www.elsevier.comrlocateranireprosci
The use of transmigration and Spermac
e
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
Ž .
morphology and transmigration rate TMR — 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
mly1and 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 tion apparature. The estimated percentage of motile cells in the target chamber of the
transmigra-Ž .
tion apparature was improved by using phosphate-buffered saline PBS as transmigration medium. Morphology was assessed both after fixation of spermatozoa in Hancock solution and after staining of smears with Spermace. Spermacedid not stain all protoplasmic droplets but proved to be more suitable for the routine examination of acrosomal morphology after thawing.
q2000 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.
Ž .
E-mail address: sabine.schaefer@vu-wien.ac.at S. Schafer .¨
0378-4320r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved.
Ž .
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
motil-Ž . Ž .
ity and transmigration rate TMR , was assayed in the bull Holzmann, 1987 , the boar ŽPilz, 1992 , the horse Renner et al., 1992 and in the dog Schafer et al., 1996 , and the. Ž . Ž
¨
. 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 consis-tently 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 forŽ
spermatozoa from wild felines Platz 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
Ž
semen from the bull, ram, goat, dog, horse, boar, cheetah, and man Oettle, 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
Ž .
background does not take up stain Oettle, 1986a; Oettle and Soley, 1985 . Another aim of our investigation was to test Spermacestain for the morphological assessment of cat
spermatozoa.
2. Materials and methods
2.1. Preparation of spermatozoa
Ž .
Fifty-one testes from tom cats 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
Ž .
2.2. Quality control of spermatozoa before deep freezing
Ž .
A phase contrast microscope magnification=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 500ml of
Ž .
Hancock solution chemical 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
Ž .
microscopy magnification=1000, oil immersion . The TMR was measured in a special
Ž .
apparature Fa 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
Ž .
warmed to 388C Fig. 1b . An amount of 100ml of semen was placed upon the unipore
Ž w x .
membrane in the probe chamber Fig. 1b arrow 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 .
sŽ
concentration of spermatozoamly1in the target chamber.
=1000=
Ž
ml.
rŽ
concentration of spermatozoamly1in the probe chamber.
=100
Ž
ml.
4
=100.2.3. Deep freezing and thawing of spermatozoa
Ž
A TES–Tris diluent TEST; 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
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dropwise to the spermatozoal suspension suspensionqdiluents1q3 . The diluted semen was placed in 0.5 ml plastic straws and allowed to equilibrate at 48C for 3 h.
Ž .
Afterwards, cryopreservation was performed in two steps: 1 straws were placed in
Ž . Ž .
nitrogen vapour y1308C for 15 min; 2 straws were immersed in liquid nitrogen.
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After storage of 2 weeks in canes with liquid nitrogen y1968C , straws were thawed in
Ž . Ž .
a warm water bath 408C for 10 s, then left in another water bath 388C 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 morphologi-cal assessment of spermatozoa, one after spermatozoal fixation in Hancock solution,
Ž
while the other was stained with Spermace Stain Enterprises, South Africa;
distribu-.
Ž . but watched under phase contrast microscopy magnification =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
Ž Ž . .
TES N-Tris hydroxymethyl methyl-2-aminoethanesulfonic acid : )99.5% titration;
Ž .
Fa Sigma, Vienna, Austria. §Tris: Tris hydroxymethyl -aminomethane GR, buffer
sub-Ž .
stance; Fa Merck, Darmstadt, Germany. TEST medium: 0.325 m TES 74.66 g q0.325
Ž . Ž . Ž .
m Tris 39.46 g q7.5% glycerol 1.9 ml q15% hen’s egg yolk 4 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
Ž .
double-distilled water. Buffer solution: 1 21.682 g Na HPO2 4=2H O and 500 ml of2
Ž .
double-distilled water; 2 22.254 g KH PO2 4 and 500 ml of double-distilled water.
Ž . Ž .
Amounts of 200 ml of 1 and 80 ml of 2 were mixed to obtain 280 ml of buffer Ž . solution. The final Hancock solution was mixed as follows: 62.5 ml formalin 37% q 150 ml sodium saline solutionq150 ml buffer solution, and 500 ml of double-distilled water.
2.6. Statistical analyses
Ž .
Data are expressed as means with the standard deviation of the mean mean"SD . Student’s t-test was used for statistical comparison of morphologically abnormal sper-matozoa 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
Ž .
mediums. Statistical significance was tested to the 95% level 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
signifi-Ž .
Fig. 1. Scheme of the functional principal of the probe chamber a and one of the four units of the
Ž . Ž .
transmigration apparature b . The probe chamber b, arrow can be opened by screwing it in the middle.
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Table 1
Parameters of the semen quality control
Conc.sconcentration of spermatozoa; Morph.smorphologically abnormal spermatozoa, SDsstandard devi-ation.
Ž .
Values in the same column with different superscripts are significantly different P-0.05 .
3 y1
Ž . Ž . Ž Ž .
Variable Motility % TMR % Conc. =10 ml Morph. %
Ž . Ž . Ž . Ž .
mean"SD range mean"SD range mean"SD range mean"SD Range
a a a
cantly. There was a positive but not significant correlation between the parameters,
Ž .
estimated motility and TMR, after thawing ns25 straws, Rs q0.06, P)0.05 .
3.2. Effects of the transmigration medium
Quality control of the 11 samples that were transmigrated both in physiological Ž
sodium chloride solution and in PBS revealed the following results means 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 ŽP-0.05 . The percentages of morphologically abnormal spermatozoa did not increase.
Ž .
significantly, neither after transmigration in sodium chloride nor in PBS P)0.05 . After transmigration, the concentration of spermatozoa in the target chambers filled with
Ž .
sodium chloride or PBS did not differ significantly P)0.05 . The TMR was slightly
Ž .
higher after transmigration in sodium chloride when compared to PBS P)0.05 .
3.3. Morphological assessment of spermatozoa
In Spermace-stained smears, spermatozoal acrosomes were stained green and the
Ž .
heads red Fig. 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
Ž .
Parameters of the semen quality control after transmigration in PBS and sodium chloride ns11 straws Conc.sconcentration of spermatozoa; Morph.smorphologically abnormal spermatozoa; NaClssodium chloride; SDsstandard deviation; TMstransmigration; TMRstransmigration rate.
Ž .
Values in the same column with different superscripts are significantly different P-0.05 .
Ž . Ž .
Fig. 2. Cat spermatozoa stained with Spermace, a normal acrosome, b 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
Ž .
Fig. 3. Comparison of morphological aberrations in Spermace and Hancock smears. PCD, proximal cytoplasmic droplets; Total, total percentage of morphologically abnormal spermatozoa.) ) )Indicates highly
Ž .
significant difference P-0.001 .
Spermace-stained smears, the average of total percentage morphologically abnormal
Ž .
spermatozoa after thawing was less than in Hancock smears Fig. 3, P)0.05 . A highly significant difference was assayed between the percentage of proximal cytoplasmatic
Ž .
droplets in Spermaceand Hancock smears P-0.001, Fig. 3 . Fig. 3 reveals the main morphological abnormalities in Spermacesmears 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%
Ž .
were obtained. Hay and Goodrowe 1993 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
Ž .
similar to that reported by Hay and Goodrowe 1993 and Lengwinat and Blottner Ž1994 . These workers used other diluents, but after thawing, percent motility also.
Ž .
decreased by 22–24. Lengwinat and Blottner 1994 increased spermatozoal motility to estimated 74% by processing swim-up after thawing.
A positive and significant correlation between the parameters, TMR and estimated
Ž . Ž .
stream improved spermatozoal viability and was the cause for the high incidence of Ž .
migrated spermatozoa. As early as 1905, Adolphi 1905 supposed that spermatozoa are forced to consistently move forward against a fluid stream if the fluid’s speed exceeds
Ž .
that of the spermatozoa. Rosenkranz and Holzmann 1995 postulated that the counter-current 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 compari-son to swim-up, the transmigration procedure was much more easy to handle and
Ž .
required only a brief procedure Auer, 1993; Rosenkranz et al., 1994 . During a study about the influence of semen preparation and culture medium on the success of IVF in
Ž .
cattle, Rosenkranz and Holzmann 1995 noticed that after swim-up treatment, capacita-tion-inducing and motility-enhancing supplements to fertilization medium were neces-sary 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
transmi-Ž grated semen has also been tested in a canine IVM and IVF system. The author Sipos,
.
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.
Ž .
In the boar, Pilz 1992 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
Ž .
piglets per sow Rs0.39 and P-0.01 and calculated a regression as follows: number of pigletss5.41q0.09=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 consis-tently move forward in a countercurrent and through a unipore membrane in the time unit.
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 Ž .
detected. Similar results were reported by Hay and Goodrowe 1993 . Morphological assessment of cat spermatozoa after thawing was additionally performed with Spermace,
because thawing frequently causes acrosomal damages and Spermaceis mainly used to
Ž .
examine acrosomal integrity. Pope et al. 1991 tested a new staining method called Ž
single stain and additionally stained smears with a modified triple stain Talbot 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 Spermacehas mainly been used to assess
morphologi-Ž
cal abberations of the acrosome or, in one case, of the midpiece Oettle and Soley, .
1985 , cytoplasmatic droplets were not even mentioned in any of the relevant publica-tions. 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 Spermac. e
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, Spermaceshould 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. Spermaceproved to be an easy and useful staining for the detection of acrosomal
abberations of cat spermatozoa after thawing.
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