. We found that HS resulted in a significant
reduction in sperm motility, APD, VSL, STR, and LIN, indicating the susceptibility of boar
sperm to high temperatures. Consistent with our results, heat stress also altered the CASA
parameters of sperm in boars [6] and buffalo bulls [15]. Unexpectedly, HS only reduced the
percentage of viable sperm, but did not harm sperm motility and other motion parameters
in rabbits [17]. This discrepancy in sperm motion characteristics might be due to differences
between species.
Metabolites are classified based on their composition as amino acids, lipids, carbo-
hydrates, nucleotides, minerals, and vitamins. We found in the positive and negative ion
mode that the proportion of lipid molecules and organic acids was maximal in native boar
sperm, respectively. Similarly, organic acids were major metabolic classes in the seminal
plasma of commercial boars [18]. A previous study showed that the major metabolic class in
bull sperm was also organic acids [19], indicating that organic acids might have important
functions in the sperm of different species.
In addition, we identified that heat-stressed sperm generated 160 or 171 differential
metabolites in the two ion modes. Studies have shown that differential metabolites related
to sperm quality mainly involved lysophosphatidylcholine [20], palmitic acid [21], and γ-
aminobutyric acid [22]. Specifically, lysophosphatidylcholine supplementation reportedly
induced an acrosome reaction of sperm in rabbits [23] and bulls [24]. Palmitic acid is the
most abundant saturated fatty acid in boar sperm plasma membranes [25], and could im-
prove boar sperm motility via mitochondrial B-oxidation [21]. γ-aminobutyric acid addition
enhanced the capacitation and acrosome reaction of sperm in several species [26–29]. Given
the association between the above-mentioned differential metabolites and sperm function-
ality, it is conceivable that these metabolites could be regarded as potential biomarkers of
heat stress for boar sperm.
Differential metabolites identified in this study are mainly enriched in the metabolic
pathways of glycerophospholipid, choline, and amino acids. These metabolic pathways
participate in the regulation of sperm quality. For instance, glycerophospholipid incorpo-
rated into sperm membranes can reduce oxidative damage and improve stallion sperm
quality [30]. Glycerophospholipid supplementation significantly alleviated oxidative stress,
and enhanced the motility and viability of fresh and post-thaw sperm in humans [31,32].
Dietary choline was required for sperm motility in Drosophila [33]. Lysine acetylation was
involved in regulating boar sperm motility and acrosome integrity [34], and inducing a
mouse sperm acrosome reaction [35]. Alanine addition improved the post-thaw motility of
striped bass sperm [36]. D-aspartic acid supplementation elevated the quality of rooster
post-thaw sperm [37], rabbit buck fresh sperm [38], and the fertilizing capacity in mice [39].
Thus, it may be possible to developing tools to mitigate the heat stress of boar sperm via
modulation of these metabolic pathways.
Numerous studies indicated that HS caused a significant reduction in human sperm
quality [40–42]. Differential metabolites and metabolic pathways identified in this study
might be relevant to human sperm quality. For example, mRNA levels of γ-aminobutyric
acid receptors in human sperm are correlated with poor sperm quality [43]. D-aspartate
could protect human sperm via preventing the decrease of motility and the increase of DNA
fragmentation and lipid peroxidation [44]. Moreover, lysine acetylation and glutarylation
in human sperm were well associated with sperm functions [45,46]. Thus, these results
could provide a useful reference for identifying the HS biomarkers of human sperm.
Genes 2022, 13, 1647 10 of 12

5. Conclusions
Our results demonstrate that HS alters the motion characteristics and metabolomic
profiles of boar sperm. These findings have implications for screening heat-tolerant animal
and human semen, and for developing strategies to mitigate HS.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/genes13091647/s1, Table S1: The percentage of major classes of
sperm metabolites identified in the positive ion mode; Table S2: The percentage of major classes of
sperm metabolites identified in the negative ion mode.
Author Contributions: Conceptualization, Z.C. and Y.Z.; methodology, H.S. and S.W.; software, S.W.;
validation, G.L. and F.M.; formal analysis, G.L. and F.M.; investigation, H.S. and S.W.; writing—
original draft preparation, H.S., S.W., Z.C. and Y.Z.; writing—review and editing, H.S., S.W., Z.C. and
Y.Z.; supervision, Y.Z.; project administration, Z.C. and Y.Z.; funding acquisition, Z.C. and Y.Z. All
authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by grants from the Special Fund for Anhui Agriculture Research
System (AHCYJSTX-04), the Joint Research Project on the Anhui Local Pigs Breeding and Utilization
(340000211260001000431), the Hefei Innovation and Entrepreneurship Support Plan for Returnee
Scholar (03082009), the Anhui Provincial Innovation and Entrepreneurship Support Plan for Re-
turnee Scholar (2020LCX015), and the Natural Science Project of Universities in Anhui Province
(KJ2019ZD17).
Institutional Review Board Statement: This study was conducted according to the guidelines
of the Basel Declaration, the recommendations of the Guide for the Care and Use of Laboratory
Animals (http://grants1.nih.gov/grants/olaw/references/phspol.htm, accessed on 12 May 2016),
and the ethics committee of Anhui Agricultural University. The protocol was approved by the ethics
committee of Anhui Agricultural University under permit no., AHAU 20101025.
Informed Consent Statement: Not applicable.
Data Availability Statement: The raw data presented in the study are deposited in the MetaboLights
repository (accession number: MTBLS5391).
Acknowledgments: We thank Xiangdong Zhang, Zhenyuan Ru, and Haiqin Luo, Mei Sheng, Xueying
Zhang for their help in technical assistance. We are grateful for Jason Knott’s English editing.
Conflicts of Interest: The authors declare no conflict of interest.

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