The relationship between the production of reactive oxygen
species (ROS) and abnormal sperm morphology in stallions
Evelyn Bulkeley, BS; Christa Darr, PhD; Stuart Meyers, DVM, PhD
Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California,
Davis, Davis, CA
Results
Background
§  Stallions, unlike many other domestic species, are selected for breeding based on
phenotypic characteristics, athletic performance, and pedigree− not reproductive
performance.
§  Mitochondrial oxidative function is absolutely essential for sperm motility3, and excess
mitochondrial ROS production can be detrimental to the sperm membrane and DNA
integrity1,4.
§  In horses, a positive correlation has been observed between pregnancy rates and the
percent of morphologically normal sperm, and a negative correlation between
pregnancy rates and morphologic features such as bent midpieces, hairpin tails, and
coiled tails6.
§  Studies by Aziz and coworkers reported a positive correlation in humans between ROS
production and sperm with amorphous heads, acrosomal damage, cytoplasmic droplets,
midpiece, and tail defects5.
§  The relationship between sperm morphology and ROS production can now be
determined using the ImageStream high speed quantitative imaging cytometer (Amnis
Corp., Seattle, WA). This instrument enables direct correlation between biomarker
intensity and sperm morphology7.
§  Investigating the relationship between ROS production and abnormal stallion sperm
morphology on an individual cell basis will allow increased precision in assessment and
subsequent management of stallion fertility.
TABLE 1
Percent Abnormal Midpieces
Percent Normal
Percent Abnormal Percent Abnormal Percent Proximal
Percent Distal
Percent Other
Cells
Heads
Tails
Droplets
Droplets
abnormal midpieces
High Low Total High Low Total High Low Total High Low Total High Low Total High Low Total
DHE DHE Count DHE DHE Count DHE DHE Count DHE DHE count DHE DHE Count DHE DHE Count
Stallion
1
25
56
60
5
4
3
12
2
3
38
22
23
10
11
6
10
5
5
2
22
66
69
3
8
5
10
2
7
45
12
10
8
7
5
12
5
4
Stallion
Stallion
Stallion
3
16
41
53
9
4
3
8
2
2
47
42
32
5
4
3
15
7
7
4
23
42
51
7
3
2
9
7
5
26
12
12
16
21
16
19
15
14
Table 1: Morphology percentages of the entire ejaculate via phase contrast microscopy (total count) and morphology
percentages of the high and low DHE cell populations via the Amnis Imagestream brightfield channel. 100 cells from
each population were counted.
A
B
Percent of Cells with
Normal Morphology
80
*
70
40
30
Method
Endpoint(s)
40
5
10
0
0
a
High DHE
Low DHE
b
4
2
2
1
High DHE
d
Low DHE
c
§ 
§ 
§ 
High DHE
0
Low DHE
High DHE
Low DHE
d
Computer Assisted Sperm Analysis (CASA)
§  Total Motility
(TM, %)
§  3 μL of semen was loaded into a chamber slide on a §  Progressive
slide warmer at 37°C.
Motility (PM, %)
§  A minimum of 500 sperm were analyzed per sample.
§  Average Path
Velocity (VAP,
μm/s)
§  Viability (SYTOX)
§  ROS production
§  Viability was measured using SytoxGreen (SYTOX), which
(DHE)
is impermeant to cells with uncompromised membranes §  Morphology of
§  Cellular ROS production was monitored using
high and low DHE
dihydroethidium (DHE), which indicates superoxide anion
populations
production.
§  High and low DHE populations were gated (Figure 1), and
morphology of those populations was analyzed in
brightfield and two fluorescent channels (Figure 2).
Figure 2: Percent of cells in the high and low DHE populations with (A) normal morphology, (B) abnormal midpieces, (C)
head abnormalities, and (D) abnormal tails. (*= p<0.05). Brightfield (Ch01) and DHE fluorescence (Ch04) images of (a)
a normal cell, (b) a cell with a bent midpiece, (c) a cell with a proximal droplet, and (d) a cell with a coiled tail.
Method Continued
Amnis Imagestream Flow Cytometry
Figure 1: Gating of ImageStream flow cytometry data for
analysis. (A) The gating of live (SYTOX positive) cells into
high and low DHE populations. (B) The mean fluorescent
intensity of the DHE channel, used to determine the
fluorescent intensity value to gate the high and low DHE
populations.
12
15
10
8
20
10
6
4
10
5
2
0
High DHE
Low DHE
High DHE
b
0
Low DHE
High DHE
Low DHE
c
6
3
0
20
14
30
*
8
4
30
16
40
Percent of Cells with
Other Midpiece
Abnormalities
25
18
50
a
C
Percent of Cells With
Distal Droplets
20
60
0
B
*
10
Sample Preparation
§ 
§  Initial gel-free
sperm
Single ejaculates were collected twice from four light
concentration
breed resident UCD stallions (n=4)
§  Resuspended
Gel-free sperm concentration was determined with a
sample to a
NucleoCounter SP-100
concentration of
the semen was then diluted 1:1 with pre-warmed (37°C)
100 million
INRA 96
cells/mL
The sperm was centrifuged at 300xg for 10 minutes, and
the sperm pellet was resuspended to a concentration of
100 million cells/mL using a modified Biggers, Whitten,
and Whittingham (BWW) media.
6
Percent of Cells with
Proximal Droplets
12
8
50
10
with Abnormal Tails
9
7
20
D Mean Percent of Cells
Mean Percent of Cells
with Abnormal Heads
10
60
20
Method
C
*
70
50
§  The aim of this study was to evaluate stallion semen using the ImageStream high-speed
quantitative imaging cytometer (Amnis Corp., Seattle, WA) to investigate the
relationship between ROS production and abnormal sperm morphology on an individual
cell basis, hypothesizing that there would be a positive correlation between increased
ROS production and sperm cells with head, midpiece, and tail defects.
Mean Percent of Cells
with Abnormal
Midpieces
80
60
Aim and Hypothesis
A
Figure 3: Percent of cells with three types of midpiece abnormalities: (A)
proximal droplets, (B) distal droplets, and (C) other. Other midpiece
abnormalities (C) include bent and thickened midpieces. (*=p<0.05). Brightfield
(Ch01) and DHE fluorescent (Ch04) images of (a) a cell with a proximal droplet,
(b) a cell with a distal droplet, and (c) a cell with a bent midpiece.
§  The high DHE population had significantly fewer morphologically normal cells
than the low DHE population (p<0.05) (Figure 2A).
§  The high DHE population had a significantly higher percent of cells with
midpiece abnormalities than the low DHE population (p<0.05) (Figure 2B).
§  There is no significant difference between the percent of cells with head
abnormalities between the high and low DHE populations (Figure 2C).
§  The high DHE population had a significantly higher percent of cells with tail
abnormalities than the low DHE population (p<0.05) (Figure 2D).
§  There was no significant difference observed between the amount of
proximal or distal droplets between the low and high DHE populations, but
the high DHE population had a significantly higher percent of “other midpiece
abnormalities” (p<0.05) (Figure 3A,B,C).
Conclusions
References
§  There is a negative relationship between the percent of morphologically normal cells and high
ROS production.
§  There is a positive relationship between the percent of cells with abnormal tails and ROS
production.
§  There is a positive relationship between the percent of cells with abnormal midpieces and ROS
production.
§  There was not a significant association between ROS production and the percent of cells with
abnormal heads.
§  Of cells with abnormal midpieces, there was no significant relationship found between cells
with proximal droplets or distal droplets and ROS production, but a significant positive
relationship was found between ROS production and “other” midpiece abnormalities (bent
midpiece, thickened midpiece, etc.).
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Acknowledgements
Research reported was supported by “Students Training in Advanced Research” (STAR) Fellowship
award from the School of Vet Med Endowment Funds, as well as the National Institutes of Health
(NIH), National Center for Research Resources as a Ruth L. Kirschstein National Research Service
Award (NRSA), grant number T32OD010931-11.