research article Bovine Frozen Semen Motility Analysis Using Two Different Laser Wavelengths
PHA Carvalho1, RODS Rossi2,
E Lopes1, APC Santos1,
JNS Sales3, GF Rabelo4,
RA Braga Jr4, JB Barreto Filho1*
1Department of Veterinary, Animal Reproduction Laboratory,
Federal University of Lavras, Lavras, Minas Gerais, Brazil
2Federal Institute of Triângulo
Mineiro, Campus Uberlândia. Sobradinho Farm S/N Zona Rural, Uberlândia, Brasil
3Veterinary Medicine, Federal University of Juiz de Fora,
Minas Gerais, Brazil
4 Department of
Automatica, Centre for Development of Applied Instrumentation, Federal
University of Lavras, Lavras, Minas Gerais, Brazil
*Corresponding author: Joao Bosco
Barreto Filho, Department of Veterinary, Animal Reproduction Laboratory,
Federal University of Lavras, Lavras, CEP 37200-000 CP3037 Minas Gerais,
Brazil. Tel: +55-3538291718; Email: barreto@ufla.br
Citation: Carvalho PHA, Rossi RODS, Lopes E, Santos APC, Sales JNS,
et al. (2019) Bovine Frozen Semen Motility Analysis Using Two Different Laser
Wavelengths. Adv Biochem Biotechnol 7: 1087. DOI: 10.29011/2574-7258.001087
Abstract
Laser
of 632 and 532 nanometers were evaluated for bovine semen kinetic analysis.
Numerical index of dynamic laser speckle (DLSI) was compared with motility
assessment by light microscopy. Straws (n = 123) were thawed and evaluated as
to velocity and cell motility. In assay one, each sample was lit by either kind
of laser (red, n = 56; green, n = 20). In the second assay 46 samples were lit
alternately by both laser sources. An index grouping velocity and motility was
used to compare the evaluations. In a third assay a single sample was
illuminated with both laser sources to assess the motility decreasing, and
regression analysis of dynamic laser speckle index, velocity and motility index
have been done. Correlations between dynamic laser speckle index, velocity and
cell motility were r = 0.594; r = 0.734 and r = 0.665; r = 0.684 for samples
illuminated by red and green laser, respectively. Correlation coefficients for
red and green laser found between DLSI index and velocity (r = 0.801; r =
0.590), cell motility (r = 0.826; r = 0.613) and velocity and motility index (r
= 0.840; r = 0.618), were significant (p < 0.01). No difference was observed
between the laser sources that were sensitive in detecting semen activity, thus
both of them can be used to generate the biospeckle phenomena and assess
motility in the bull frozen semen.
Introduction
Semen
analysis intends to determine the fertility potential of the ejaculate by
conventional methods. Sperm motility is considered to be a good fertilization
predictor, and high positive correlations were observed between spermatozoa
motility and fertility in the human species [1], in the stallion [2], ovine [3]
and cattle [4]. On the contrary, poor correlations were mentioned in some
species [5].
In
sperm motility analysis the semen kinetics evaluation by routine methods
usually implies a subjective component [6,7] and an uncertainty degree [8].
Thus, methodology and the precision of the measurement are important to semen
evaluation, as well as the choice of the right attribute, such as progressive
motility [9]. In the last few years, the use of computer-assisted technologies
improved the accuracy of semen analysis, however, these are high cost
techniques and not suitable in many situations [10,11]. In addition, many
studies observed positive and even high correlations between motility
evaluation by conventional methods and computer-aided systems [12,13].
Many
efforts have been done to increase the objectivity of the ancillary
methodologies used to analyze semen samples [14,15]. Optical metrology is one
such approach and was pioneering presented by Ross, et al. using laser
illumination, the Doppler effect evaluation and correlation with semen motility
[16].
The
earliest works on spermatozoa (fish and rabbit sperm) showed that laser light
scattering was strongly influenced by mobile cells, indicating its value as an
assay of per cent motility [17]. First models (point scatterers) proposed by
Harvey & Woolford assumed that spermatozoa behave as point particles moving
with constant velocity [18]. However, these models proved to be inadequate,
because sperm cells, particularly from rams and bulls, are large and far from
being spherical in form. The interpretation of correlation functions on light
scattered from bovine motile sperm requires a model which considers the size,
shape, and unusual swimming trajectories of these cells.
In
addition, it was found that the spermatozoa exhibited a strong orientation
effect, and the light scattered intensity was dominated by those cells swimming
in a narrow angle which were almost perpendicular to the scattering vector. Further
studies have pointed to the importance of immotile cells present in the
ejaculate, interactions or collisions between motile and immotile cells, the
distribution of spermatozoa within the scattering cell, spatial orientation
effects and the shape of the bull spermatozoon in order to understand the
spectrum or autocorrelation function of the scattered light by semen [19].
Other
methodologies using the optical approach are the dynamic laser speckle or
biospeckle laser techniques that was initially adapted in blood flow [20,21]
and after applied to many phenomena related or not to a flow movement [22,23].
Previous report of our laboratory [24] showed the reliable use of the
biospeckle laser as an approach to evaluate semen kinetic parameters. Nevertheless,
different laser wavelengths can influence the activity of spermatozoa [25-27],
compromising the DLSI outcomes.
The
objective of this study was to evaluate the biospeckle technique using two
distinct wavelengths as a tool to measure the quality of bovine frozen semen
samples under the view of motility parameters.
Materials and
Methods
Semen Samples
Semen
of the bulls were diluted in citrate-yolk extender and frozen according to
routine procedures. Briefly, ejaculates were diluted in yolk extender (lactose
7,4 nmol x L-1; egg-yolk 20% - v/v; added with 105 UI of penicillin K and 105
µg of diidroestreptomyicin), cooled at 5 °C for approximately 90 minutes,
filled into 0.5 ml straws (IMV technologies, France) and after 4 hours at 5 °C
for equilibration they were frozen. Samples were kept in liquid nitrogen (-196
°C) until the time of evaluation. Each sample was thawed in a water-bath at 37
°C, for 30 seconds, and then analyzed. The average sperm cell concentration
used in all experiments was 10 million viable cells, and total concentration
was 30 to 35 x 106 sperm cells per straw.
Biospeckle Setup
Experimental
equipment consisted of low-power Helium-Neonium (He-Ne) and Solid State laser
(power and wavelengths were 10 and 3 mW and 632 and 532 nm, respectively), a
beam splitter, a mirror, a color digital Charge-Coupled Detector Device (CCD)
and a computer with an image processor (Figure 1). A density filter was also
used to ensure the same light intensity in the sample submitted to both laser
sets in experiments II and III. The time history speckle patterns (512 images)
were constructed using a CCD camera of 640x480 pixels. The speckle grains were
always larger than the pixels; the backscattering configuration was the adopted
to illuminate and to get the images in a frame rate of 0.08 seconds, which were
processed forming the Time History Speckle Pattern of 512 x 512 pixels. That
was the database adopted in the biospeckle image analysis and to obtain the
Inertial Moment (IM), (numerical biospeckle index) based on a second order
statistical result, with the use of an intensity dispersion matrix as a secondary
image derived from the Time History Speckle Pattern (THSP), in accordance with
Arizaga, et al. [28].
Experimental
Design
In
experiment I, the straws (n=76) were thawed at 37 oC for 30 seconds and 10 µL
were evaluated microscopically to measure sperm velocity (V) in a relative
scale (1 - slow to 5 - fast movements) and Percent Sperm Cell Motility (PSCM)
in slides with cover slips (15 x 15 mm), warmed in a hot platinum device [29].
In this study, 76 samples were illuminated by the red (n=56) and green (n=20)
laser without a sequence, which means that specimens were lit by red laser and
in other set of illuminations they were lit by the green one. Each sample was
lit by either kind of laser (red and green), after a 30 seconds period of
stabilization in the slide before biospeckle readings. The two laser sets in
the visible range were analyzed by the biospeckle routine analysis. Inertial
moment values were compared to routine semen evaluation using two evaluators,
both trained in the same laboratory, who analyzed V and PSCM by light
microscopy.
In
experiment II, 46 samples were illuminated by red and green lasers alternately,
using the same set up and without moving the sample. Samples were submitted to
the same period of stabilization described in the first experiment. The results
accounted by IM were compared to V, PSCM and the Velocity and Motility Index
(VMI) proposed in this work. Velocity and motility index was proposed as a form
of direct comparison of a single measure evaluated by light microscopy and the
IM generated by BSL. According to VMI, the same value, obtained in the same
metrics, was given to V (measured in a 0 to 5 scale; folded by 20) and PSCM
(measured in percentage; 0-100%) because up to this experiment the influence of
each parameter in BSL analysis was unknown. This index was composed in
accordance with the equation:
VMI = ½ (20V + PSCM) (Eq. 1)
In
experiment III, one single sample was illuminated alternately, throughout time,
with red and green laser in the same intensity, and the evaluations provided by
IM and VMI were compared. Eight illuminations were performed at room
temperature allowing two-minute intervals (0, 2, 4, 6, 8, 10, 12 and 14 minutes
after thawing). To each BSL results obtained, another light microscopy
evaluation was done. The choice of light microscopy was due to the short time
elapsed among the evaluations.
Statistical
Analysis
In
the first and second experiments, IM values - for the combinations found among
the classes of V and PSCM investigated with the two sources of laser (red and
green) - were evaluated by means of an exploratory analysis, involving the
calculation of the mean and standard deviation. In the second experiment, the
analyses included VMI data. In these experiments, three classes of PSCM (C1
< 30%; C2 – 30<50%; C3>50%) and V (C1< 2; C2 – 2<3 and C3>3)
were established to functional analysis. The correlation coefficient between IM
and the variables V, PSCM and VMI, as ascribed by two raters, was obtained and
tested by the Spearman correlation test, with a nominal significance level of
5%. The adjustment of a simple linear regression equation performed for the IM
as related to the VMI for the sources of red and green laser. Three classes of
VMI were also established (C1< 50; C2 – 50< 70; C3>70) as mentioned
before. In order to assess decreasing motility throughout the time a temporal
regression analysis of IM and VMI was done in the third assay. For all
statistic analysis, the statistical software Sisvar® version 4.0 [30] and R®
version 3.4.3 [31] were utilized.
Results
Experiment I
The
results from red laser illumination of the 56 samples showed positive
correlations among V, PSCM and the IM. Apparently, correlations between PSCM
and IM were greater than V and IM (Table 1). The green laser illumination of 20
samples also exhibited similar behavior (Table 2). Spearman coefficients
relating inertial moment to percent sperm cell motility and velocity for two
evaluators are presented. Tables 3 and 4 classified the features analyzed in
groups of inertial moment ranges considering red and green laser illuminations.
The inertial moment was influenced by both parameters.
Experiment II
Table
5 shows that the index can represent both features, once its value is in the
same metrics of V and PSCM, and positive correlations were verified. Similar
values were observed even to V and PSCM separately or grouped in the VMI index.
The classification in classes allowed a relationship of IM values with some
defined ranges of VMI (Table 6). Figure 2 presents curves fitted to IM versus
VMI values under red and green lasers, although fitted curves expressed a
linear pattern.
Experiment III
The
behavior of semen motility in time under the two wavelengths is presented in
Figure 3 with the evolution of the IM in time fitted by a second order curve
with R2 about 0.98 to green and 0.96 to red laser. The VMI behavior is observed
in Figure 4. In both evaluations it was observed a decrease in semen activity.
Discussion
Experiment I
Variations
between estimates made by the two evaluators were kept within 10% limits and so
average values were used to subsequent analysis. Several studies uphold the
reliability of light microscopy data to examine sperm motility, used as control
of IM results in our work.
Coefficient
of Variation (CV) of 21% for sperm motility evaluation by manual methods was
verified [32], close to that observed (24%) in Computer Assisted Sperm Analysis
(CASA) [33]. Nevertheless, a CV of 44.3% was observed among 26 technicians who
estimated sperm cell concentration in a single semen sample using light
microscopy [34]; similar results were obtained for motility and morphology
evaluation. Interestingly, CVs of 39.5% and 45.0% were obtained in an inter
laboratory evaluation when using CASA and subjective (light microscopy)
analyses, respectively [13]. The small variation observed between the two
technicians in this study was due to the standard training received in our
laboratory and was considered precise enough to support our results.
Inertial
moment values ranged from 78 to 311 and 60 to 111 in red and green laser
analyses, respectively. For both laser sources, increasing velocity and percent
sperm cell motility values corresponded to progressively higher average IM
values in the BSL analysis. Previous work of our laboratory (data not
published), using undiluted raw semen of the ram, showed IM values ranging
approximately from 1000 to 3000 in a different setup. IM was strongly
influenced by velocity and percent sperm motility in this species. It was not
possible to affirm that other features of the ejaculate, like seminal plasma
differences, were responsible for interfering with the IM behavior.
Whereas
mean values presented a clear tendency, individual relations expressed some
stable states mainly in the velocity feature. Otherwise, the percent sperm
motility values were reliable in all configurations, which suggest a strong
relation between PSCM and IM, allowing the conclusion that the laser technique
is better to evaluate activity related to the movement. Further studies
comparing computer-assisted sperm analysis and the BSL technique, using bovine
frozen semen, indicated that IM values were related to motile and progressive
percentual count, progressive concentration as well as curvilinear velocity
(unpublished data).
The
results of the present study allowed the conclusion that the inertial moment
presented coherent values to V and PSCM to both evaluators under the two light
sources.
Experiment II
Apparently
no effect of stimulation on the spermatozoa was observed caused by the incidence
of either red or green wavelength, as we can see by the decreasing of the VMI
and IM values throughout the time of analysis (Figures 2 and 3), which
indicates that short time incidence of laser on the specimen was not sufficient
to alter their biological properties. Some authors [25] observed significant
(p<0.05) velocity increase from five minutes of irradiation with the green
laser (532nm; 0.38 J/cm2), while others [27], using red laser (660 nm; 4.0
Joules; power from 30-100 mW; 133 seconds), also observed an increment
(p<0.05) of sperm progressive motility in the bull. In our study these kind
of improvement was not detectable, probably because of the short time of
exposition.
The
regression curve between inertial moment and the VMI index showed that 83% of
IM value variations could be explained by variations that occur in the VMI
index. The off-set between the laser sources can be attributed to the
back-scattering configuration, with the back ground and the liquid absorbing
more the real wavelength without interfering with the dynamics of the
phenomenon monitored, thus presenting the same tendency to both cases (laser
sources).
Results
showed that the index proposed in this work is a reliable tool to
simultaneously compare V and PSCM with IM. Its parametric characteristic can be
pointed out as a positive factor.
Experiment III
The off-set between the evolution of red and green laser curve over a sample in the third experiment reinforces the hypothesis about the absorptive property of the light by the background-sperm. When light microscopy evaluation was compared to IM data over the time it has been observed that spermatozoa motility decreasing judged by the BSL was less intense in relation to the human estimates. This fact can be interpreted as the human tendency to force a continuous decrease in the result of VMI, whereas the objective results of IM curves point to an expected or reliable tendency of physical phenomena. Besides, the BSL analysis is not subjective and inertial moment values are more precise to assess the behavior of a biological phenomenon.
In
conclusion, evaluation of semen motility by the IM could be a reliable and
objective approach to estimate fertility in the frozen semen.
Acknowledgment
The
authors wish to express their gratitude to Dr Fernando Vilela (ABS-Pecplan,
Uberaba, Brazil), for the technical assistance. Reported research was supported
financially by FAPEMIG (Grant EDT – CVZ 94/07) and CNPq.
Figure 1: Equipment set-up.
Figure 2: Mean values of Inertial
Moment (IM) obtained by the types of red (RIM) and green (GIM) laser related to
Velocity and Motility Index (VMI).
Figure 3: Inertial Moment (IM) values obtained by red
and green lasers light related to the time.
Figure 4: Velocity and Motility Index (VMI) values obtained by microscopy evaluation for samples by red and green lasers light related to the time.
|
|
V A1 |
V A2 |
PSCM A1 |
PSCM A2 |
IM |
|
V A1 |
1.0000 |
|
|
|
0.5906 |
|
(p<0.0001) |
|||||
|
V A2 |
0.8362 |
1.0000 |
|
|
0.5436 |
|
(p<0.0001) |
(p<0.0001) |
||||
|
V |
---- |
---- |
---- |
---- |
0.5945 |
|
Me |
(p=0.0002) |
||||
|
PSCM |
0.8767 |
---- |
1.0000 |
|
0.6747 |
|
A1 |
(p<0.0001) |
(p<0.0001) |
|||
|
PSCM |
----- |
0.8631 |
0.9228 |
1.0000 |
0.6469 |
|
A2 |
(p<0.0001) |
(p<0.0001) |
(p<0.0001) |
||
|
PSCM |
---- |
---- |
---- |
---- |
0.6651 |
|
Me |
(p<0.0001) |
Table 1: Spearman correlation coefficients estimate with their respective
significances for all the variables velocity (V) and Percent Sperm Cell
Motility (PSCM) ascribed by two raters (A1 and A2) and for their means (Me),
and Inertial Moment (IM) on the basis of 56 samples of bovine frozen semen
analyzed under red laser.
|
|
V A1 |
V A2 |
PSCM A1 |
PSCM A2 |
IM |
|
V A1 |
1.0000 |
|
|
|
0.6811 |
|
(p=0.0009) |
|||||
|
V A2 |
0.8709 |
1.0000 |
|
|
0.7999 |
|
(p<0.0001) |
(p<0.0001) |
||||
|
V |
---- |
---- |
---- |
---- |
0.7342 |
|
Me |
(p=0.0002) |
||||
|
PSCM |
0.7679 |
---- |
1.0000 |
|
0.6563 |
|
A1 |
(p<0.0001) |
(p=0.0017) |
|||
|
PSCM |
----- |
0.8461 |
0.9378 |
1.0000 |
0.7579 |
|
A2 |
(p<0.0001) |
(p<0.0001) |
(p=0.0001) |
||
|
PSCM |
---- |
---- |
---- |
---- |
0.6840 |
|
Me |
(p=0.0009) |
Table 2: Spearman correlation coefficients estimate with their respective
significances for all the variables velocity (V) and percent sperm motility
(PSCM) ascribed by two raters (A1 and A2) and for their means (Me), and
Inertial Moment (IM) on the basis of 20 samples of bovine frozen semen analyzed
under green laser.
|
|
|
Percent sperm cell motility classes* |
Mean |
||
|
|
|
1 |
2 |
3 |
|
|
Velocity classes* |
1 |
101.79 |
145.16 |
---- |
106.13 |
|
(13.06) |
|
|
(18.43) |
||
|
2 |
100.19 |
145.75 |
172.75 |
135.40 |
|
|
(15.77) |
(37.40) |
|
(38.51) |
||
|
3 |
---- |
158.96 |
180.21 |
176.79 |
|
|
|
(41.44) |
(48.49) |
(47.45) |
||
|
Mean |
|
101.29 |
149.84 |
179.94 |
|
|
|
(13.28) |
(36.57) |
(47.57) |
|
|
|
*Classes of percent sperm motility: 1 (≤30); 2 (30 a 50); 3 (>50).
Classes of velocity: 1 (≤2); 2 (2 a 3); 3 (>3). |
|||||
|
|
|
Percent sperm cell motility classes* |
Mean |
||
|
1 |
2 |
3 |
|||
|
Velocity classes* |
1 |
64.96 |
65.82 |
---- |
65.13 |
|
(6.04) |
(5.25) |
||||
|
2 |
75.58 |
66.37 |
70.50 |
68.59 |
|
|
|
(3.95) |
(4.88) |
|||
|
3 |
---- |
80.85 |
87.74 |
85.44 |
|
|
|
(5.62) |
(16.02) |
(13.43) |
||
|
Mean |
|
67.08 |
71.73 |
85.28 |
|
|
|
(7.07) |
(8.53) |
(16.02) |
|
|
|
*Classes of percent sperm motility: 1 (≤30); 2 (30 a 50); 3 (>50).
Classes of velocity: 1 (≤2); 2 (2 a 3); 3 (>3). |
|||||
|
|
V |
PSCM |
VMI |
IM Red. |
IM Green |
|
V |
1.0000 |
|
|
|
0.5906 |
|
(p<0.0001) |
|||||
|
PSCM |
0.9506 |
1.0000 |
|
|
0.6132 |
|
(p<0.0001) |
(p<0.0001) |
||||
|
VMI |
---- |
---- |
1.0000 |
|
0.6186 |
|
(p<0.0001) |
|||||
|
IM Red |
0.8019 |
0.8267 |
0.8403 |
1.0000 |
---- |
|
(p<0.0001) |
(p<0.0001) |
(p<0.0001) |
|||
|
IM Green |
0.5906 |
0.6132 |
0.6186 |
---- |
1.0000 |
|
(p<0.0001) |
(p<0.0001) |
(p<0.0001) |
Table 5: Spearman correlation coefficients estimate with their respective significances for all the variables velocity (V), Percent Sperm Motility (PSCM), Inertial Moment (IM) and Velocity and Motility Index (VMI) on the basis of 46 samples of bovine frozen semen analyzed under red and green lasers light.
|
Laser light |
*VMI
classes |
||
|
|
1 |
2 |
3 |
|
Red |
130.19 (31.26) |
205.94 (69.36) |
402.61 (261.25) |
|
Green |
187.75 (58.24) |
248.29 (130.9) |
545.69 (362.15) |
|
*Classes of VMI: 1 (≥50); 2 (50 a 70); 3 (>70). |
|||
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