Ann. Anim. Sci., Vol. 9, No. 1 (2009) 43 – 49
Membrane integrity, energy status and motility
of rabbit spermatozoa stored for 2 days at 15°C*
Piotr Gogol, Agnieszka Wierzchoś-Hilczer
Department of Biotechnology of Animal Reproduction, National Research Institute
of Animal Production, 32-083 Balice n. Kraków, Poland
Abstract
The effects of semen storage on membrane functional integrity, ATP content and motility of rabbit spermatozoa were investigated. The ejaculates were diluted with a commercial rabbit semen
extender Galap. Semen was stored at 15°C and changes in quality were evaluated after 2, 24 and
48 hours of preservation. Membrane integrity was assessed with the HOS test and ATP content
using a bioluminescence method. The time of storage had a significant effect on all studied parameters. However, motility and ATP content were significantly more affected than membrane integrity. After 48-h of preservation, sperm motility percentage and ATP content dramatically decreased
by over 50%, while percentage of spermatozoa positively reacting to the HOS test decreased by
approximately 14%. A significant correlation was found between the percentage of motile spermatozoa and intracellular ATP content (r = 0.59). HOS test score was correlated with neither sperm
motility percentage nor sperm ATP content. The present results indicate that compared to membrane integrity, assessed with the HOS test, sperm ATP content and motility are more sensitive
indicators of the effect of storage time on rabbit semen. The significant correlation between ATP
content and the percentage of motile spermatozoa indicates that ATP assessment may be useful as
an additional, objective laboratory test.
Key words: rabbit, semen storage, HOS test, ATP, sperm motility
In commercial practice rabbit semen used for artificial insemination is stored at
15°C following the addition of an appropriate diluent. Results obtained with deep
frozen semen are not yet satisfying. Many extenders have been developed for rabbit
semen storage in liquid state. Of these, an extender containing Tris, citric acid and
glucose, as well as commercial extenders such as Galap (IMV, France) have found
wide application in practice (Alvarino, 2000). Nevertheless the diluents are not able to
completely prevent changes to a range of essential features for sperm functions which
rapidly occur under in vitro conditions.
This work was conducted as part of NRIAP statutory activity, project no. 3423.1.
44
P. Gogol and A. Wierzchoś-Hilczer
Therefore, studies are under way to improve extenders for storage of rabbit semen
at above-zero temperatures. Efforts are made to increase semen storage time without
reducing fertility (Maertens and Luzi, 1995; Alvarino, 2000; Roca et al., 2000).
The correct choice of the optimum semen preservation method is based on the use
of accurate and objective evaluation of sperm, which enables structural and biochemical changes responsible for poorer semen quality to be identified.
The classical method of semen evaluation is based on the application of a number
of tests that are simple to perform and relatively inexpensive. In general, they measure
the progressive motility, the percentage of viable cells and the morphology.
Once it was recognized that the standard spermiogram does not provide reliable
diagnostic information about fertility, it was necessary to improve the analytic procedures. The new sperm assays try to explore the functional capacity of the spermatozoa.
Among sperm functional tests the study of plasmalemma is of particular importance since a biochemically active membrane is required in the process of capacitation, the acrosome reaction and the binding of the spermatozoon to the oocyte surface
(Correa and Zavos, 1994). These tests of membrane function, like the hypoosmotic
swelling test (HOS test) yield information regarding sperm membrane functionality.
In the HOS test, viable sperm with a functional tail membrane, show a curling flagellum, while non-viable sperm maintain tail linearity. The HOS test has been widely
used in both human andrology (Hossain et al., 1998) and domestic species (Rota et al.,
2000; Perez-Llano et al., 2001; Janett et al., 2003), but its use for rabbit spermatozoa
has been limited (Ducci et al., 2002).
Another functional test exploring the cellular metabolic activity is ATP measurement. ATP is required for sperm motility as a cellular energy source. It can be suggested that the irreversible loss of motility may be a consequence of a decrease in
ATP production to a level incapable of supporting motility. The ATP content of bird
spermatozoa was used as a semen quality parameter, as it was correlated with fertility
(Wishart, 1995). The literature results concerning the relation between sperm ATP
content, motility and fertility for mammals (human and domestic species) are contradictory (Januskauskas and Rodriguez-Martinez,1995; Gumińska et al., 1997; Minelli
et al., 1999; Tardif et al., 1999). These contradictory results may be caused by large
experimental differences.
The aim of the study was to check some parameters (membrane functional integrity, ATP content and motility) in rabbit spermatozoa during 48 h of storage at 15°C,
which would be used to predict the fertilizing potential of the semen.
Material and methods
Animals and experimental procedure
Semen of 9 sexually mature New Zealand White male rabbits was used in the
experiment. The semen was collected by means of an artificial vagina at weekly intervals. Just after collection, ejaculate volume was determined and sperm concentration
calculated using a Buerker chamber. Ejaculates with at least 0.6 ml volume and sperm
Membrane integrity, energy status and motility of rabbit spermatozoa
45
concentration in excess of 300 million/ml were qualified for the study. Each qualified
ejaculate was diluted at a 1:10 ratio with rabbit semen extender Galap (IMV, France)
and stored for up to 48 h at 15ºC. Aliquots were removed after 2 (T2), 24 (T24) and
48 h (T48) of storage for assessment of semen quality.
Hypoosmotic swelling test
The hypoosmotic swelling test (HOS test) was performed by mixing 100 μl of semen sample with 900 μl of 60 mOsmol fructose solution (Ducci et al., 2002). After 30
minutes of incubation at 37°C, a drop of 10 μl was smeared on a microscope slide and
observed in a phase contrast microscope at 400x magnification. A total of 200 cells
were observed. The spermatozoa were classified as positive or negative based on the
presence or absence of a coiled tail.
Adenosine triphosphate measurement
The adenosine triphosphate (ATP) from spermatozoa cells was determined
using the ViaLight Plus kit according to the manufacturer’s instructions (Cambrex
Bio Science Rockland, Inc., USA). The kit is based upon the bioluminescent measurement of ATP in mammalian cells. The bioluminescent method utilizes an enzyme,
luciferase, which catalyses the formation of light from ATP and luciferin. The emitted light intensity is linearly related to the ATP concentration and is measured using
a luminometer.
Prior to assay, samples composed of 10 μl of diluted semen were mixed with 100
μl Cell Lysis Reagent and incubated at room temperature for 5 minutes to extract ATP
from cells. Following the addition of 100 μl ATP Monitoring Reagent via automated
dispensers, luminescence was measured using an AutoLumat LB953 luminometer.
Sperm ATP content from each probe was assessed in duplicate.
Assessment of sperm motility
Samples of semen were placed in a water bath at 37°C and after 15 minutes of
incubation motility was assessed using a phase contrast microscope (100x magnification) and a heating plate (37°C). The motility was expressed as percentage of progressively motile spermatozoa.
Statistical analysis
Data were subjected to variance analysis according to the GLM procedure of the
Statistical Analysis System (SAS, version 8.2).
The significance of differences between means was tested by the least squares
method using the LSMEANS procedure. The correlations between sperm quality parameters were calculated using Spearman’s rank method.
Results
The time of storage had a significant effect on percentage of spermatozoa with
coiled tails, intracellular ATP concentration and percentage of motile spermatozoa
46
P. Gogol and A. Wierzchoś-Hilczer
(Table 1). Mean percentage of rabbit spermatozoa showing coiling (positive reaction to the HOS test) decreased gradually with increasing storage time. Significant
differences between T2 versus T48 were observed but not between T2 v T24 and
T24 v T48. Mean percentage of spermatozoa with intact membrane and motile spermatozoa at T2 was similar (69.5 and 71.3, respectively). After 24 and 48 h of semen
preservation, the decrease in motility was considerably higher than the decrease in the
HOS test score.
Table 1. Effect of storage time on sperm parameters ( ± SE; n = 24)
x
2h
HOS-test1
ATP (%)2
Motility (%)
69.54±2.75 A
24 h
48 h
65.33±1.67
58.04±3.28 B
100±0 A
70.97±4.27 B
43.15±3.90 C
71.25±2.02 A
52.50±4.27 B
15.42±3.66 C
Percentage of spermatozoa with coiled tails.
ATP level at 2 h was assumed to be 100%.
A, B, C – values in rows with different letters differ significantly (P<0.01).
1
2
Figure 1. Percentage decrease in sperm parameters after 48-h of storage
During 48-h of preservation, sperm motility percentage and ATP content dramatically decreased by over 50% while percentage of spermatozoa positively reacting to
the HOS test decreased by approximately 14% (Figure 1).
A significant correlation was found between the percentage of motile spermatozoa
and intracellular ATP content (r = 0.59; P<0.0001). HOS test score was correlated
with neither sperm motility percentage nor sperm ATP content.
Discussion
Under the conditions of this study it was found that intracellular ATP content in
relation to cell membrane integrity, evaluated here with the HOS test is a much more
Membrane integrity, energy status and motility of rabbit spermatozoa
47
sensitive indicator of the effect of storage time on rabbit spermatozoa preserved in
liquid state.
After 24 and 48 h of semen storage, the percentage of sperm with an intact membrane in the ejaculates studied was much higher than the percentage of progressively
motile spermatozoa. This attests to a relatively high resistance of rabbit sperm cell
membranes to damage. This is in agreement with our previous studies (Gogol and
Bochenek, 2003) and with the findings of Castellini et al. (2000) and Roca et al.
(2000) who investigated the viability and plasma membrane integrity of rabbits using 6-carboxyfluorescein diacetate and propidium iodide. They demonstrated that reduced sperm motility in the diluted semen was more significant than the accompanying reduction in the percentage of spermatozoa with an intact cell membrane. Similar
to our study, the percentage of membrane-intact spermatozoa was higher than the
percentage of motile spermatozoa.
The decreased motility of spermatozoa observed in the present study could result
from the damage made to axoneme with only moderate loss of functional integrity of
cell membranes. Damage like this may occur due to the action of free radicals under
oxidative stress. Spermatozoa are particularly vulnerable to oxidative damage during
in vitro storage when the production of free radicals could be significantly enhanced
as a result of metabolic changes (Hammerstedt, 1993). Castellini et al. (2000) found
that peroxidation is one of the main causes of rabbit sperm deterioration during conservation. There are several possible mechanisms behind the decreased motility of
spermatozoa connected with oxidative stress. The most often cited is peroxidation
of membrane lipids (Aitken et al., 1993). Mammalian spermatozoa are cells highly
sensitive to oxidative damage due to their high content of easily peroxidable polyunsaturated fatty acids and the lower enzymatic activity of the antioxidative system
(Jones et al., 1979). The attack of free radicals on the unsaturated fatty acid rich lipids
of sperm cell membranes leads to irreversible reduction of membrane fluidity and to
the damage of cell membrane related ATP-ases, which are responsible for regulation
of intracellular level of ions necessary to maintain normal sperm motility (Rao et al.,
1989).
The decrease in sperm motility induced by free radicals without the accompanying increase in lipid peroxidation (De Lamiranda and Gagnon, 1992; Baumber et al.,
2000) indicates that there is a mechanism whereby free radicals can affect the motility
of spermatozoa regardless of lipid peroxidation. Also Armstrong et. al. (1999) reported that low concentrations of H2O2 caused inhibition of sperm motility and a reduction
in sperm ATP levels without any elevation in lipid peroxidation. It is supposed that
H2O2 disturbs important biochemical functions of the cell such as increased formation
of oxidized intracellular sulfhydryls, a rapid decrease in ATP level and a consequent
depression of glycolytic flux. These processes take place before any signs of the loss
of cell membrane integrity and increased lipid peroxidation (Hyslop et al., 1988). De
Lamiranda and Gagnon (1992) suggest that ROS are responsible for the loss of spermatozoal motility through decreased phosphorylation of axonemal proteins required
for sperm movement.
In summary, the present results indicate a limited usefulness of the HOS test for
evaluation of cell membranes in studies on the effect of storage time on rabbit sperma-
48
P. Gogol and A. Wierzchoś-Hilczer
tozoa and point to the necessity of other methods enabling detection of subtle damage
in spermatozoal structure and function.
In relation to membrane functional integrity, evaluated here with the HOS test,
sperm ATP content and motility are much more sensitive indicators of the effect of
storage on rabbit semen preserved in liquid state.
The significant correlation between ATP concentration and the percentage of motile spermatozoa indicates that ATP assessment may be useful as an additional, objective laboratory test. Further studies will be necessary to ascertain whether the results
of our method used to quantify ATP content in spermatozoa could be correlated with
semen fertility.
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Accepted for printing 12 II 2009
PIOTR GOGOL, AGNIESZKA WIERZCHOŚ-HILCZER
Integralność błon komórkowych, stan energetyczny i ruchliwość plemników królika
przechowywanych przez 2 dni w temperaturze 15°C
STRESZCZENIE
Badano wpływ czasu przechowywania nasienia na stan funkcjonalny błony komórkowej, zawartość
ATP i ruchliwość plemników królika. Badane ejakulaty rozcieńczano komercyjnym rozcieńczalnikiem Galap i przechowywano w temperaturze 15°C. Plemniki oceniano po 2, 24 i 48 godzinach konserwacji. Błony
komórkowe oceniano testem hypoosmotyczym (test HOS), a poziom ATP metodą bioluminescencyjną.
Wykazano istotny wpływ czasu konserwacji na wszystkie badane parametry jakości nasienia. W trakcie
konserwacji poziom ATP oraz odsetek plemników ruchliwych obniżyły się o ponad 50%, podczas gdy
odsetek plemników z nieuszkodzoną błoną komórkową obniżył się w tym samym czasie o około 14%.
Stwierdzono istotną korelację pomiędzy odsetkiem plemników ruchliwych a wewnątrzkomórkowym poziomem ATP (r=0,59). Wyniki testu HOS nie były skorelowane ani z odsetkiem plemników ruchliwych,
ani z poziomem ATP. Przeprowadzone badania wykazały, że ruchliwość i poziom ATP są w porównaniu do integralności błon komórkowych, ocenianej przy użyciu testu HOS, znacznie bardziej czułymi
wskaźnikami wpływu czasu konserwacji na plemniki królika. Istotna korelacja pomiędzy zawartością
ATP i ruchliwością plemników wskazuje, że pomiar ATP może być wykorzystany jako dodatkowy,
obiektywny test jakości nasienia królika.