Vol. 7, No. 3
221
ORIGINAL RESEARCH
Characteristics of sperm motility in boar semen
diluted in different extenders and stored
for seven days at 18ºC
Mark J. Estienne1, Allen F. Harper and Jennifer L. Day
Department of Animal and Poultry Sciences, Virginia Polytechnic
Institute and State University, Blacksburg, VA, USA
Received: 3 October 2007; accepted: 3 November 2007
SUMMARY
Although numerous extenders exist for diluting boar semen, little research
has been conducted comparing commercial extenders with regard to maintaining sperm motility during storage. The objective was to use a computer-assisted sperm analysis system to assess motility of boar spermatozoa
diluted in Beltsville Thawing Solution, Merck-III, Androhep-lite, Sperm
Aid, MR-A, Modena, X-Cell, VSP, and Vital. Ejaculates from boars (n=10)
were collected and sub-samples were diluted (35×106 spermatozoa/ml) in
the different extenders and stored for seven days at 18ºC. Extender by day
interactions were detected (p<0.01) and on each day post collection, there
were numerically small, but statistically significant differences in characteristics of sperm motility among extenders. For example, on day 7, the
percentages of motile and progressively motile spermatozoa were highest
(p<0.05) in X-Cell (90.7%) and Modena (63.9%), respectively. The average velocity measured over the actual point-to-point track followed by the
Corresponding author: Virginia Tech-Tidewater Agricultural Research and Extension Center, 6321
Holland Road, Suffolk, VA 23437, USA; E-mail: [email protected]
1
Copyright © 2007 by the Society for Biology of Reproduction
222
Semen extenders and sperm motility in boars
sperm cell (VCL; 198.2 µm/s) and path velocity of the smoothed cell path
(VAP; 106.4 µm/s) were highest (p<0.05) in Vital and Modena, respectively.
Average velocity measured in a straight line from the beginning to the end
of the track (VSL; 78.3 µm/s), average value of the ratio VSL/VAP (straightness; 73.2) and average value of the ratio VSL/VCL (linearity; 44.1) on day
7 were highest in Androhep-lite. In summary, changes in sperm motility during storage were affected by the extender utilized, but with the exception of
Sperm Aid, all extenders maintained a high degree of sperm motility through
7 days of storage. Reproductive Biology 2007 7 3:221-231.
Key words: sperm motility, extender, boar
INTRODUCTION
Similar to commercial swine industries in many other countries, artificial insemination (AI) is the predominant method used for breeding in the U.S. and at
least 70% of sows and gilts are mated using this technique [8]. Semen used for
AI is typically collected from boars housed at off-site studs and is ultimately
diluted in any one of a variety of commercially available extenders. These extenders are used to create multiple insemination doses from a single ejaculate
and contain buffers and nutrients that provide spermatozoa with an environment that maintains viability for three or more days post-collection [6].
Although numerous extenders are available, relatively few trials have
been conducted comparing products with regard to maintenance of various
indicators of sperm quality such as motility during storage [1, 6, 7, 11]. Thus,
the objective of this experiment was to compare, using a computer-assisted
sperm analysis (CASA) system, characteristics of sperm motility in boar semen diluted in different extenders and stored for seven days at 18ºC.
MATERIALS AND METHODS
This research was conducted during July at the Virginia Tech- Tidewater
Agricultural Research and Extension Center in Suffolk, VA, USA. The barn
Estienne et al.
223
housing experimental animals was curtain-sided and serviced by a thermostatically-controlled sprinkler cooling system. Mature, Yorkshire×Landrace
boars (428±12 days of age; mean±SE; n=10) were individually housed in
pens (4.5 m2) that had a combination of solid concrete and steel rod flooring and were each equipped with a nipple drinker. A separate pen (5.8 m2)
contained an artificial sow (Minitube of America, Inc.; Verona, WI, USA)
that facilitated semen collection via the gloved hand technique. Boars were
fed daily 2.3 to 2.7 kg of a fortified, corn and soybean meal-based diet that
met or exceeded the recommendations for the various nutrients [9].
Experimental Protocol
The experimental protocol was approved by the Animal Care Committee
of Virginia Polytechnic Institute and State University. Semen was collected
from each boar by the same trained technician. During collection, semen
was filtered (US BAG; Minitube of America, Inc.) to remove the gel fraction
of the ejaculate. Gel-free volume of semen was determined gravimetrically
using a top-loading balance (Acculab; Minitube of America, Inc.).
Semen samples were diluted in Beltsville Thawing Solution (BTS; Swine
Genetics International, Ltd., Cambridge, IA, USA) at 37ºC to achieve a ratio of
semen to extender of 1 to 30. Samples were then loaded in a Leja standard count
4 chamber slide (IMV USA, Maple Grove, MN, USA) and sperm concentration and characteristics of sperm motility determined using a computer-assisted
sperm analysis system (CASA; Integrated Visual Optical System, Version 12;
Hamilton Thorne Research, Beverly, MA, USA). Starting CASA values for
boar sperm analysis were consistent with manufacturer recommendations. The
following characteristics of sperm motility were determined: percentages of
motile and progressively motile spermatozoa, path velocity of the smoothed
cell path (VAP), average velocity measured in a straight line from the beginning
to the end of the sperm track (VSL), average velocity measured over the actual
point to point track followed by the sperm cell (VCL), straightness (average
value of the ratio VSL/VAP) which measured the departure of the sperm cell
path from a straight line, and linearity (average value of the ratio VSL/VCL)
which measured the departure of the cell track from a straight line.
224
Semen extenders and sperm motility in boars
Sub-samples of each whole ejaculate were diluted in nine different extenders (tab. 1) and stored in plastic artificial insemination bottles (Minitube of America, Inc.). Each bottle contained 3 billion spermatozoa in 85
ml of semen and extender (35×106 spermatozoa/ml). Extended semen was
stored in a semen storage unit (Minitube of America, Inc.) at 18ºC for 7
days (day of collection=day 0). On each of day 0 through 7, an aliquot of
each sample was removed and warmed to 37ºC for 30 minutes. The sample was then analyzed for characteristics of sperm motility as described
above.
Table 1. Commercially available products used to determine the effects of boar semen
extender on characteristics of sperm motility during storage for seven days at 18ºC
Extender
Beltsville Thawing
Solution (BTS)
Merck III (M-III)
Androhep-lite
Claimed
Preservation Source
Period (days)
Lot No.
3
IMV USA; Maple
Grove, MN, USA
3313B71
Not reported
Minitube of
America, Inc.;
Verona, WI, USA
5238016
6
Minitube of
America, Inc.
0110883571
Sperm Aid
3-7
International Boar
Semen; Eldora,
IA, USA
0515031
X-Cell
<7
IMV USA
0552B24
MR-A
6-7
International Boar
Semen
E019-CADU0204
Modena
5-7
International Boar
Semen
02306503
Vital
<5
IMV USA
1932B94
VSP
<4
IMV USA
3422B37
Estienne et al.
225
Statistical analyses
Data were analyzed by ANOVA using the GLM procedure of SAS (SAS Institute, Inc., Cary, NC, USA). The model included extender, boar, day, extender
by boar, extender by day, and boar by day as possible sources of variation.
The effects of extender and day were tested using boar by extender and boar
by day, respectively, as the error terms. The effects of boar, extender by day,
boar by extender, and boar by day were tested using boar by extender by day
(residual) as the error term. Significant effects of extender by day and boar by
extender were detected and subsequent analyses were then conducted. Within
a specific day or boar, one-way ANOVA was used to determine the main effect
of extender. Means were then compared using Tukey’s studentized range test.
RESULTS
Results of the statistical analysis are displayed in Table 2. There were effects of extender, boar, boar by extender, extender by day, and boar by day
Table 2. Effects (p-values) of extender, boar (n=10), day of storage, and two way
interactions on characteristics of sperm motility in semen stored for seven days at 18ºC
Item:
ExtendBoar
er
Day
Boar ExtendBoar
× Exer
× Day
tender × Day
Motility, %
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
Progressive motility, %
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
VCL, µm/s1
<0.01
<0.01
0.03
<0.01
<0.01
<0.01
VAP, µm/s2
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
VSL, µm/s3
<0.01
<0.01
0.01
<0.01
<0.01
<0.01
Straightness, %4
<0.01
<0.01
0.16
<0.01
<0.01
<0.01
Linearity, %5
<0.01
<0.01
0.45
<0.01
<0.01
<0.01
average velocity measured over the actual point to point track followed by the cell, 2path velocity of the
smoothed cell path, 3average velocity measured in a straight line from the beginning to the end of track,
4
STR= average value of the ratio VSL/VAP; measured the departure of the cell path from a straight line,
5
LIN= average value of the ratio VSL/VCL; measured the departure of the cell track from a straight line
1
Day 0
Motility, %
Progressive motility,%
VCL, µ/s1
VAP, µ/s2
VSL, µ/s3
Straightness, %4
Linearity, %5
Day 3
Motility, %
Progressive motility,%
VCL, µ/s1
VAP, µ/s2
VSL, µ/s3
Straightness, %4
Linearity, %5
Day 5
Motility, %
Progressive motility,%
VCL, µ/s1
VAP, µ/s2
VSL, µ/s3
Straightness, %4
Linearity, %5
Item
  85.5a,b
  60.5
181.3
104.6
  80.1a
  73.1a
  44.4a
  79.9b
  49.4
173.2
  94.0
  69.8a,b
  69.8b
  39.9b
  90.2a
  61.3
167.4
  92.0
  66.4b
  69.5b
  39.9b
  90.1a,b
  59.6
158.8
  86.7
  62.2b
  69.3b,c
  39.7b,c
  91.5a
  65.0
196.6
105.2
  72.8a,b
  66.4c
  36.9c,d
  91.8a
  63.7
191.3
102.6
  70.0a,b
  65.7d
  36.8d
  88.7a,b
  62.7
189.0
104.7
  76.1a,b
  69.5b
  39.9b
  89.5a,b
  63.0
187.4
104.4
  75.8a,b
  69.5b
  40.2b
  91.0a
  58.8
199.4
105.3
  69.2a,b
  63.0d
  34.6d
  91.1a,b
  56.9
190.6
100.7
  64.8b
  61.8e
  34.1e
   94.2
   69.6a
196.5a,b,c
102.9a,b,c
  71.2b,c
  66.8b,c
  36.7c,d
  85.6a,b
  56.5
186.1
101.2
  73.3a,b
  68.7b,c
  39.0b,c
  82.4b
  48.4
161.2
  86.9
  63.8b
  69.2b,c
  38.9b,c,d
  93.2
  71.7a
195.7a,b,c
107.0a,b
  78.6a,b
  71.0a,b
  40.5a,b,c
  89.0a,b
  58.8
177.3
  96.6
  68.4a,b
  68.0b,c
  38.6b,c
  87.2a,b
  62.7
181.7
104.5
  79.6a
  72.7a
  43.6a
  94.1
  73.1a
205.7a,b
111.4a,b
  81.4a,b
  70.6a,b
  39.6a,b,c
  85.7a,b
  55.5
173.9
  95.1
  68.5a,b
  68.5b,c
  39.4b,c
  93.2
  71.5a
172.8b,c
  95.8b,c
  70.6b,c
  71.7a,b
  41.6a,b
  89.6a,b
  59.0
166.3
  91.0
  64.4b
  68.5b,c
  39.1b,c,d
  91.0
  53.8b
168.9c
  86.2c
  57.9c
  63.5c
  33.7d
  94.6
  69.9a
189.6a,b,c
  99.8b,c
  70.4b,c
  67.8b,c
  37.4b,c,d
  93.2
  70.7a
189.9a,b,c
102.1a,b,c
  74.3b,c
  70.2a,b
  39.3a,b,c
  96.0
  78.7a
214.0a
121.7a
  93.0a
  73.9a
  43.9a
Merck- Andro- SpermX-Cell MR-A Modena Vital
III
hep-lite Aid
BTS
  87.9a,b
  60.1
195.6
106.1
  74.9a,b
  67.4b,c
  38.0b,c
  88.2a,b
  59.1
190.4
103.4
  72.3a,b
  66.8c,d
  37.6c,d
93.8
71.0a
184.8a,b,c
100.5b,c
  73.1b,c
  70.5a,b
  39.9a,b,c
VSP
0.07
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
p
1.2
1.4
3.7
1.8
1.4
0.9
0.8
<0.01
0.10
0.03
0.06
<0.01
<0.01
<0.01
0.9 0.05
1.5 0.10
4.4 0.01
2.4 0.01
1.9 <0.01
1.0 <0.01
0.8 <0.01
0.4
2.2
3.0
3.8
3.2
1.0
0.9
SE
Table 3. Effects of extender type on characteristics of sperm motility on selected days after semen was collected from
boars (n=10) and stored at 18ºC
226
Semen extenders and sperm motility in boars
  88.9a
  58.3a
181.3a,b
  98.1a,b
  69.3a,b
  67.5b
  38.0b,c
  84.0a,b
  53.5a,b
184.2a,b
  98.6a,b
  70.2a,b
  67.5b
  37.7b,c
  85.5a
  59.5a
177.4a,b
102.1a,b
  78.3a
  73.2a
  44.1a
  74.5b
  40.7b
162.2b
  86.7b
  63.7b
  68.7b
38.8b,c
  90.7a
  61.2a
172.6a,b
  94.4a,b
  67.6a,b
  68.9b
  39.2b,c
  90.4a
  62.8a
192.8a,b
102.7a,b
  71.3a,b
  66.6b
  36.7c,d
  89.4a
  63.9a
193.1a,b
106.4a
  77.4a
  69.5b
  39.8b
  90.4a
  57.2a,b
198.2a
104.7a,b
  68.4a,b
  62.4c
  34.2d
  89.2a
  60.0a
194.5a,b
104.9a,b
  73.5a,b
  67.1b
  37.4b,c
1.7
2.3
3.9
2.0
1.5
0.9
0.9
<0.01
<0.01
0.02
0.03
0.01
<0.01
<0.01
Merck-III
69.5b,c
67.4b,c
67.6b
.
68.1b,c
67.1b,c
71.0a,b
67.8b
69.3a,b
69.0a,b
BTS
69.4b,c
68.3b,c
67.4b
.
69.0b
68.0b,c
71.1a,b
68.5a,b
67.8b
68.1b
74.8a
71.5a
72.0a
.
71.9a
72.9a
74.1a
73.3a
72.0a
73.8a
68.3c,d
66.3c,d
67.3b
.
68.3b,c
67.0b,c
70.0b
71.8a,b
69.3a,b
70.1a,b
70.9b,c
69.4a,b
69.4a,b
.
69.9a,b
67.1b,c
72.0a,b
69.9a,b
70.1a,b
70.5a,b
65.1d,e
64.3d,e
67.3b
.
68.1b,c
66.0c
69.4b
67.4b
67.5b
65.5b,c
71.9a,b
68.4b,c
68.0b
.
69.5b
69.0b
70.8a,b
69.1a,b
69.9a,b
68.3b
63.5e
61.5e
63.5c
.
66.1c
60.9 d
64.8 c
62.1 c
62.6 c
61.9 c
AndroSpermModehepX-Cell MR-A
Vital
Aid
na
lite
69.4b,c
65.6c,d
67.1b
.
69.0b
67.0b,c
68.5b
67.4 b
67.6 b
67.4 b
VSP
1.1
0.9
0.7
.
0.5
1.0
0.8
1.0
0.8
1.1
SE
<0.01
<0.01
<0.01
.
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
p
1
average value of the ratio of the average velocity measured in a straight line from the beginning to the end of the track [VSL] to the path velocity
of the smoothed cell path [VAP], 2data inadvertently lost for Boar No. 28, a,b,c,dvalues within a row with different superscripts differ (p<0.05)
Ejaculate
Boar Volume, Sperm
Conc.,
ml
106/ml
25
167.4
190
26
227.9
215
27
253.1
150
282
214.2
144
29
182.7
160
32
135.0
132
33
138.9
176
34
228.7
186
35
170.8
182
36
215.8
145
Table 4. Ejaculate characteristics and straightness1 of sperm motility in semen from different boars (n=10) diluted in
different extenders
2
a,b,c,d
values within a row with different superscripts differ (p<0.05), 1average velocity measured over the actual point to point track followed by the cell.
path velocity of the smoothed cell path, 3average velocity measured in a straight line from the beginning to the end of track, 4STR= Average value
of the ratio VSL/VAP; measured the departure of the cell path from a straight line, 5LIN= Average value of the ratio VSL/VCL; measured the
departure of the cell track from a straight line
Day 7
Motility, %
Progressive motility,%
VCL, µ/s1
VAP, µ/s2
VSL, µ/s3
Straightness, %4
Linearity, %5
Table 3. Continued from previous page.
Estienne et al.
227
228
Semen extenders and sperm motility in boars
(p<0.01). An effect of day was evident for the percentages of motile and
progressively motile spermatozoa, VAP and VSL (p<0.01), as well as VCL
(p=0.03); however, there was no effect of day on straightness or linearity
(p>0.1). Because extender by day was significant, we conducted subsequent analyses, during which the effects of extender on characteristics of
sperm motility on specific days after collection were determined. For example, Table 3 shows characteristics of sperm motility for semen stored in
the different extenders as evaluated on days 3, 5, and 7 post-collection. For
each characteristic of sperm motility, there were in general numerically
small, but statistically significant, differences among extenders. Perhaps
noteworthy, however, is that on each of days 3, 5, or 7, VSL and straightness and linearity of sperm motility was highest (p<0.05) for semen extended in Androhep-lite. On day 7, semen extended in Androhep-lite had a
VSL of 78.3 µm/s, straightness of 73.2%, and linearity of 44.1%.
On day 7, the percentages of motile and progressively motile spermatozoa were highest (p<0.05) in X-Cell (90.7%) and Modena (63.9%), respectively. The VCL (198.2 µm/s) and VAP (106.4 µm/s) were highest
(p<0.05) in Vital and Modena, respectively.
There was an effect of boar by extender (p<0.01) for each characteristic
of sperm motility (tab. 2) as illustrated by the straightness of sperm motility in semen collected from different boars and diluted in the different
extenders (tab. 4). For each boar there were numerically small, but statistically significant differences in straightness among extenders. Although the
absolute ranking of extenders (high to low) with regard to straightness of
sperm motility varied among animals, for each boar Androhep-lite was the
highest (p<0.05) and Vital, the lowest (p<0.05).
DISCUSSION
Numerous semen extenders are available for use by commercial swine
producers. In the current investigation, we compared nine such extenders
with regard to sperm motility characteristics during storage for seven days
at 18ºC. We focused on sperm motility because it indicates active metabo-
Estienne et al.
229
lism and integrity of membranes, and is of great importance for fertilizing
capacity [4]. In commercial boar studs, sperm motility assessment is a
technique used routinely for semen evaluation [10]. It is acknowledged,
however, that like any on-farm laboratory test, assessment of sperm motility
does not provide a completely accurate or quantitative measure of semen fertility per se. At subjectively determined sperm motilities greater than 60%, no
relationship existed between sperm motility and farrowing rates and number
of pigs born live [2]. Reproductive performance was reduced, however, in
sows inseminated with semen the sperm motility of which was less than 60%,
suggesting that subjective appraisals of the percentage of sperm cells displaying motility can at least be used to identify ejaculates of overtly poor quality.
Visual motility assessment tends to be highly subjective and in many boar
studs is being replaced with more objective, detailed and repeatable assessments using automated systems [12]. Moreover, Holt et al. [3] demonstrated
that up to 24% of the variance in litter size due to boars on commercial swine
farms could be explained by differences in sperm motion characteristics determined using CASA technology. Boar spermatozoa that exhibited increased
VSL and track linearity were associated with larger litter sizes.
As expected, there were numerous differences among extenders with
regard to characteristics of sperm motility over storage time. X-Cell is generally classified as a long-term extender with a manufacturer claimed preservation period of up to seven days. Interestingly, shorter-term extenders
used in this study, such as BTS which has a claimed preservation period of
three days displayed an ability to maintain many characteristics of sperm
motility that was not dramatically different from longer-term extenders.
For example, the percentage of motile and progressively motile spermatozoa on days 3, 5 or 7 was similar for BTS and X-Cell. Consistent with our
results, Vyt et al. [11] reported that the percentage of dead spermatozoa in
semen extended in long-term (Mulberry III, Androhep and Acromax) and
short-term (Kobidil and BTS) diluents, did not vary significantly during
storage for seven days. In that study, the two short-term extenders preserved good sperm motility scores for three to four days, and although
there was a subsequent decline, motility was still evident seven days after
collection. Additionally, Laforest and Allard [7] compared semen preserva-
230
Semen extenders and sperm motility in boars
tion with MR-A, BTS, Modena, and Androhep and found no differences in
motility scores or fertility due to extender or day of storage. Using CASA
technology, De Ambrogi et al. [1] reported no effects of extender by day
on total motility, linear motility, VSL, VAP or VCL for semen extended in
BTS, MR-A or X-Cell, suggesting the diluents maintained characteristics
of sperm motility in a similar fashion during storage at 17ºC for four days.
Kuster and Althouse [6] compared Androhep and X-Cell extenders and
found no difference in the percentage of motile spermatozoa from days 0
to 6 post-collection. However, gilts inseminated with Androhep-diluted semen showed a decrease in farrowing rate compared with gilts inseminated
with X-Cell diluted semen when stored for 5 to 6 days prior to use.
Based on results of the current study and previous research [1, 7, 11] it
would appear that there are at most only subtle differences among extenders with regard to maintaining the percentage of spermatozoa exhibiting
motility during storage. However, our results suggest that other, more detailed characteristics of sperm motility may be impacted by the extender
employed. For example, among the extenders examined, Androhep-lite
had the greatest VCL, and straightness and linearity of sperm movement
throughout the study. These findings certainly warrant further investigation because as previously mentioned, semen containing boar spermatozoa
exhibiting increased VSL and track linearity was associated with larger
litter sizes after artificial insemination [3].
Kommisrud et al. [5] reported a significant influence of boar on motility in semen stored for 5 days in BTS, suggesting variability exists among
boars with regard to the ability to maintain sperm motility during storage.
In the current investigation, significant effects of the interaction between
boar and extender, and boar and day, were detected for each characteristic
of sperm motility. These results suggest that there is at least subtle variation among boars in terms of identifying extenders expected to be most
successful in maintaining characteristics of sperm motility during storage.
In summary, changes in sperm motility during storage were affected by
the extender utilized, but with the exception of Sperm Aid, all extenders
maintained a high degree of sperm motility during storage. In general, even
extenders classified as short-term were reasonably successful at maintaining
Estienne et al.
231
motility up to seven days post-collection. Subtle differences in characteristics of sperm motility stored in various extenders and how these differences
equate to swine fecundity will be addressed in future research.
ACKNOWLEDGEMENTS
This research was supported by a grant from the Virginia Pork Industry
Board. The authors thank Dr. David Notter for his statistical expertise
during data analysis.
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