Directory UMM :Data Elmu:jurnal:A:Animal Reproduction Science:Vol59.Issue3-4.May2000:
Animal Reproduction Science 59 Ž2000. 201–211
www.elsevier.comrlocateranireprosci
The use of transmigration and Spermace stain to
evaluate epididymal cat spermatozoa
S. Schafer
¨ ) , A. Holzmann
Clinic for Obstetrics, Gynaecology and Andrology, UniÕersity of Veterinary Medicine-Vienna,
Veterinarplatz
1, Vienna A-1210, Austria
¨
Received 30 July 1999; received in revised form 26 November 1999; accepted 13 December 1999
Abstract
Epididymal spermatozoa of domestic cats were diluted with TEST medium and frozen. The
parameters — estimated percentage of motile spermatozoa, concentration of spermatozoa, cell
morphology and transmigration rate ŽTMR. — were evaluated before freezing and after thawing.
Transmigration is a new method to measure the percentage of spermatozoa that consistently move
forward, and has not been investigated with cat spermatozoa until now. Estimated percentage of
motile spermatozoa averaged 65%, TMR was 76%, concentration of spermatozoa was 30,000
mly1 and the incidence of morphologically abnormal spermatozoa averaged 58% before freezing.
After thawing, the estimated number of motile spermatozoa declined by 22%, but TMR remained
at 76%. The TMR did not correlate with estimated motility but mostly was higher than the latter,
which is postulated to be caused by the mobilizing effect of the countercurrent in the transmigration apparature. The estimated percentage of motile cells in the target chamber of the transmigration apparature was improved by using phosphate-buffered saline ŽPBS. as transmigration
medium. Morphology was assessed both after fixation of spermatozoa in Hancock solution and
after staining of smears with Spermace. Spermace did not stain all protoplasmic droplets but
proved to be more suitable for the routine examination of acrosomal morphology after thawing.
q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Cat; Epididymal spermatozoa; Quality; Cryopreservation; Transmigration
)
Corresponding author. Tel.: q43-1-25077-5412; fax: q43-1-25077-5490.
E-mail address: sabine.schaefer@vu-wien.ac.at ŽS. Schafer
¨ ..
0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 4 3 2 0 Ž 0 0 . 0 0 0 7 3 - 7
202
S. Schafer,
¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
1. Introduction
The transmigration is an objective method to assess the percentage of spermatozoa
that consistently move forward in a countercurrent and through a unipore membrane in
the time unit. The transmigration apparature was invented in 1986 by Holzmann, and
since that time has successfully been tested with spermatozoa from different animal
species. A positive and significant correlation between the parameters, estimated motility and transmigration rate ŽTMR., was assayed in the bull ŽHolzmann, 1987., the boar
ŽPilz, 1992., the horse ŽRenner et al., 1992. and in the dog ŽSchafer
¨ et al., 1996., and the
latter parameter turned out to be the more objective method and additionally the
technique was strikingly easy to handle. One aim of the present study was to proof
transmigration for the measurement of the percentage of cat spermatozoa that consistently move forward, both with fresh and frozen cat semen, because this has not yet been
done with spermatozoa of the cat species; additionally, two different transmigration
mediums were compared. Spermatozoa from testes collected during castration were
cryopreserved. Epididymal spermatozoa from tom cats can be used as a model for
spermatozoa from wild felines ŽPlatz et al., 1978; Goodrowe and Hay, 1993; Goodrowe
et al., 1988; Hay and Goodrowe, 1993; Lengwinat and Blottner, 1994; Lengwinat et al.,
1992; Pope et al., 1994; Roth et al., 1994..
Spermace stain has previously been used for the staining of fresh and extended
semen from the bull, ram, goat, dog, horse, boar, cheetah, and man ŽOettle, 1986a,b;
Oettle and Soley, 1985; Oetjen, 1988; Chan et al., 1996; Hay et al., 1996; Viggiano et
al., 1996; Watkins et al., 1996.. This metachromatic stain is mainly used to make
various degrees of acrosomal damages visible, and additionally, some of the changes
that occur during acrosomal reaction can be seen. It is described as a quick, reliable and
easy procedure that can also be used for diluted and frozen semen, because the smear’s
background does not take up stain ŽOettle, 1986a; Oettle and Soley, 1985.. Another aim
of our investigation was to test Spermace stain for the morphological assessment of cat
spermatozoa.
2. Materials and methods
2.1. Preparation of spermatozoa
Fifty-one testes from tom cats Ž Felis catus L.. collected during routine castration
were rinsed with a sterile 0.9% sodium chloride solution not later than 5 h after
castration. After dissection of the cauda epididymides and part of the ductus deferens, all
visible blood vessels were excised, or the blood squeezed out of the vessels with a fine
scalpel blade. Samples were placed in a sterile Petri dish and incised five to six times
with a fine scalpel blade. Afterwards, the tissue was overlaid dropwise by 500 ml of
phosphate-buffered saline ŽPBS, 388C.. The resulting spermatozoal suspension was
carefully transferred into a warmed glass tube and the semen quality evaluated.
S. Schafer,
¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
203
2.2. Quality control of spermatozoa before deep freezing
A phase contrast microscope Žmagnification =100–400. was used for the estimation
of motility. The concentration of spermatozoa was measured in a microcellcounter. For
morphological assessment, at least three drops of semen fluid were added to 500 ml of
Hancock solution Žchemical compound in Section 2.5.. One drop of this mixture was put
on a slide and covered. Morphology of spermatozoa was assessed under phase contrast
microscopy Žmagnification =1000, oil immersion.. The TMR was measured in a special
apparature ŽFa Biegler, Vienna, Austria., as invented and described by Holzmann
Ž1987.. The TMR is defined as the percentage of spermatozoa that migrates through a
membrane with defined pore width in a countercurrent and in the time unit. A sterile
syringe with 1000 ml volume containing physiological sodium chloride solution was
prepared as target chamber, put into the transmigration apparature, and automatically
warmed to 388C ŽFig. 1b.. An amount of 100 ml of semen was placed upon the unipore
membrane in the probe chamber ŽFig. 1b warrowx and a.. The pore width of the unipore
membrane was 8 mm, the speed of the countercurrent was 5 ml hy1 . After 3 min of
transmigration of spermatozoa from the probe chamber through the unipore membrane
and into the target chamber, the target chamber was removed and the spermatozoal
concentration measured. The TMR was calculated as follows:
TMR Ž % . s Ž concentration of spermatozoa mly1 in the target chamber . = 1000
= Ž ml . r Ž concentration of spermatozoa mly1 in the probe chamber .
=100 Ž ml . 4 = 100.
2.3. Deep freezing and thawing of spermatozoa
A TES–Tris diluent ŽTEST; Lengwinat and Blottner, 1994; chemical compound in
Section 2.5. was used as freezing medium. It was modified by adding 450 ng of
gentamycin per milliliter of diluent. The diluent was warmed to 388C and added
dropwise to the spermatozoal suspension Žsuspensionq diluents 1 q 3.. The diluted
semen was placed in 0.5 ml plastic straws and allowed to equilibrate at 48C for 3 h.
Afterwards, cryopreservation was performed in two steps: Ž1. straws were placed in
nitrogen vapour Žy1308C. for 15 min; Ž2. straws were immersed in liquid nitrogen.
After storage of 2 weeks in canes with liquid nitrogen Žy1968C., straws were thawed in
a warm water bath Ž408C. for 10 s, then left in another water bath Ž388C. for further 5
min.
2.4. Quality control of spermatozoa after thawing
After thawing, the parameters — estimated motility, morphology, concentration of
spermatozoa and TMR —were investigated. Two smears were prepared for morphological assessment of spermatozoa, one after spermatozoal fixation in Hancock solution,
while the other was stained with Spermace ŽStain Enterprises, South Africa; distribution: Fertility Technologies, Natick, MA, USA.. Spermace smears were not covered,
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¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
S. Schafer,
¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
205
but watched under phase contrast microscopy Žmagnification =1000, oil immersion..
Eleven additional samples were divided after thawing and transmigrated both in
physiological sodium chloride solution and in PBS. In these samples, we additionally
estimated motility of spermatozoa in the target chamber after transmigration.
2.5. Preparation of TEST medium and Hancock solution
TES Ž N-TrisŽhydroxymethyl.methyl-2-aminoethanesulfonic acid.: ) 99.5% titration;
Fa Sigma, Vienna, Austria. §Tris: TrisŽhydroxymethyl.-aminomethane GR, buffer substance; Fa Merck, Darmstadt, Germany. TEST medium: 0.325 m TES Ž74.66 g. q 0.325
m Tris Ž39.46 g. q 7.5% glycerol Ž1.9 ml. q 15% hen’s egg yolk Ž4 ml., and 1000 ml of
double-distilled water; 450 ng gentamycin mly1 of diluent. pH: 7.5; osmolality: 360
mosM kgy1 . Hancock solution: sodium saline solution: 9.01 g NaCl and 500 ml of
double-distilled water. Buffer solution: Ž1. 21.682 g Na 2 HPO4 = 2H 2 O and 500 ml of
double-distilled water; Ž2. 22.254 g KH 2 PO4 and 500 ml of double-distilled water.
Amounts of 200 ml of Ž1. and 80 ml of Ž2. were mixed to obtain 280 ml of buffer
solution. The final Hancock solution was mixed as follows: 62.5 ml formalin Ž37%. q
150 ml sodium saline solutionq 150 ml buffer solution, and 500 ml of double-distilled
water.
2.6. Statistical analyses
Data are expressed as means with the standard deviation of the mean Žmean " SD..
Student’s t-test was used for statistical comparison of morphologically abnormal spermatozoa in Spermace and Hancock smears as well as for the comparison of the
parameters estimated motility, morphology, concentration of spermatozoa and TMR in
samples before freezing and after thawing and after transmigration in two different
mediums. Statistical significance was tested to the 95% level Ž P - 0.05..
3. Results
3.1. Semen quality control
The means and standard deviations of the following results of the semen quality
control before freezing and after thawing are demonstrated in Table 1. Cryopreservation
and thawing of semen impaired estimated motility markedly but not significantly. Before
freezing, TMR was never below 50% and thawing did not change the TMR signifiFig. 1. Scheme of the functional principal of the probe chamber Ža. and one of the four units of the
transmigration apparature Žb.. The probe chamber Žb, arrow. can be opened by screwing it in the middle.
Before each measurement, a sterile unipore membrane Ža, arrow. is placed between the two parts of the probe
chamber. Before transmigration, the probe chamber is automatically let down to close the system.
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¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
Table 1
Parameters of the semen quality control
Conc.s concentration of spermatozoa; Morph.s morphologically abnormal spermatozoa, SDsstandard deviation.
Values in the same column with different superscripts are significantly different Ž P - 0.05..
Variable
Motility Ž%.
mean"SD Žrange.
TMR Ž%.
mean"SD Žrange.
Conc. Ž=10 3 mly1
mean"SD Žrange.
Morph. Ž%.
mean"SD ŽRange.
Before freezing
Ž ns 21 probes.
After thawing
Ž ns 25 straws.
65"10 a
Ž50–80.
47"14a
Ž20–70.
76"14a
Ž57–99.
76"23a
Ž11–94.
30"15
Ž14–53.
20"5
Ž12–35.
58"13a
Ž31–77.
57"12 a
Ž37–88.
cantly. There was a positive but not significant correlation between the parameters,
estimated motility and TMR, after thawing Ž n s 25 straws, R s q0.06, P ) 0.05..
3.2. Effects of the transmigration medium
Quality control of the 11 samples that were transmigrated both in physiological
sodium chloride solution and in PBS revealed the following results Žmeans and standard
deviations in Table 2.: After transmigration, the difference between the percentage of
motile spermatozoa in PBS and sodium chloride averaged 7% in favour of PBS
Ž P - 0.05.. The percentages of morphologically abnormal spermatozoa did not increase
significantly, neither after transmigration in sodium chloride nor in PBS Ž P ) 0.05..
After transmigration, the concentration of spermatozoa in the target chambers filled with
sodium chloride or PBS did not differ significantly Ž P ) 0.05.. The TMR was slightly
higher after transmigration in sodium chloride when compared to PBS Ž P ) 0.05..
3.3. Morphological assessment of spermatozoa
In Spermace-stained smears, spermatozoal acrosomes were stained green and the
heads red ŽFig. 2a,b.. Best results were obtained when smears were not allowed to dry
for longer than 3 min and were fixed for a minimum of 5 min. When smears were left
for less than 2 min in solution A, spermatozoal heads were stained greenish blue instead
Table 2
Parameters of the semen quality control after transmigration in PBS and sodium chloride Ž ns11 straws.
Conc.s concentration of spermatozoa; Morph.s morphologically abnormal spermatozoa; NaClssodium
chloride; SDsstandard deviation; TMs transmigration; TMR s transmigration rate.
Values in the same column with different superscripts are significantly different Ž P - 0.05..
Variable
Motility Ž%.
mean"SD
TMR Ž%.
mean"SD
Conc. Ž=10 3 mly1 .
mean"SD
Morph. Ž%.
mean"SD
After thawing
TM in NaCl
TM in PBS
42.7"11.9 a
18.2"8.7 b
25.4"10.4 c
–
79.8"19.6 a
75.4"10.6 a
26.2"1.3 a
2.3"1.1b
2.1"1b
64.8"16.6 a
67.8"18.6 a
66.8"7.5a
S. Schafer,
¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
207
Fig. 2. Cat spermatozoa stained with Spermace, Ža. normal acrosome, Žb. acrosomal abnormality.
of red. In Hancock smears, spermatozoa were not stained but only fixed and morphology
could only be distinguished because of different grey shadows.
The difference between the percentage abnormal morphology in semen fixed with
Hancock solution before freezing and after thawing averaged 1% ŽTable 1, P ) 0.05.. In
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¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
Fig. 3. Comparison of morphological aberrations in Spermace and Hancock smears. PCD, proximal
cytoplasmic droplets; Total, total percentage of morphologically abnormal spermatozoa. ) ) ) Indicates highly
significant difference Ž P - 0.001..
Spermace-stained smears, the average of total percentage morphologically abnormal
spermatozoa after thawing was less than in Hancock smears ŽFig. 3, P ) 0.05.. A highly
significant difference was assayed between the percentage of proximal cytoplasmatic
droplets in Spermace and Hancock smears Ž P - 0.001, Fig. 3.. Fig. 3 reveals the main
morphological abnormalities in Spermace smears in comparison with those in Hancock
smears.
4. Discussion
After careful preparation of epididymides and vasa deferentia from tom cats and
overlaying them with PBS, cat spermatozoa with an average estimated motility of 65%
were obtained. Hay and Goodrowe Ž1993. used PBS and BSA as transport medium for
cat epididymides and vasa deferentia, respectively, transferred it in three different
diluents and macerated the tissue. After dilution of the suspension with modified KRB
and incubation, percent motility averaged 76. During our investigations, we also
estimated motilities up to 80%, but assessed higher standard deviations than did the
mentioned authors. After thawing, the estimated motility declined by 22% — a result
similar to that reported by Hay and Goodrowe Ž1993. and Lengwinat and Blottner
Ž1994.. These workers used other diluents, but after thawing, percent motility also
decreased by 22–24. Lengwinat and Blottner Ž1994. increased spermatozoal motility to
estimated 74% by processing swim-up after thawing.
A positive and significant correlation between the parameters, TMR and estimated
motility, was assayed in the bull ŽHolzmann, 1987., the boar ŽPilz, 1992., the horse
ŽRenner et al., 1992., and the dog ŽSchafer
¨ et al., 1996., but not in the present study.
The transmigration has not been processed with cat spermatozoa until now, so comparative data were not available. After thawing, the TMR still averaged 76%, but percent
motility declined by 22. A possible explanation could be that swimming against a fluid
S. Schafer,
¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
209
stream improved spermatozoal viability and was the cause for the high incidence of
migrated spermatozoa. As early as 1905, Adolphi Ž1905. supposed that spermatozoa are
forced to consistently move forward against a fluid stream if the fluid’s speed exceeds
that of the spermatozoa. Rosenkranz and Holzmann Ž1995. postulated that the countercurrent during transmigration exerted a mobilizing effect on bull spermatozoa and
additionally triggered acrosomal capacitation. However, concerning cat spermatozoa,
there was a positive, but not significant, correlation between estimated motility and
TMR and this absence of significance is supposed to depend on insufficient probe
numbers. Percentage of transmigrated spermatozoa in most cases exceeded that of
estimated spermatozoa presumably because of a stimulating effect of the countercurrent,
which depends on tactile irritation of spermatozoa, and these present results make the
technique interesting for other use than it originally was provided for. For example, the
transmigration of semen has been used for the preparation of bull and dog spermatozoa
for the IVF: when fertilization capacity of transmigrated semen was tested in a bovine
IVF system and compared with swim-up semen treatment results, fertilization rates in
the transmigration groups exceeded those in swim-up groups, both for fresh semen as
well as for frozen–thawed semen, but this was not statistically significant. In comparison to swim-up, the transmigration procedure was much more easy to handle and
required only a brief procedure ŽAuer, 1993; Rosenkranz et al., 1994.. During a study
about the influence of semen preparation and culture medium on the success of IVF in
cattle, Rosenkranz and Holzmann Ž1995. noticed that after swim-up treatment, capacitation-inducing and motility-enhancing supplements to fertilization medium were necessary to obtain sufficient fertilization results. Transmigrated semen required no additional
supplementation of the fertilization medium. Additionally, Rosenkranz and Holzmann
Ž1997. showed that bovine spermatozoa penetrated the oocyte during IVF earlier after
preparation with transmigration than after swim-up. Fertilization capacity of transmigrated semen has also been tested in a canine IVM and IVF system. The author ŽSipos,
1997. does not report about any differences concerning the fertilization rate after semen
preparation with transmigration or centrifugation, but emphasizes that only 5 min are
required for the transmigration procedure. Because of capacitation of spermatozoa
during transmigration, no further preparation is needed before IVF. IVF with epididymal
cat spermatozoa after transmigration has not yet been assayed, but because of hitherto
existing results from other species, could be the aim of consecutive studies.
In the boar, Pilz Ž1992. investigated whether there is a relation between boar semen
characteristics and fertility of artificially inseminated sows. She assessed a highly
significant correlation between the TMR of diluted fresh semen and the number of
piglets per sow Ž R s 0.39 and P - 0.01. and calculated a regression as follows: number
of pigletss 5.41 q 0.09 = TMR. The advantages concerning the use of transmigrated
semen for the artificial insemination of cats have not yet been assessed; in the present
study, transmigration of cat spermatozoa was only investigated as a new, objective and
easy technique for the measurement of the percentage of cat spermatozoa that consistently move forward in a countercurrent and through a unipore membrane in the time
unit.
The use of PBS as a transmigration medium revealed some advantage in comparison
to physiological sodium chloride solution, as far as estimated spermatozoal motility was
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¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
significantly higher after transmigration in PBS; it is therefore recommended to use PBS
as transmigration medium. The transmigration procedure did not increase the number of
spermatozoa with morphological aberrations, regardless of which solution was used.
Before deep freezing of spermatozoa, only 1–6% of acrosomal deficiencies could be
detected. Similar results were reported by Hay and Goodrowe Ž1993.. Morphological
assessment of cat spermatozoa after thawing was additionally performed with Spermace,
because thawing frequently causes acrosomal damages and Spermace is mainly used to
examine acrosomal integrity. Pope et al. Ž1991. tested a new staining method called
single stain and additionally stained smears with a modified triple stain ŽTalbot and
Chacon, 1981.. They reported that more acrosomal deficiencies were detected in smears
stained with triple stain than in single-stain probes. Spermace did not exert this effect
on acrosomal integrity, but in comparison with Hancock smears, significantly less
cytoplasmatic droplets were detected with Spermace, which has not been reported
before. One reason could be that Spermace has mainly been used to assess morphological abberations of the acrosome or, in one case, of the midpiece ŽOettle and Soley,
1985., cytoplasmatic droplets were not even mentioned in any of the relevant publications. Cytoplasmatic droplets are remainders of the Golgi apparature; maybe some of
these droplets are already too degenerated and therefore not selectively stained as is the
smear’s background. This has previously been described for stallion semen; Oetjen
Ž1988. reported that only a light green or red shadow was visible from the Spermace
smear’s background, which is of importance when using diluted semen. From the
present study, we know that in the cat as in other species, Spermace should preferably
be used to examine acrosomal integrity of spermatozoa.
5. Conclusions
At least careful extraction of cat spermatozoa with PBS resulted in spermatozoal
suspensions with an average estimated motility of 65%. After deep freezing and
thawing, percent motility declined by 22. TMR of cat spermatozoa correlated positively
but not significantly with estimated motility, but in most cases was higher than the latter,
before freezing as well as after thawing. This is supposed to depend on the mobilizing
effect of the countercurrent during transmigration. Transmigration of spermatozoa with
sodium chloride caused higher impairment of percent motility than transmigration with
PBS. Spermace proved to be an easy and useful staining for the detection of acrosomal
abberations of cat spermatozoa after thawing.
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Watkins, A.M., Chan, P.J., Patton, W.C., Jacobson, J.D., King, A., 1996. Sperm kinetics and morphology
before and after fractionation on discontinuous Percoll gradient for sex preselection: computerized
analyses. Arch. Androl. 37 Ž1., 1–5.
www.elsevier.comrlocateranireprosci
The use of transmigration and Spermace stain to
evaluate epididymal cat spermatozoa
S. Schafer
¨ ) , A. Holzmann
Clinic for Obstetrics, Gynaecology and Andrology, UniÕersity of Veterinary Medicine-Vienna,
Veterinarplatz
1, Vienna A-1210, Austria
¨
Received 30 July 1999; received in revised form 26 November 1999; accepted 13 December 1999
Abstract
Epididymal spermatozoa of domestic cats were diluted with TEST medium and frozen. The
parameters — estimated percentage of motile spermatozoa, concentration of spermatozoa, cell
morphology and transmigration rate ŽTMR. — were evaluated before freezing and after thawing.
Transmigration is a new method to measure the percentage of spermatozoa that consistently move
forward, and has not been investigated with cat spermatozoa until now. Estimated percentage of
motile spermatozoa averaged 65%, TMR was 76%, concentration of spermatozoa was 30,000
mly1 and the incidence of morphologically abnormal spermatozoa averaged 58% before freezing.
After thawing, the estimated number of motile spermatozoa declined by 22%, but TMR remained
at 76%. The TMR did not correlate with estimated motility but mostly was higher than the latter,
which is postulated to be caused by the mobilizing effect of the countercurrent in the transmigration apparature. The estimated percentage of motile cells in the target chamber of the transmigration apparature was improved by using phosphate-buffered saline ŽPBS. as transmigration
medium. Morphology was assessed both after fixation of spermatozoa in Hancock solution and
after staining of smears with Spermace. Spermace did not stain all protoplasmic droplets but
proved to be more suitable for the routine examination of acrosomal morphology after thawing.
q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Cat; Epididymal spermatozoa; Quality; Cryopreservation; Transmigration
)
Corresponding author. Tel.: q43-1-25077-5412; fax: q43-1-25077-5490.
E-mail address: sabine.schaefer@vu-wien.ac.at ŽS. Schafer
¨ ..
0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 4 3 2 0 Ž 0 0 . 0 0 0 7 3 - 7
202
S. Schafer,
¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
1. Introduction
The transmigration is an objective method to assess the percentage of spermatozoa
that consistently move forward in a countercurrent and through a unipore membrane in
the time unit. The transmigration apparature was invented in 1986 by Holzmann, and
since that time has successfully been tested with spermatozoa from different animal
species. A positive and significant correlation between the parameters, estimated motility and transmigration rate ŽTMR., was assayed in the bull ŽHolzmann, 1987., the boar
ŽPilz, 1992., the horse ŽRenner et al., 1992. and in the dog ŽSchafer
¨ et al., 1996., and the
latter parameter turned out to be the more objective method and additionally the
technique was strikingly easy to handle. One aim of the present study was to proof
transmigration for the measurement of the percentage of cat spermatozoa that consistently move forward, both with fresh and frozen cat semen, because this has not yet been
done with spermatozoa of the cat species; additionally, two different transmigration
mediums were compared. Spermatozoa from testes collected during castration were
cryopreserved. Epididymal spermatozoa from tom cats can be used as a model for
spermatozoa from wild felines ŽPlatz et al., 1978; Goodrowe and Hay, 1993; Goodrowe
et al., 1988; Hay and Goodrowe, 1993; Lengwinat and Blottner, 1994; Lengwinat et al.,
1992; Pope et al., 1994; Roth et al., 1994..
Spermace stain has previously been used for the staining of fresh and extended
semen from the bull, ram, goat, dog, horse, boar, cheetah, and man ŽOettle, 1986a,b;
Oettle and Soley, 1985; Oetjen, 1988; Chan et al., 1996; Hay et al., 1996; Viggiano et
al., 1996; Watkins et al., 1996.. This metachromatic stain is mainly used to make
various degrees of acrosomal damages visible, and additionally, some of the changes
that occur during acrosomal reaction can be seen. It is described as a quick, reliable and
easy procedure that can also be used for diluted and frozen semen, because the smear’s
background does not take up stain ŽOettle, 1986a; Oettle and Soley, 1985.. Another aim
of our investigation was to test Spermace stain for the morphological assessment of cat
spermatozoa.
2. Materials and methods
2.1. Preparation of spermatozoa
Fifty-one testes from tom cats Ž Felis catus L.. collected during routine castration
were rinsed with a sterile 0.9% sodium chloride solution not later than 5 h after
castration. After dissection of the cauda epididymides and part of the ductus deferens, all
visible blood vessels were excised, or the blood squeezed out of the vessels with a fine
scalpel blade. Samples were placed in a sterile Petri dish and incised five to six times
with a fine scalpel blade. Afterwards, the tissue was overlaid dropwise by 500 ml of
phosphate-buffered saline ŽPBS, 388C.. The resulting spermatozoal suspension was
carefully transferred into a warmed glass tube and the semen quality evaluated.
S. Schafer,
¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
203
2.2. Quality control of spermatozoa before deep freezing
A phase contrast microscope Žmagnification =100–400. was used for the estimation
of motility. The concentration of spermatozoa was measured in a microcellcounter. For
morphological assessment, at least three drops of semen fluid were added to 500 ml of
Hancock solution Žchemical compound in Section 2.5.. One drop of this mixture was put
on a slide and covered. Morphology of spermatozoa was assessed under phase contrast
microscopy Žmagnification =1000, oil immersion.. The TMR was measured in a special
apparature ŽFa Biegler, Vienna, Austria., as invented and described by Holzmann
Ž1987.. The TMR is defined as the percentage of spermatozoa that migrates through a
membrane with defined pore width in a countercurrent and in the time unit. A sterile
syringe with 1000 ml volume containing physiological sodium chloride solution was
prepared as target chamber, put into the transmigration apparature, and automatically
warmed to 388C ŽFig. 1b.. An amount of 100 ml of semen was placed upon the unipore
membrane in the probe chamber ŽFig. 1b warrowx and a.. The pore width of the unipore
membrane was 8 mm, the speed of the countercurrent was 5 ml hy1 . After 3 min of
transmigration of spermatozoa from the probe chamber through the unipore membrane
and into the target chamber, the target chamber was removed and the spermatozoal
concentration measured. The TMR was calculated as follows:
TMR Ž % . s Ž concentration of spermatozoa mly1 in the target chamber . = 1000
= Ž ml . r Ž concentration of spermatozoa mly1 in the probe chamber .
=100 Ž ml . 4 = 100.
2.3. Deep freezing and thawing of spermatozoa
A TES–Tris diluent ŽTEST; Lengwinat and Blottner, 1994; chemical compound in
Section 2.5. was used as freezing medium. It was modified by adding 450 ng of
gentamycin per milliliter of diluent. The diluent was warmed to 388C and added
dropwise to the spermatozoal suspension Žsuspensionq diluents 1 q 3.. The diluted
semen was placed in 0.5 ml plastic straws and allowed to equilibrate at 48C for 3 h.
Afterwards, cryopreservation was performed in two steps: Ž1. straws were placed in
nitrogen vapour Žy1308C. for 15 min; Ž2. straws were immersed in liquid nitrogen.
After storage of 2 weeks in canes with liquid nitrogen Žy1968C., straws were thawed in
a warm water bath Ž408C. for 10 s, then left in another water bath Ž388C. for further 5
min.
2.4. Quality control of spermatozoa after thawing
After thawing, the parameters — estimated motility, morphology, concentration of
spermatozoa and TMR —were investigated. Two smears were prepared for morphological assessment of spermatozoa, one after spermatozoal fixation in Hancock solution,
while the other was stained with Spermace ŽStain Enterprises, South Africa; distribution: Fertility Technologies, Natick, MA, USA.. Spermace smears were not covered,
204
S. Schafer,
¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
S. Schafer,
¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
205
but watched under phase contrast microscopy Žmagnification =1000, oil immersion..
Eleven additional samples were divided after thawing and transmigrated both in
physiological sodium chloride solution and in PBS. In these samples, we additionally
estimated motility of spermatozoa in the target chamber after transmigration.
2.5. Preparation of TEST medium and Hancock solution
TES Ž N-TrisŽhydroxymethyl.methyl-2-aminoethanesulfonic acid.: ) 99.5% titration;
Fa Sigma, Vienna, Austria. §Tris: TrisŽhydroxymethyl.-aminomethane GR, buffer substance; Fa Merck, Darmstadt, Germany. TEST medium: 0.325 m TES Ž74.66 g. q 0.325
m Tris Ž39.46 g. q 7.5% glycerol Ž1.9 ml. q 15% hen’s egg yolk Ž4 ml., and 1000 ml of
double-distilled water; 450 ng gentamycin mly1 of diluent. pH: 7.5; osmolality: 360
mosM kgy1 . Hancock solution: sodium saline solution: 9.01 g NaCl and 500 ml of
double-distilled water. Buffer solution: Ž1. 21.682 g Na 2 HPO4 = 2H 2 O and 500 ml of
double-distilled water; Ž2. 22.254 g KH 2 PO4 and 500 ml of double-distilled water.
Amounts of 200 ml of Ž1. and 80 ml of Ž2. were mixed to obtain 280 ml of buffer
solution. The final Hancock solution was mixed as follows: 62.5 ml formalin Ž37%. q
150 ml sodium saline solutionq 150 ml buffer solution, and 500 ml of double-distilled
water.
2.6. Statistical analyses
Data are expressed as means with the standard deviation of the mean Žmean " SD..
Student’s t-test was used for statistical comparison of morphologically abnormal spermatozoa in Spermace and Hancock smears as well as for the comparison of the
parameters estimated motility, morphology, concentration of spermatozoa and TMR in
samples before freezing and after thawing and after transmigration in two different
mediums. Statistical significance was tested to the 95% level Ž P - 0.05..
3. Results
3.1. Semen quality control
The means and standard deviations of the following results of the semen quality
control before freezing and after thawing are demonstrated in Table 1. Cryopreservation
and thawing of semen impaired estimated motility markedly but not significantly. Before
freezing, TMR was never below 50% and thawing did not change the TMR signifiFig. 1. Scheme of the functional principal of the probe chamber Ža. and one of the four units of the
transmigration apparature Žb.. The probe chamber Žb, arrow. can be opened by screwing it in the middle.
Before each measurement, a sterile unipore membrane Ža, arrow. is placed between the two parts of the probe
chamber. Before transmigration, the probe chamber is automatically let down to close the system.
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¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
Table 1
Parameters of the semen quality control
Conc.s concentration of spermatozoa; Morph.s morphologically abnormal spermatozoa, SDsstandard deviation.
Values in the same column with different superscripts are significantly different Ž P - 0.05..
Variable
Motility Ž%.
mean"SD Žrange.
TMR Ž%.
mean"SD Žrange.
Conc. Ž=10 3 mly1
mean"SD Žrange.
Morph. Ž%.
mean"SD ŽRange.
Before freezing
Ž ns 21 probes.
After thawing
Ž ns 25 straws.
65"10 a
Ž50–80.
47"14a
Ž20–70.
76"14a
Ž57–99.
76"23a
Ž11–94.
30"15
Ž14–53.
20"5
Ž12–35.
58"13a
Ž31–77.
57"12 a
Ž37–88.
cantly. There was a positive but not significant correlation between the parameters,
estimated motility and TMR, after thawing Ž n s 25 straws, R s q0.06, P ) 0.05..
3.2. Effects of the transmigration medium
Quality control of the 11 samples that were transmigrated both in physiological
sodium chloride solution and in PBS revealed the following results Žmeans and standard
deviations in Table 2.: After transmigration, the difference between the percentage of
motile spermatozoa in PBS and sodium chloride averaged 7% in favour of PBS
Ž P - 0.05.. The percentages of morphologically abnormal spermatozoa did not increase
significantly, neither after transmigration in sodium chloride nor in PBS Ž P ) 0.05..
After transmigration, the concentration of spermatozoa in the target chambers filled with
sodium chloride or PBS did not differ significantly Ž P ) 0.05.. The TMR was slightly
higher after transmigration in sodium chloride when compared to PBS Ž P ) 0.05..
3.3. Morphological assessment of spermatozoa
In Spermace-stained smears, spermatozoal acrosomes were stained green and the
heads red ŽFig. 2a,b.. Best results were obtained when smears were not allowed to dry
for longer than 3 min and were fixed for a minimum of 5 min. When smears were left
for less than 2 min in solution A, spermatozoal heads were stained greenish blue instead
Table 2
Parameters of the semen quality control after transmigration in PBS and sodium chloride Ž ns11 straws.
Conc.s concentration of spermatozoa; Morph.s morphologically abnormal spermatozoa; NaClssodium
chloride; SDsstandard deviation; TMs transmigration; TMR s transmigration rate.
Values in the same column with different superscripts are significantly different Ž P - 0.05..
Variable
Motility Ž%.
mean"SD
TMR Ž%.
mean"SD
Conc. Ž=10 3 mly1 .
mean"SD
Morph. Ž%.
mean"SD
After thawing
TM in NaCl
TM in PBS
42.7"11.9 a
18.2"8.7 b
25.4"10.4 c
–
79.8"19.6 a
75.4"10.6 a
26.2"1.3 a
2.3"1.1b
2.1"1b
64.8"16.6 a
67.8"18.6 a
66.8"7.5a
S. Schafer,
¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
207
Fig. 2. Cat spermatozoa stained with Spermace, Ža. normal acrosome, Žb. acrosomal abnormality.
of red. In Hancock smears, spermatozoa were not stained but only fixed and morphology
could only be distinguished because of different grey shadows.
The difference between the percentage abnormal morphology in semen fixed with
Hancock solution before freezing and after thawing averaged 1% ŽTable 1, P ) 0.05.. In
208
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¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
Fig. 3. Comparison of morphological aberrations in Spermace and Hancock smears. PCD, proximal
cytoplasmic droplets; Total, total percentage of morphologically abnormal spermatozoa. ) ) ) Indicates highly
significant difference Ž P - 0.001..
Spermace-stained smears, the average of total percentage morphologically abnormal
spermatozoa after thawing was less than in Hancock smears ŽFig. 3, P ) 0.05.. A highly
significant difference was assayed between the percentage of proximal cytoplasmatic
droplets in Spermace and Hancock smears Ž P - 0.001, Fig. 3.. Fig. 3 reveals the main
morphological abnormalities in Spermace smears in comparison with those in Hancock
smears.
4. Discussion
After careful preparation of epididymides and vasa deferentia from tom cats and
overlaying them with PBS, cat spermatozoa with an average estimated motility of 65%
were obtained. Hay and Goodrowe Ž1993. used PBS and BSA as transport medium for
cat epididymides and vasa deferentia, respectively, transferred it in three different
diluents and macerated the tissue. After dilution of the suspension with modified KRB
and incubation, percent motility averaged 76. During our investigations, we also
estimated motilities up to 80%, but assessed higher standard deviations than did the
mentioned authors. After thawing, the estimated motility declined by 22% — a result
similar to that reported by Hay and Goodrowe Ž1993. and Lengwinat and Blottner
Ž1994.. These workers used other diluents, but after thawing, percent motility also
decreased by 22–24. Lengwinat and Blottner Ž1994. increased spermatozoal motility to
estimated 74% by processing swim-up after thawing.
A positive and significant correlation between the parameters, TMR and estimated
motility, was assayed in the bull ŽHolzmann, 1987., the boar ŽPilz, 1992., the horse
ŽRenner et al., 1992., and the dog ŽSchafer
¨ et al., 1996., but not in the present study.
The transmigration has not been processed with cat spermatozoa until now, so comparative data were not available. After thawing, the TMR still averaged 76%, but percent
motility declined by 22. A possible explanation could be that swimming against a fluid
S. Schafer,
¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
209
stream improved spermatozoal viability and was the cause for the high incidence of
migrated spermatozoa. As early as 1905, Adolphi Ž1905. supposed that spermatozoa are
forced to consistently move forward against a fluid stream if the fluid’s speed exceeds
that of the spermatozoa. Rosenkranz and Holzmann Ž1995. postulated that the countercurrent during transmigration exerted a mobilizing effect on bull spermatozoa and
additionally triggered acrosomal capacitation. However, concerning cat spermatozoa,
there was a positive, but not significant, correlation between estimated motility and
TMR and this absence of significance is supposed to depend on insufficient probe
numbers. Percentage of transmigrated spermatozoa in most cases exceeded that of
estimated spermatozoa presumably because of a stimulating effect of the countercurrent,
which depends on tactile irritation of spermatozoa, and these present results make the
technique interesting for other use than it originally was provided for. For example, the
transmigration of semen has been used for the preparation of bull and dog spermatozoa
for the IVF: when fertilization capacity of transmigrated semen was tested in a bovine
IVF system and compared with swim-up semen treatment results, fertilization rates in
the transmigration groups exceeded those in swim-up groups, both for fresh semen as
well as for frozen–thawed semen, but this was not statistically significant. In comparison to swim-up, the transmigration procedure was much more easy to handle and
required only a brief procedure ŽAuer, 1993; Rosenkranz et al., 1994.. During a study
about the influence of semen preparation and culture medium on the success of IVF in
cattle, Rosenkranz and Holzmann Ž1995. noticed that after swim-up treatment, capacitation-inducing and motility-enhancing supplements to fertilization medium were necessary to obtain sufficient fertilization results. Transmigrated semen required no additional
supplementation of the fertilization medium. Additionally, Rosenkranz and Holzmann
Ž1997. showed that bovine spermatozoa penetrated the oocyte during IVF earlier after
preparation with transmigration than after swim-up. Fertilization capacity of transmigrated semen has also been tested in a canine IVM and IVF system. The author ŽSipos,
1997. does not report about any differences concerning the fertilization rate after semen
preparation with transmigration or centrifugation, but emphasizes that only 5 min are
required for the transmigration procedure. Because of capacitation of spermatozoa
during transmigration, no further preparation is needed before IVF. IVF with epididymal
cat spermatozoa after transmigration has not yet been assayed, but because of hitherto
existing results from other species, could be the aim of consecutive studies.
In the boar, Pilz Ž1992. investigated whether there is a relation between boar semen
characteristics and fertility of artificially inseminated sows. She assessed a highly
significant correlation between the TMR of diluted fresh semen and the number of
piglets per sow Ž R s 0.39 and P - 0.01. and calculated a regression as follows: number
of pigletss 5.41 q 0.09 = TMR. The advantages concerning the use of transmigrated
semen for the artificial insemination of cats have not yet been assessed; in the present
study, transmigration of cat spermatozoa was only investigated as a new, objective and
easy technique for the measurement of the percentage of cat spermatozoa that consistently move forward in a countercurrent and through a unipore membrane in the time
unit.
The use of PBS as a transmigration medium revealed some advantage in comparison
to physiological sodium chloride solution, as far as estimated spermatozoal motility was
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¨ A. Holzmannr Animal Reproduction Science 59 (2000) 201–211
significantly higher after transmigration in PBS; it is therefore recommended to use PBS
as transmigration medium. The transmigration procedure did not increase the number of
spermatozoa with morphological aberrations, regardless of which solution was used.
Before deep freezing of spermatozoa, only 1–6% of acrosomal deficiencies could be
detected. Similar results were reported by Hay and Goodrowe Ž1993.. Morphological
assessment of cat spermatozoa after thawing was additionally performed with Spermace,
because thawing frequently causes acrosomal damages and Spermace is mainly used to
examine acrosomal integrity. Pope et al. Ž1991. tested a new staining method called
single stain and additionally stained smears with a modified triple stain ŽTalbot and
Chacon, 1981.. They reported that more acrosomal deficiencies were detected in smears
stained with triple stain than in single-stain probes. Spermace did not exert this effect
on acrosomal integrity, but in comparison with Hancock smears, significantly less
cytoplasmatic droplets were detected with Spermace, which has not been reported
before. One reason could be that Spermace has mainly been used to assess morphological abberations of the acrosome or, in one case, of the midpiece ŽOettle and Soley,
1985., cytoplasmatic droplets were not even mentioned in any of the relevant publications. Cytoplasmatic droplets are remainders of the Golgi apparature; maybe some of
these droplets are already too degenerated and therefore not selectively stained as is the
smear’s background. This has previously been described for stallion semen; Oetjen
Ž1988. reported that only a light green or red shadow was visible from the Spermace
smear’s background, which is of importance when using diluted semen. From the
present study, we know that in the cat as in other species, Spermace should preferably
be used to examine acrosomal integrity of spermatozoa.
5. Conclusions
At least careful extraction of cat spermatozoa with PBS resulted in spermatozoal
suspensions with an average estimated motility of 65%. After deep freezing and
thawing, percent motility declined by 22. TMR of cat spermatozoa correlated positively
but not significantly with estimated motility, but in most cases was higher than the latter,
before freezing as well as after thawing. This is supposed to depend on the mobilizing
effect of the countercurrent during transmigration. Transmigration of spermatozoa with
sodium chloride caused higher impairment of percent motility than transmigration with
PBS. Spermace proved to be an easy and useful staining for the detection of acrosomal
abberations of cat spermatozoa after thawing.
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